Cell Biology and Physiology of CLC Chloride Channels and ...

P1: OTA/XYZ P2: ABCJWBT335-c110038 JWBT335/Comprehensive Physiology June 12, 2012 16:4 Printer Name: Yet to Come

Cell Biology and Physiology of CLC ChlorideChannels and TransportersTobias Stauber,1 Stefanie Weinert,1 and Thomas J. Jentsch*1

ABSTRACT:Proteins of the CLC gene family assemble to homo- or sometimes heterodimers and eitherfunction as Cl– channels or as Cl–/H+-exchangers. CLC proteins are present in all phyla. Detailedstructural information is available from crystal structures of bacterial and algal CLCs. Mammalsexpress nine CLC genes, four of which encode Cl– channels and five 2Cl–/H+-exchangers. Twoaccessory β-subunits are known: (1) barttin and (2) Ostm1. ClC-Ka and ClC-Kb Cl– channels needbarttin, whereas Ostm1 is required for the function of the lysosomal ClC-7 2Cl–/H+-exchanger.ClC-1, -2, -Ka and -Kb Cl– channels reside in the plasma membrane and function in the controlof electrical excitability of muscles or neurons, in extra- and intracellular ion homeostasis, and intransepithelial transport. The mainly endosomal/lysosomal Cl–/H+-exchangers ClC-3 to ClC-7may facilitate vesicular acidification by shunting currents of proton pumps and increase vesicularCl– concentration. ClC-3 is also present on synaptic vesicles, whereas ClC-4 and -5 can reachthe plasma membrane to some extent. ClC-7/Ostm1 is coinserted with the vesicular H+-ATPaseinto the acid-secreting ruffled border membrane of osteoclasts. Mice or humans lacking ClC-7or Ostm1 display osteopetrosis and lysosomal storage disease. Disruption of the endosomalClC-5 Cl–/H+-exchanger leads to proteinuria and Dent’s disease. Mouse models in which ClC-5 or ClC-7 is converted to uncoupled Cl– conductors suggest an important role of vesicularCl– accumulation in these pathologies. The important functions of CLC Cl– channels were alsorevealed by human diseases and mouse models, with phenotypes including myotonia, renal lossof salt and water, deafness, blindness, leukodystrophy, and male infertility. C© 2012 AmericanPhysiological Society. Compr Physiol 2:1701-1744, 2012.

IntroductionThe gene family of CLC chloride channels and transporterswas discovered by the expression cloning of the Torpedoelectric organ Cl– channel ClC-0 in 1990 (179). CLC pro-teins are found in all phyla, from bacteria to men, andcomprise nine members in mammals. Currently three mam-malian CLC proteins are known to assemble with auxil-iary β-subunits (99, 204) that modify their transport activityand influence their intracellular localization. While severalCLCs are chloride channels like its founding member ClC-0(179, 399, 410, 419, 456), many others rather mediate elec-trogenic 2Cl–/H+-exchange (2, 135, 275, 322, 362, 448). Thepresence of different classes of transport activities in homol-ogous proteins offers excellent opportunities for structure-function studies, in particular since crystal structures of CLCproteins from bacteria (92, 93) and algae (108) have beenelucidated. These crystal structures confirmed that CLC chan-nels and transporters are (homo)dimers with two translocationpathways, as previously suggested by biophysical analysisof native (259, 261) and cloned (21) Torpedo channels, andrather stringently demonstrated by site-directed mutagenesis(227, 258, 449). CLC proteins can also form functional het-erodimers with subunits of the same homology branch (225),but the biological relevance of these mixed dimers remainsunclear. Each subunit encloses its own permeation pathway

(92,449) that leads to the “double-barrel” appearance of CLCCl– channels in single-channel recordings. At least in CLCchannels, each pore can be opened and closed (“gated”) byan individual “protopore” gate that is largely or totally in-dependent from the other subunit, but there is also a com-mon gate that acts on both pores simultaneously (259, 261).Voltage-dependent gating is not only observed with CLCchannels but also with CLC exchangers (206, 300). So far,no charged amino acids providing potential protein-intrinsicgating charges have been identified in any CLC protein. Asgating of CLC channels in general depends on Cl–- and H+-concentrations, permeating anions were proposed as gatingcharge (332) and these channels may be directly gated byprotons (221). The negatively charged side chain of a highlyconserved glutamate protrudes into the permeation pathwayand competes with Cl– for a binding site (92, 93). This “gatingglutamate” plays a pivotal role in the gating of CLC Cl– chan-nels (93,103,437) and in coupling H+ to Cl– transport in CLC

*Correspondence to [email protected] fur Molekulare Pharmakologie (FMP) andMax-Delbruck-Centrum fur Molekulare Medizin (MDC), Berlin,Germany

Published online, July 2012 (comprehensivephysiology.com)

DOI: 10.1002/cphy.c110038

Copyright C© American Physiological Society

Volume 2, July 2012 1701


Cell Biology and Physiology of CLC Chloride Channels and Transporters Comprehensive Physiology

ClC-7/Ostm1

ClC-6

ClC-5

ClC-4

ClC-3

ClC-Kb/barttin

ClC-Ka/barttin

ClC-2

ClC-1

Kidney(also: intestine...)

Expression

Skeletalmuscle

Kidney, ear

Kidney, ear

Broad (brain, kidney, liver...)

Broad (brain, kidney, muscle...)

Nervous system

Broad

Broad

Human disease

Myotonia congenita

Bartter III(renal saltloss)

Dent’s disease

Osteopetrosis,retinal degeneration, lysosomal storage (NCL)

Mouse model

Myotonia congenita(adr mouse)

Diabetes insipidus

Degeneration:retina/hippocampus

Defect in renalendocytosis

Function

Acidification of synaptic vesicles,endosomes

Cl– accumulation into lysosomes/acidification of resorption lacuna

Stabilization of membrane potential

Transepithelialtransport

Transepithelialtransport

Cl– accumulation into endosomes/acidification of endosomes

Acidification of late endosomes?

Degeneration of retina and testes/leukodystrophy

Intra/extracellularion homeostasis

?

Osteopetrosis,retinal degeneration, lysosomal storage (NCL)

Lysosomal storage (NCL)

Loss of barttin or both ClC-Ks:Bartter IV(renal salt loss and deafness)C

l– ch

ann

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of

the

pla

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mem

bra

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Cl−

/H+

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of

intr

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sicl

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Same as for ClC-7Same as for ClC-7

Figure 1 The mammalian CLC family of chloride channels and transporters. The CLC family comprises nine members in mammals. Thisoverview depicts their tissue expression (and of the appropriate β-subunits (in red)), cellular function, and known human and mouse pathologies.The members of the first branch of the CLC family, ClC-1, -2, -Ka, and -Kb, are plasma membrane Cl– channels. By contrast, the members of thesecond (ClC-3, -4, and -5) and third (ClC-6 and -7) subfamilies are Cl–/H+-exchangers that localize predominantly to intracellular compartmentsof the endosomal/lysosomal pathway.

2Cl–/H+-exchangers (2, 322, 362). Eukaryotic CLC proteinshave two so-called cystathione-β-synthase (CBS) domains intheir large cytoplasmic C-terminus, which may have a rolein common gating (47, 100, 115). In some CLCs, ATP andother nucleotides can bind at the interface between CBS1 andCBS2 of either subunit (257) which can have an influence ongating of CLC channels (25) or the activity of CLC Cl–/H+-exchangers (482). This may confer some kind of metabolicregulation on anion transport.

The nine mammalian CLC family members can be dividedinto three homology groups (Fig. 1). The first branch con-sists of ClC-1, ClC-2, and ClC-Ka and -Kb, all Cl– channelsthat reside mainly in the plasma membrane. Their functionsinclude the regulation of muscular and neuronal excitabil-ity (345, 392, 397), extracellular ion homeostasis (35, 38),and transepithelial transport (38, 249, 382). Members of thetwo remaining branches of the CLC family (ClC-3 to -5,and ClC-6 and -7, respectively) rather reside predominantlyon intracellular membranes, mainly of endosomes and lyso-somes (140,197,327,402), but also of synaptic vesicles (402).They are important for vesicular ion homeostasis by provid-ing countercurrents for proton pumping (141, 291) and ac-cumulating Cl– into the lumen (448) in a secondary activetransport process (174). Their important cellular functions

are evident from severely reduced endocytosis with a lackof ClC-5 (324) and impaired lysosomal protein degradationwhen ClC-7 is missing (445). Five of the nine human CLCgenes are mutated in genetic disease (194,197,223,366,382),as are both known accessory β-subunits, barttin (33) (whichassociates with ClC-Ka and -Kb (99)) and Ostm1 (57), theβ-subunit of ClC-7 (204). These diseases, as well as sponta-neous and engineered genetic mouse models for these diseases(57,197,324,397) or for CLCs not yet known to underlie hu-man disease (38, 249, 291, 327, 402), have shed considerablelight on the physiological importance of chloride transport.Human and mouse phenotypes include neurodegeneration(183,204,402) and lysosomal storage disease (183,204,327),leukodystrophy (35), blindness (38, 197, 204, 402), deafness(33,343,366), osteopetrosis (57,197), renal salt and water loss(33,249,382), proteinuria (223,324), kidney stones (223), andmale infertility (38). This unexpectedly broad spectrum of dis-ease phenotypes—and there are more to come—demonstratesimpressively the previously unrecognized importance of chlo-ride transport for cells and the organism as a whole.

In this review, we focus on the physiological and cell bio-logical roles of mammalian CLC chloride channels and trans-porters, with individual chapters for each mammalian CLC.The equally interesting structure-function aspects are the

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Comprehensive Physiology Cell Biology and Physiology of CLC Chloride Channels and Transporters

focus of a parallel review by Alessio Accardi (1-4) andwill be mentioned here only as far as they are needed forunderstanding the biological roles of CLC proteins. Forthe interesting roles of CLC transporters in plants, readersare referred to a recent excellent review (480). As entrypoints to the exciting fields of CLCs in yeast and wormsmay serve a recent article on the single yeast CLC gef1p(41) and a report on the regulation of CLCs in C. elegans(104). CLC Cl– channels and transporters were the topicof several other excellent reviews over the past few years(3, 90, 91, 173-175, 178, 222, 260, 325, 330, 479), which mayconvey different perspectives and other details.

CLC Cl– Channels of The PlasmaMembraneClC-1—the major skeletal muscle Cl– channelthat is mutated in myotoniaThe skeletal muscle Cl– channel ClC-1 was the first mam-malian voltage-gated Cl– channel to be identified at the molec-ular level (399) because it is the closest ortholog of the Tor-pedo electric organ Cl– channel ClC-0 (179). The electricorgan has been derived in evolution from skeletal muscleand prominently expresses other ion channels like muscle-type acetylcholine receptors. ClC-1 is almost exclusively ex-pressed in skeletal muscle with only trace amounts beingfound in the heart (399). In mice, ClC-1 is upregulated afterbirth (399). ClC-1 expression levels are higher in fast than inslow muscle and are strongly modulated by its electrical ac-tivity, as revealed by denervation and the analysis of myotonicmouse mutants (189).

Basic biophysical properties

ClC-1 currents are already present at the resting membranepotential of skeletal muscle, but increase further upon de-polarization (399). Consistent with its homodimeric archi-tecture, ClC-1 displays typical “double-barreled” currents insingle-channel recordings with two equally spaced currentsteps (360) but with a much smaller single-channel conduc-tance of about 1.5 pS (333, 360, 449) (compared to ∼10 pSof ClC-0 (21)). A further difference is that the very slowcommon gate of ClC-0 is activated by hyperpolarizationand the much faster protopore gate activated by depolariza-tion, whereas both these gates are fast and depolarization-activated in ClC-1 (360). These features render a detailedbiophysical analysis of ClC-1 gating more difficult than thatof ClC-0.

Like other CLC channels, gating of ClC-1 depends onanions and pH and has been studied extensively (1, 4, 14,15, 26, 56, 87, 88, 349-352, 360). Intracellular ATP and othernucleotides inhibit ClC-1 by shifting its current-voltage rela-tionship to more positive potentials probably by affecting thecommon gate (25, 27, 416, 417). This effect is enhanced by

low intracellular pH (416) and is lost upon oxidation (475),which was probably the reason why another group did notfind effects of ATP on ClC-1 gating (483). The inhibition ofClC-1 by ATP relies on its CBS domains as shown by site-directed mutagenesis (27, 417). The ATP-binding site of theCBS domains of ClC-5 that was revealed by crystallography(257) was used to specifically design ClC-1 mutations inter-fering with the effect of ATP on gating (417). It is believedthat the effect of ATP and intracellular pH on ClC-1 gating isimportant during muscle fatigue (27, 311-313) by enhancingmuscle excitability.

Role of ClC-1 in muscle physiology and myotonia

Whereas the resting conductance of most cells is dominatedby K+, skeletal muscle displays an unusually high Cl– con-ductance (about 80% at rest (42)) that is mediated by ClC-1.It has been proposed that muscles use Cl– channels for re-polarizing their action potentials, because during prolongedexercise repolarizing K+-efflux would lead to an accumu-lation of K+ in the small volumes of t-tubules that pene-trate muscle fibers. Since extracellular K+ concentration isin the range of 5 mmol/L, K+ efflux could lead to a changein extracellular [K+] that may depolarize the muscle mem-brane. By contrast, the same charge transfer would lead to amuch smaller relative change of extracellular Cl– (which hasa much higher extracellular concentration in the 120 mmol/Lrange) and therefore avoid detrimental effects on the restingpotential. This consideration, however, hinges on the local-ization of ClC-1 to t-tubular membranes. Such a localizationwas inferred from a decrease of muscular Cl–-conductance,but not of K+ conductance, when tubules were disrupted byglycerol treatment (302). However, ClC-1 could not be de-tected by immunohistochemistry in t-tubules, but rather onthe sarcolemma (142,307). Two recent studies (85,229) havere-investigated this issue, but come to opposite conclusions.These articles have been commented (102, 484) and it seemsthat there is no final answer yet.

Loss of ClC-1 function leads to myotonia (397), a musclehyperexcitability in which the muscle does not relax properlyafter voluntary contraction. This is due to a train of muscle ac-tion potentials that persists after the excitatory input throughsynaptic transmission at the motor endplate has ceased. Theseso-called “myotonic runs” can be readily recorded frompatients with myotonia. Work from Bryant and colleagueshad demonstrated in the 1960s and 1970s that skeletal muscleCl– conductance is reduced in myotonic goats and a subsetof human patients with myotonia (44, 219, 220). The role ofClC-1 in myotonia was first shown for myotonic adr mice(397), followed by humans (194), and later by goats (22) anddogs (340). In humans, myotonia congenita can be inheritedas a recessive trait (Becker type) and as a dominant disorder(Thomsen’s disease). Dominantly inherited myotonia iscaused by dominant negative mutants that retain their abilityto associate with wildtype (WT) subunits in WT/mutantheteromeric channels in which the mutant subunit impairs the




function also of the WT subunit (398). As ClC-1 is a homod-imeric channel with two largely independent pores, mutantsthat impair permeation or change the protopore gating areunlikely to have dominant effects on associated WT subunitsin WT/mutant heterodimers. Many mutations found in dom-inant myotonia were found to shift the voltage-dependenceof ClC-1 dimers to positive voltages where they can nolonger participate in action potential repolarization (334). Asexpected, these mutations affected the common gate, therebyaffecting also the WT subunit of heterodimers (360). Agree-ing with this observation, many of the mutations found indominant myotonia are located close to the interface betweenboth subunits of the dimer (87). Human dominant mutationsdiffered in the extent of the shift in voltage-dependence, andmutations that caused a more moderate shift were found inboth dominant and recessive myotonia (201). Of course, othermechanisms of dominant negative effects are possible, likeretention of WT/mutant dimers in the endoplasmic reticulum(ER). It should be realized, however, that in theory dominantnegative effects with dimers can decrease currents to min-imally 25%, whereas much larger dominant negative effectsare possible, for example, with tetrameric K+-channels (downto 6.25%). Accordingly, myotonia in patients with dominantThomsen’s disease is less severe than in the recessive Becker-type myotonia where ClC-1 currents may be abolishedcompletely.

Interestingly, Dr. Thomsen, who first described myoto-nia in 1876 (413), was himself affected by that disorderand his CLCN1 mutation has been identified by study-ing his descendants (398). Many different CLCN1 mu-tations have been identified in the meantime (128, 129,193, 200, 201, 233, 268, 326) and summarized in an excellentreview (330).

Myotonia is also a cardinal symptom of myotonic dys-trophy, which is a multisystem disorder caused by nucleotiderepeat expansions in untranslated regions of two genes. Inthose patients, myotonia may be caused by aberrant splicingof ClC-1 that leads to a large decrease in ClC-1 protein levels(58, 238). In a mouse model for myotonic dystrophy, a mor-pholino antisense oligonucleotide targeting a ClC-1 splice sitecould increase ClC-1 protein levels and decrease myotonicdischarges (455).

ClC-1 can be rather specifically inhibited by quite highconcentrations of 9-anthracene-carboxylic acid (9-AC), andthe inhibitor-binding site has been mapped (101). ClC-1can also be inhibited by several other (nonselective)molecules like zinc (88), niflumic acid (213), and 2-(p-chlorophenoxy)propionic acid analogues (212).

ClC-2—many physiological roles of a broadlyexpressed plasma membrane Cl– channelClC-2 is expressed in the plasma membranes of cells frommany tissues, including the brain, intestine, kidney, liver,and heart (410). Like other CLCs, ClC-2 forms homodimericchannels with two identical, largely independent pores and has

a Cl–>I– selectivity sequence. The ClC-2 single-pore conduc-tance of approximately 3 pS is independent from the neigh-boring subunit, as demonstrated with concatemers covalentlylinking ClC-0 to ClC-2 (449). ClC-2-like single-channel cur-rents have been described in cultured cortical astrocytes (290)and hippocampal neurons (443), although they were believedto be mediated by ClC-3 in the latter case.

Basic biophysical properties of ClC-2

The slow activation of ClC-2 by hyperpolarization results instrongly inwardly rectifying macroscopic currents (410). Thethreshold and kinetics of voltage-dependent activation de-pends on the expression system (308) and is influenced by thecholesterol content of membranes (155). Akin to other CLCchannels like the well-studied ClC-0 from Torpedo electricorgan (59,221,332), voltage-dependent gating of ClC-2 is in-fluenced by the concentrations of Cl– and H+. In particular theactivation by a rise in intracellular Cl– concentration, whichshifts the voltage-dependence to a more depolarized voltagerange (285, 331, 358), may be of physiological importance.With low [Cl–]i, also extracellular [Cl–] affects ClC-2 gating(358). ClC-2 is activated by mild extracellular acidification(180, 345), but decreasing extracellular pH further decreasescurrents (16,285). This decrease in current amplitude could beattributed to the titration of an extracellular histidine, whereasthe activation by acidic pH may involve protonation of the“gating glutamate” (284) that is most likely also involved inthe effect of [Cl–] (358).

ClC-2 can also be activated by osmotically induced cellswelling (138, 180). Both activations by swelling and acidicpH depend on the presence of a cytoplasmic amino-terminaldomain that can be transplanted to a site between the twocytosolic CBS domains without loss of function (138, 180).This leads to nearly constitutively open ClC-2 channels whenstudied by two-electrode voltage-clamp in Xenopus oocytes(138, 180) and perforated patch measurements of transfectedhuman embryonic kidney (HEK) cells (427), but surpris-ingly neither in excised patches from oocytes (331) nor inwhole cell recordings of HEK cells (427). This suggests thatan unknown diffusible intracellular factor that is lost in thelatter measurements affects the gating of the deletion mu-tant. The kinetics of ClC-2 gating may be slightly changedby intracellular ATP, which may bind to its CBS domains(286). Surprisingly, ClC-2 still traffics normally to the plasmamembrane and displays hyperpolarization-activated gatingwhen both CBS domains are deleted (126). Using mutage-nesis and heterologous expression, the structure-function re-lationship of ClC-2 has been investigated in detail by severalgroups (126,284,285,358,469,485) (see also contribution byAccardi (1-4)).

Some reports suggest that ClC-2 may be a target forphosphorylation (120, 124, 303, 309), but it is unclearwhether this is of physiological relevance. cAMP-dependentphosphorylation of ClC-2 by PKA does not affect itstransport properties (309). We also ignore whether any of




the proposed interaction partners of ClC-2 (Hsp90 (156),cereblon (158), and the dynein motor complex (80)) are ofbiological relevance. Rapid cycling to and from an endocyticcompartment may regulate the plasma membrane residenceof ClC-2. It depends on a tyrosine-based internalization motifbetween intramembrane helices D and E (67).

ClC-2 is involved in various physiological processes

Native ClC-2-like hyperpolarization-activated Cl– currentshave been observed in whole-cell recordings of several mam-malian cell types, including Sertoli cells (38), sympathetic(64) and hippocampal (345, 391) neurons, rod bipolar cells(97), astrocytes (109, 236, 290), carotid chemoreceptor cells(315), hepatocytes (202), erythrocytes (39, 166), trabecularmeshwork cells (66), colon epithelial cells (168), pancreaticacinar cells (52), as well as salivary acinar (276, 308) andduct (346) cells. In some of these cases (38, 166, 236, 276,345, 346), the identity of these currents with ClC-2 hasbeen confirmed by using cells from Clcn2–/– mice ascontrol.

Several reports showed that native ClC-2-like currentscould be activated by mild extracellular acidification like inheterologous expression (180), for example, in hippocam-pal neurons (345), astrocytes (235), carotid chemoreceptors(315), and parotid acinar cells (16). Swelling activation ofClC-2-like currents was confirmed, for example, in pancre-atic acinar cells (52), the T84 colon carcinoma cell line (121),erythrocytes (166), sympathetic neurons (64), and trabecularmeshwork cells (66). However, although ClC-2 is activatedby cell swelling in salivary acinar cells, no effect on vol-ume regulation could be detected when comparing cells fromWT and knockout (KO) mice (276). In fact, it was evidentfrom the beginning (138, 410) that ClC-2 is distinct fromVRAC (Volume Regulated Anion Channel) (157, 297), theubiquitously expressed swelling activated Cl– channel that isbelieved to be important for regulatory volume decrease. Con-trasting with ClC-2, VRAC is outwardly rectifying, conductsI– better than Cl– and has a larger single-channel conductance.The physiological relevance of swelling-activation of ClC-2remains obscure.

Clcn2–/– mice: retinal and testicular degenerationand leukodystrophy suggest role in extracellular ionhomeostasis

There had been speculation that ClC-2 might be a pathwayfor Cl– secretion across epithelia in parallel to CFTR, or thatit might play a role in gastric acid secretion. However, thesespeculations were not confirmed by our analysis of Clcn2–/–

mice (38), which unexpectedly showed testicular and reti-nal degeneration (38) as well as leukodystrophy (35). Thesephenotypes were confirmed by other groups using an inde-pendently generated mouse model (68, 276) and a mouseline stemming from an 1-ethyl-1-nitrosourea (ENU) muta-genesis screen (94). The early degeneration of photorecep-

tors and germ cells was tentatively attributed to an impairedtransepithelial transport across retinal pigment epithelial cellsand Sertoli cells, respectively (38). Indeed, short-circuit cur-rents across the retinal pigment epithelium were reduced inClcn2–/– mice (38). Both photoreceptors and germ cells arelocated behind a blood-organ barrier and depend on transep-ithelial transport for the supply of nutrients or the removalof metabolites. The activation of ClC-2 by mild extracellularacidification (138) might play a role in regulating the ioniccomposition in the narrow clefts between supporting cells andphotoreceptors and germ cells, respectively (38).

Likewise, a dysregulation of extracellular ion concentra-tion was proposed as mechanism underlying the spongiformvacuolation of white matter tracts in Clcn2–/– mice (35). Vac-uoles appear in the myelin sheaths of central, but not periph-eral, neurons a few weeks after birth and continue to grow insize (Fig. 2). The morphology of axons, even of those withseverely vacuolated myelin sheaths, appeared normal and noneuronal cell death could be detected (35). The reason for thediscrepancy to a recent report (68) describing neuronal cellloss in old Clcn2–/– mice is not entirely clear. Consistent witha myelination defect, nerve conduction velocity was slowedand no obvious neurological phenotype (with the exceptionof blindness) was detected (38).

In the brain, ClC-2 immunoreactivity was detected both inneurons and glia (35, 379), for instance in pyramidal cells ofthe hippocampus and in Bergmann glia. Importantly, ClC-2was found in astrocytic endfeet that contact the endotheliumof brain capillaries, where it colocalizes with the Kir4.1K+ channel and the aquaporin 4 water channel. In addition,ClC-2 labeling was found close to the plasma membrane ofoligodendrocytes (35), a localization shared by Kir4.1 and thegap junction protein connexin 47 (Cx47). These proteins arebelieved to have a role in “potassium siphoning,” a processby which glial cells remove extracellular K+ from neuronsand equilibrate it with the extracellular space through endfeetthat contact capillaries. Extracellular K+ would otherwiseincrease in the narrow clefts surrounding neurons dueto K+ exit during the repolarization of action potentials.Importantly, loss of Kir4.1 and the disruption of both Cx32and Cx47 resulted in myelin vacuolation resembling thatseen in Clcn2–/– mice (255, 277). Optic nerve vacuolationincreased with neuronal activity and could be blocked bypreventing optic nerve activity with tetrodotoxin injection(256). Likewise no vacuolation of the optic nerve was seen inClcn2–/– mice whose optic nerves are electrically silent dueto their retinal degeneration (35). These observations suggestthat glial ClC-2 has a role in regulating the extracellularion composition in the fluid space surrounding neurons. Nodisease-causing CLCN2 mutation could be identified in a het-erogeneous group of 150 patients with leukodystrophy (35).As the leukodystrophy of Clcn2–/– mice is strikingly similarto that observed in megalencephalic leukoencephalopathywith subcortical cysts, 18 patients with that disease and lack-ing MLC1 mutations were examined for CLCN2 mutations.However, no disease-causing mutation could be found (365).




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Figure 2 Spongiform vacuolation in the white matter of ClC-2 knockout (KO) mice. Semithin brainsections of the middle cerebellar peduncle of 2-, 5-, and 14-month-old wildtype (WT) and ClC-2 KOmice show abundant vacuoles (asterisks) in the cerebellar white matter of 5- and 14-month-old KO butnot WT animals. C, capillaries; scale bar, 20 μm. [Image adapted from, reference (35), with permission.]

Regulation of intraneuronal Cl– concentration andneuronal excitability

The expression of ClC-2 in neurons led to the speculationthat it serves to lower intraneuronal Cl– concentration tothe levels necessary for synaptic inhibition through GABAA-and glycine receptors (391). Obviously, and in contrast tothe neuronal K-Cl-cotransporter KCC2, the ClC-2 Cl– chan-nel can lower [Cl–]i only to its electrochemical equilibriumvalue. Transfection of ClC-2 into dorsal root ganglion neu-rons, which do not normally express ClC-2 or the major Cl–

extruder KCC2, resulted in a large shift of the Cl– equilibriumpotential and prevented the excitatory action of GABA (392).It was therefore speculated that the loss of ClC-2 may leadto neuronal hyperexcitability and epilepsy, but Clcn2–/– micedid neither show spontaneous epilepsy, nor a reduced seizurethreshold (35, 38). In old Clcn2–/– mice, however, seizureswere induced more easily by GABAA receptor blockade thanin WT mice, but these seizures were attributed to neurode-generation and inflammation rather than to a change in theintracellular Cl– concentration (68). A widely cited reportclaiming that CLCN2 mutation cause idiopathic generalizedepilepsy in humans has been retracted (147) because of sev-eral severe flaws (188) and there is no evidence that CLCN2mutations underlie epilepsy in humans (35, 283, 286).

The role of ClC-2 in regulating neuronal Cl– concen-tration and its impact on synaptic transmission was investi-gated in detail in two recent studies (114, 345). Rinke et al.(345) studied hippocampal CA1 pyramidal cells which areknown to express ClC-2 (379, 387) and measured robust in-wardly rectifying chloride currents with typical features ofClC-2, including slow gating, stimulation by mild extracel-lular acidification, and reduced amplitude with iodide. Theabsence of these currents in Clcn2–/– mice confirmed that

they were mediated by ClC-2. These currents were not seenat early postnatal ages and were fully developed around post-natal day 10 (P10). WT neurons extruded Cl– more rapidlythan those from Clcn2–/– mice when they were loaded withCl– through activated GABAA receptors, demonstrating thatClC-2 is an efficient Cl– extruder when [Cl–]i is above elec-trochemical equilibrium. Rather surprisingly, comparison ofmembrane resistance between WT and Clcn2–/– cells suggeststhat ClC-2 contributes about 40% to the resting membraneconductance (345). As a consequence from their higher in-put resistance, Clcn2–/– pyramidal cells showed increased ex-citability compared to WT cells. However, this increased ex-citability of pyramidal cells did not lead to an increased basalsynaptic transmission in field recordings, and input/output re-lation was rather decreased (345). This surprising observationwas explained by increased feedforward inhibition throughGABAergic interneurons, which may be hyperexcitable. Thedecreased hippocampal excitability agrees well with the lackof epilepsy or increased seizure thresholds in Clcn2–/– mice(35, 38).

The study by Foldy et al. (114) investigated the regulationof inhibitory basket cell synapses on hippocampal pyramidalcells by ClC-2. Whereas fast-spiking, parvalbumin-positivebasket cell synapses were closely associated with ClC-2 thatprevented neuronal Cl– loading as suggested previously (387),cholecystokinin-positive basket cells were less influenced byClC-2. These experiments suggest different Cl–-regulatorymechanisms at these synapses that show differential distribu-tion on somata and dendrites in pyramidal cells, and the exis-tence of intraneuronal Cl– gradients, at least during synapticactivity. It will be interesting to fine-map the spatial localiza-tion of different types of inhibitory synapses in comparisonwith Cl–-extruders such as KCC2 and ClC-2, and to perform




perforated-patch measurements that are less likely to interferewith [Cl–]i than the whole-cell patch-clamp technique used inthose studies (114, 345).

ClC-2 in transepithelial transport

ClC-2 is also prominently expressed in several epithelia, in-cluding those affected in cystic fibrosis (CF) (410) and ithas been speculated that ClC-2 might provide an alternativepathway for Cl– secretion by partially substituting for the Cys-tic Fibrosis Transmembrane Conductance Regulator (CFTR)(372, 410). This issue was explicitly addressed by crossingCftr mouse mutants with Clcn2–/– mice (470). CFTR KOmice, or mice carrying the equivalent of the most frequenthuman CF mutation �F508, do not show the typical lungsymptoms of most human CF patients, but rather a severeintestinal phenotype resembling meconium ileus in CF in-fants and die early from intestinal obstruction. If ClC-2 pro-vides an apical Cl– conductive pathway in parallel to CFTR,additional disruption of ClC-2 might worsen the intestinalphenotype and even lead to a lung phenotype. However, themorphology of these tissues was indistinguishable betweenmice mutant for only CFTR and CFTR/ClC-2 double mu-tants, and Cftr�F508/�F508/Clcn2–/– mice survived even betterthan Cftr�F508/�F508 littermates (470). Moreover, under certainexperimental conditions, short-circuit currents across colonicepithelia were larger rather than smaller in Clcn2–/– mice com-pared to WT mice. These data suggested that ClC-2 is rathera basolateral Cl– channel in intestinal epithelia.

Due to poor specificity of antibodies and a lack of appro-priate controls in many studies, there are conflicting reportsas to the subcellular localization of ClC-2. Although somereports suggested that ClC-2 resides in apical membranes orclose to tight junctions (143), several groups now demon-strated convincingly that ClC-2 indeed localizes to basolat-eral membranes of intestinal epithelia (colon and jejunum)(54, 55, 218, 314) by immunohistochemistry that was in partcontrolled using Clcn2–/– tissue (314) (Zdebik and Jentsch,unpublished results). Transfection of ClC-2 into Caco-2 in-testinal cells and Madin Darby canine kidney (MDCK) ep-ithelial cells also resulted in a basolateral localization of theprotein (314). This localization depends on a dileucine sort-ing motif in the second CBS domain of ClC-2 that apparentlyinteracts with the μ1B, but not the μ1A subunit of the AP-1sorting complex (314). Moreover, ClC-2 is expressed in in-testinal surface epithelia (54, 55, 168, 314) and not in the se-cretory crypts that coexpress NKCC1 and CFTR, suggestingthat ClC-2 is involved in Cl– resorption rather than secretion(470). In this respect it is important to note that lubiprostone,a drug used in treating constipation, was claimed to exert itseffect by stimulating intestinal Cl– and fluid secretion throughdirect activation of apical ClC-2 Cl– channels (11, 48). How-ever, this claim is based on a single publication that reportedeffects of this drug on currents that differ substantially fromtypical ClC-2 currents (71). In view of the basolateral local-ization of ClC-2 in reabsorptive intestinal epithelia, ClC-2

activation may rather be useful in diarrhea than in constipa-tion. Lubiprostone may rather work through the activation ofprostaglandin receptor subtypes (20,72), as already suggestedby its chemical structure, and might exert its effect on con-stipation by indirectly activating CFTR (13, 31) or by othereffects (75).

Neither immunohistochemistry with KO-controlled anti-bodies (314), nor Western blotting detected the ClC-2 proteinin the stomach (161). In contrast to earlier speculation (237),Clcn2–/– mice showed no impairment of gastric acid secretion(38). KO-controlled immunohistochemistry detected ClC-2in basolateral membranes, acinar and duct cells of salivaryglands, which also displayed typical ClC-2 currents (346).Nevertheless, Clcn2–/– mice displayed no significant alter-ation in saliva secretion (346).

Pharmacology

ClC-2 Cl– channels are rather poorly inhibited by nonspecific“anion transport inhibitors” such as 4,4′-diisothiocyano-2,2′-stilbenedisolfonic acid (DIDS), 9-AC, N-phenylanthranilicacid (DPC), and 5-nitro-2-(3-phenylpropylamino)benzoicacid (NPPB) (410, 411), with more potent inhibition observedwith Cd2+ and in particular with Zn2+ which inhibits in the10 to 100 μmol/L concentration range (64). However, thesesubstances are hardly specific. A venom component from thescorpion L. quinquestriatus hebraeus has more recently beenshown to partially inhibit ClC-2 (411). It was subsequentlypurified, shown to be identical to leuroperide II and renamedGaTx2 (412). It acts at subnanomolar concentration and slowschannel opening. GaTx2 lacks effects on single-channel con-ductance and does not affect open channels (412). The incom-plete inhibition of ClC-2 by this “gating modifier” limits itsusefulness as a pharmacological probe for ClC-2 function.

ClC-K/barttin channels: transepithelialtransport in kidney and inner earTwo closely related CLC channels that are almost exclusivelyexpressed in kidney and inner ear (ClC-Ka and ClC-Kb inhumans, or ClC-K1 and -K2 in rodents) (5, 187, 419) haveprobably evolved from a recent gene duplication. The genesencoding these isoforms are located side by side on humanchromosome 1p36 (40,354,382) where they are separated byapproximately 11 kb of DNA (382). The different terminologyfor human and rodent genes has been chosen because it wasimpossible to assign species homologs by sequence compar-ison. The degree of amino acid identity (roughly 90% (187))was slightly higher within a species (e.g., between rodent ClC-K1 and ClC-K2) than across species, a fact that also precludedthe generation of antibodies that reliably distinguish betweenboth subunits. However, based on their physiological rolesand their expression pattern along the nephron, it is now clearthat rodent ClC-K1 is the ortholog of human ClC-Ka, and thatClC-K2 corresponds to ClC-Kb. This classification is furtherbolstered by the position of these genes on the chromosomes:ClC-Kb and ClC-K2 are located closer to the telomere thanClC-Ka and ClC-K1.




The β-subunit barttin modifies biophysicalproperties and influences trafficking and stability ofClC-K α-subunits

In heterologous expression in Xenopus oocytes or trans-fected mammalian cells only rodent ClC-K1 gave currents(419, 437). Currents initially reported (5) for ClC-K2 areprobably endogenous to Xenopus oocytes because a mu-tant deleting important stretches in the transmembrane partyielded similar currents. Chimeras between ClC-K1 and ClC-K2 needed a part of the transmembrane region of ClC-K1(roughly helices N to R) for functional expression (437). Thebiophysical properties of this chimera differed from thoseof ClC-K1 and were thought to reflect more the proper-ties of ClC-K2. ClC-K1 currents are nearly ohmic and, incomparison to other CLCs, display little gating relaxations.They display a characteristic Br–>Cl–>I– halide selectivitysequence (419,437). When valine 166 was replaced by a glu-tamate to conform to the consensus sequence at this position,ClC-K1 was converted to an inward rectifying channel withrobust-gating relaxations (437). It is now clear that we hadinserted a “gating glutamate,” whose more general role inthe gating of CLC channels became apparent after the crys-tallization of bacterial CLC proteins (92, 93). ClC-K chan-nels are unique among mammalian CLCs in not displayingthis gating glutamate. It appears that Nature has eliminatedthe “gating glutamate” in ClC-K channels to create channelsthat are open over a broad voltage range—a feature that maybe desirable for transepithelial transport. One should pointout, however, that gating is abolished neither in ClC-K1, norin ClC-K/barttin channels, as is evident from macroscopiccurrents as well as from single-channel analysis. Likewise,mutating the “gating glutamate” of the well-studied Torpedochannel ClC-0 to alanine abolishes the voltage-dependenceof gating, but not gating itself, as is evident from the open-ing and closing of single channels (93). The gating of ClC-K/barttin channels has been the subject of several studies(110, 132, 245, 319, 367).

The failure to observe currents upon heterologous expres-sion of ClC-K2, -Ka, and -Kb (187) was puzzling, in par-ticular since immunohistochemistry revealed that they residein the plasma membrane of specific nephron segments, andbecause mutations in CLCNKB in the salt-losing nephropathyBartter syndrome III clearly indicated a role in transepithelialtransport (382). These observations raised the suspicion thatClC-K proteins may need an accessory β-subunit for func-tion (437). Indeed, this turned out to be the case. Hildebrandtand colleagues (33) identified the gene (BSND) underlyingBartter syndrome type IV, an autosomal recessive diseasethat combines severe renal salt loss with congenital deafness(203). It was shown shortly thereafter that barttin, the 320-residue protein with two transmembrane proteins encodedby the BSND gene, led to robust Cl– currents when coex-pressed with either ClC-Ka or -Kb in Xenopus oocytes (99).The voltage-dependence differed between ClC-Ka/barttin andClC-Kb/barttin (99), but both channels were inhibited by ex-

tracellular acidification and activated by raising extracellular[Ca2+] (99). The Ca2+-binding site has recently been mappedat the interface of both ClC-K α-subunits (132). Interestingly,an intracellular mutation identified in Bartter III patients alsoaffects ClC-Kb/barttin Ca2+-sensitivity through a long-rangeeffect (245). The physiological significance of the stimula-tion by extracellular Ca2+ is unclear, as is the modulation byextracellular pH.

Barttin increased the surface expression of ClC-K (but notother CLC) subunits in Xenopus oocytes (99, 436) and sim-ilarly affects the subcellular localization of ClC-K channelsin transfected mammalian cells (148,170,367). In transfectedepithelial MDCK cells, ClC-K/barttin channels localize tothe basolateral membrane (170) just like they do in the kidney(with the exception of the thin limb (420)) and the stria vas-cularis (99). A mutant barttin identified in Bartter IV (E88X),however, allowed trafficking to both basolateral and apicalmembranes of transfected cells (170), showing that no domi-nant sorting signals are present in the α-subunits. In tissues ofBsnd–/– mice, ClC-K α-subunits are almost undetectable byimmunhistochemistry (343), suggesting that they need barttinfor protein stability. In the absence of their cognate β-subunit,ClC-K subunits may be retained and degraded in the ER by aquality control mechanism.

In addition to be needed for the proper trafficking andstability of ClC-K subunits, barttin also changes the func-tional properties of ClC-K α-subunits. Comparison of ClC-K1 and ClC-K1/barttin suggested that in the presence of theβ-subunit the stimulatory effect of extracellular Ca2+ reachedits maximum already at 1.8 mmol/L (436). However, this ob-servation conflicts with a detailed investigation which showedthat the stimulatory effect of Ca2+ on ClC-Ka/barttin andClC-Kb/barttin does not even saturate at 50 mmol/L Ca2+

(132). Experiments with ClC-1/ClC-Kb concatemeric chan-nels (367) revealed that barttin is needed to turn on the iontransport activity of the α-subunit. The transmembrane part ofbarttin is needed for its association with ClC-K α-subunits andfor their transport to the plasma membrane, whereas an ad-ditional short cytoplasmic stretch immediately following thesecond transmembrane domain is needed for the activation oftransport activity (367). Further away from the plasma mem-brane barttin displays a tyrosine residue (Y98) which is in asequence context compatible with either a Y-based endocyto-sis motif or a PY-motif for WW-domain containing E3 ubiqui-tin ligases (99). Mutating this tyrosine to alanine increased thesurface residence and conductance upon expression in Xeno-pus oocytes (99), an observation that is compatible with eitherhypothesis. It was reported that the E3 ubiquitin ligase Nedd4is involved in downregulating ClC-K/barttin through this mo-tif (96), but unpublished preliminary experiments from ourlaboratory do not support this conclusion. Coimmunoprecip-itation experiments and immunolocalization experiments intransfected cells were used in an attempt to define the regionsof ClC-K subunits that interact with barttin (405). It was sug-gested that helices B and J interact with the transmembranepart of barttin. The precise stoichiometry of ClC-K/barttin




remains to be determined, although an obvious guess wouldbe 2:2 for a homodimeric channel. It remains unclear whyrodent ClC-K1 yields currents also without barttin.

Nonstationary noise analysis was performed (367) withClC-K1(V166E) because its voltage- and time-dependent gat-ing (437) renders it better amenable for this type of analy-sis. These measurements suggested that barttin modulates thesingle-channel conductance, increasing it from approximately6.5 to 19.5 pS (367). These values are in reasonable agreementwith a later study using single-channel patch-clamping (∼10pS without and ∼22 pS with barttin) (110). Surprisingly, thisaugmentation of the single-channel conductance of ClC-K1was only observed in the mutant in which the V166E mutationhad introduced a “gating glutamate” (110). The unitary con-ductance of WT ClC-K1 was determined to approximately33 pS in the absence or presence of barttin (110). Barttinincreased macroscopic ClC-K1/barttin currents by openingthe common gate (110). Single-channel recordings had thetypical “double-barreled” appearance of the Torpedo channelClC-0, but differed markedly from basolateral Cl– channelsin cells of distal convoluted tubules, connecting tubules, andcortical colleting duct, which were speculated to representClC-K/barttin (226, 289). These native channels displayedseveral typical hallmarks of ClC-K/barttin (appropriate pH-and Ca2+-sensitivity), but their ion selectivity did not fit per-fectly and they displayed a single-channel conductance ofapproximately 9 pS. Moreover, compared to heterologouslyexpressed ClC-K1/barttin (110), their gating was slower byat least one order of magnitude (226). Correlating heterolo-gously expressed ClC-K/barttin with native kidney channelsremains a challenge.

ClC-K/barttin in renal NaCl reabsorption andgeneration of osmotic gradients

As expected for an obligatory β-subunit, barttin colocalizeswith ClC-K subunits in all cells where these channels are ex-pressed (99). This includes basolateral membranes of the thinand thick limb of Henle’s loop and of intercalated cells inthe kidney, and basolateral membranes of marginal cells ofthe potassium-secreting, multilayered epithelium of the striavascularis in the inner ear (99). The renal expression patternagrees with that of several previous studies examining ClC-Kexpression along the nephron by immunohistochemistry (190,191, 253, 420, 424), in situ hybridization (468), and RT-PCRof microdissected nephron segments (187, 406, 424). In con-trast to other nephron cells where ClC-K resides exclusivelyin the basolateral membrane, ClC-K1 was found in both api-cal and basolateral membranes of the thin limb of Henle’sloop (420). The lack of isoform-specific antibodies has pre-cluded a detailed analysis of the differential distribution ofboth isoforms along the nephron. However, the lack of ClC-Kstaining in the thin limb of Henle’s loop showed that ClC-K1, but not ClC-K2, is expressed in that nephron segment,and that ClC-K2 is expressed in basolateral membranes ofthe thick ascending limb (TAL), the connecting tubule, and

α-intercalated cells (191). Furthermore, transgenic mice ex-pressing enhanced green-fluorescent protein (EGFP) underthe control of the human CLCNKB promoter (192) confirmedthese results and established that ClC-K2 and ClC-Kb are or-thologs in rodents and human, respectively. The latter micealso showed that ClC-Kb is expressed in marginal cells of thestria vascularis and in dark cells of the vestibular organ (192),consistent with the immunohistochemical labeling for ClC-K(99,353) and barttin (99). However, the additional labeling ofother cells in the inner ear, for example, fibrocytes and satel-lite cells of the spiral ganglion (232) has not been observed inKO-controlled immunohistochemistry. It seems possible thatthe CLCNKB promoter elements used to drive EGFP (192)does not fully recapitulate native ClC-Kb expression.

The physiological importance of ClC-K channels becameclear from human mutations in CLCNKB (encoding ClC-Kb)in Bartter syndrome type III (382) and of BSND (encodingbarttin) in Bartter syndrome type IV (33), as well as frommouse models in which Clcnk1 (249) or Bsnd (343) weredisrupted.

The salt-losing nephropathy Bartter syndrome (for re-views see (150, 274, 377)) is genetically heterogeneous. Acommon functional denominator is the impairment of saltresorption in the TAL of Henle’s loop. Like in many otherepithelia, transcellular transport is driven by the basolat-eral Na,K-ATPase (Fig. 3A) that lowers intracellular Na+-concentration. The sodium gradient is used by the NaK2Cl-cotransporter NKCC2 to accumulate Cl– in the cell in a sec-ondary active transport process. Cl– then leaves the cell acrossthe basolateral surface through Cl– channels embodied byClC-Kb/barttin Cl– channels. Together with the outward Na+

transport by the Na,K-ATPase, this results in net NaCl reab-sorption in the TAL. However, NKCC2 not only accumulatesCl– in the cytoplasm but also accumulates K+. For the trans-port to function, K+ ions must be recycled over the apicalmembrane of TAL cells through the renal outer medullarypotassium channel (ROMK) (Kir.1; encoded by the KCNJ1gene) K+ channels. In strong support of this transport model,mutations in NKCC2 have been identified in Bartter syndrometype I (383), mutations in KCNJ1 in Bartter syndrome typeII (384), mutations in CLCNKB in Bartter syndrome typeIII (382), and mutations in BSND in Bartter syndrome typeIV (33). Furthermore, there are activating mutations in theextracellular Ca2+-sensing receptor that lead to Bartter-likesymptoms (429, 447) (also called Bartter V). Activation ofthis G-protein-coupled receptor suppresses TAL transport.

Disease-causing mutations in CLCNKB include deletions,missense mutations, and nonsense mutations resulting in atruncation of the protein (123, 196, 382), which is also thecase for BSND (32, 33). Several of these mutants were stud-ied electrophysiologically and by immunocytochemistry inheterologous expression (99, 148, 170).

Generally associated with hypokalemic alkalosis, sev-eral forms of Bartter syndrome can be distinguished clini-cally based on their time of onset, secondary elevation ofprostaglandin E, and different effects on the loss of certain




ATP 2K+

3Na+

2Cl–

K+Na+

NKCC2Cl–ClC-Kb

barttin

Apical

ROMKK+

TAL of Henle’s loop (kidney)

ATP 2K+

3Na+

Cl–

Cl–

2Cl–

K+Na+

NKCC1K+ KCNQ1

KCNE1

ClC-Kb

barttin

ClC-Ka

barttin

Apical

Marginal cells of thestria vascularis (cochlea)

BasolateralBasolateral

(B)(A)

Figure 3 Roles of ClC-K/barttin in transepithelial transport. (A) Schematic representation of renalNaCl reabsorption in the thick ascending limb (TAL) of Henle. Active transport by the basolateral Na,K-ATPase drives NaCl uptake by the apical NKCC2 transporter. Cotransported K+ is recycled throughthe apical ROMK channel and Cl– leaves the cell through basolateral ClC-Kb/barttin. (B) Schematicrepresentation of K+ secretion in the stria vascularis of the cochlea. K+ is taken up by the basolateralNKCC1 transporter and the Na,K-ATPase. While K+ exits through the apical KCNQ1/KCNE1 channel,Cl– is recycled by basolateral ClC-Ka/barttin and ClC-Kb/barttin channels.

ionic species such as K+ and Ca2+ (150, 274, 377). The dif-ferential distribution of the ion transport proteins affected inthe different forms of Bartter syndrome certainly contributesto these different phenotypes. For instance, ROMK (Ki1.1) isalso present in apical membranes of Na+-reabsorbing prin-cipal cells of the distal nephron where it contributes to K+-secretion and the so-called “Na-K-exchange.” ClC-Kb/barttinis also present in basolateral membranes of distal tubular acid-secreting α-intercalated cells (99) where it may indirectlyfacilitate acid-secretion by recycling Cl– for the basolateralCl–/HCO3

–-exchanger AE1 like the KCl-cotransporter KCC4which is expressed in the same membrane (36). The most ob-vious example for a difference in phenotypes is given byBartter syndrome type IV, which is most often caused byloss-of-function mutations in BSND that encodes barttin (33).Since barttin is a functionally required β-subunit of both ClC-Ka and ClC-Kb (99), loss of barttin results in a loss of bothClC-Ka/barttin and ClC-Kb/barttin Cl– channels. This entailsa more severe renal phenotype than the loss of ClC-Kb in Bart-ter III, and leads to an entirely new phenotype, that is, congeni-tal sensorineural deafness. Of note, identical phenotypes wereobserved in two families in which both CLCNKA and CLC-NKB were disrupted (292,366). Because of their adjacent lo-calization on the chromosome deletions can affect both genes.

The localization of ClC-K/barttin channels to marginalcells of the stria vascularis suggested that deafness in BartterIV results from an impairment of strial K+-secretion (99). Theendolymph of the cochlear scala media has a highly unusualion composition in which Na+ is almost completely replacedby K+. Together with the equally unusual endocochlear

potential of +80 to +100 mV, this high K+-concentrationprovides a large electrochemical driving force for K+-entrythrough mechanosensitive channels of sensory hair cells(471). The transport model for K+-secretion by marginalcells of the stria vascularis, which is located in the lateralwall of the scala media, includes an apical K+ channel that iscomposed of ion-conducting KCNQ1 (Kv7.1) α-subunits andKCNE1 β-subunits (Fig. 3B). Loss of either subunit leads todeafness (280, 370). K+ is taken up through the basolateralmembrane of marginal cells by the combined action of theNa,K-ATPase and the NaK2Cl-cotransporter NKCC1. Thelatter transporter needs basolateral Cl– channels for recyclingCl–, akin to the role of ROMK in apical K+-recycling forNKCC2 in the TAL. These Cl– channels were proposed to beembodied by ClC-Ka/barttin and ClC-Kb/barttin (99). Withdeletion of only one of these channels, as with CLCNKBmutations in Bartter III, the other ClC-K/barttin channel canstill provide sufficient transport activity. Only disruption ofboth channels leads to deafness.

The renal phenotype in Bartter IV is more severe than inBartter III because of the additional loss of ClC-Ka/barttinfunction. There are no patients with mutations only in CLC-NKA, but the orthologous Clcnk1 has been disrupted in mice(249). Consistent with the localization of ClC-K1 in the thinlimb of Henle’s loop, which is involved in creating the hy-perosmolarity of the renal medulla, Clcnk1–/– mice displayednephrogenic diabetes insipidus (249). Experiments with iso-lated perfused thin limbs proved that ClC-K1 provides thehigh Cl– permeability of that nephron segment (249). Papil-lary osmolarity was significantly lower in Clcnk1–/– than in




WT mice (7), explaining the inability to adequately concen-trate the urine in the distal nephron.

To explore the pathophysiology of Bartter syndrome IV,we disrupted Bsnd in mice (343). Constitutive disruption ledto severe dehydration due to renal salt and fluid loss and micedid not survive longer than a few days. To study the role ofClC-K/barttin channels in hearing and deafness, Bsnd wasselectively eliminated in the inner ear, but not in the kidney.Immunohistochemistry of cochlear stria vascularis revealedthat not only labeling for barttin, but also for the ClC-K α-subunits was abolished. This suggests that the ClC-K proteinis unstable without its β-subunit.

Total loss of ClC-K/barttin Cl– channels leadsto deafness

Agreeing with the congenital deafness of patients with Bart-ter IV, inner-ear-specific Bsnd KO mice displayed a profoundhearing loss (of ∼60 dB) that was already present at 3 weeksof age, 1 week after the time when mice begin to hear. Thehearing loss remained stable over time (343). In mouse mod-els with severely impaired strial K+-secretion like Nkcc1–/–

(77) or Kcne1–/– (430) mice, Reissner’s membrane that sepa-rates the scala media from the scala vestibuli collapses. Sur-prisingly, this was not found in inner-ear-specific Bsnd KOmice, suggesting that strial K+- and fluid-secretion is notseverely impaired. Indeed, measurements with ion-selectivemicroelectrodes showed that the K+ concentration in the en-dolymph of the scala media was normal. By contrast, theendocochlear potential was strongly reduced (from a WTvalue of ∼100 mV to about 15 mV). The reduction in en-docochlear potential is expected to diminish, but not abolishthe mechanotransduction currents of sensory cochlear haircells, which are predominantly carried by K+ ions. To de-termine the impact of the reduced apical driving force forK+-entry into sensory outer hair cells (OHCs), we measuredotoacoustic emissions. OHCs display electromotility and am-plify sound by contracting at the same frequencies as incom-ing sound, thereby generating sound themselves. The lackof these otoacoustic emissions in inner-ear-specific Bsnd KOmice showed that mechanical sound amplification by OHCs,which increases the hearing sensitivity by about 60 dB (348),is abolished in these mice. This result agrees surprisingly wellto the approximately 60-dB hearing loss of Bsnd–/– mice andsuggests that the reduction of electrochemical driving forcefor the mechanosensitive channels of inner hair cells, whichare noncontractile and directly generate the electrical signalsthat are conveyed to the brain, is not a major factor in deafnessassociated with Bartter IV.

Otoacoustic emissions were measured in young micewhose OHCs showed no morphological abnormalities. How-ever, the lack of strial ClC-K/barttin channels also leads toprogressive OHC degeneration, with OHCs in high-frequencybasal turns being lost about 6 weeks after birth. OHCs in thelow-frequency apical turns can survive for many months. Thephysical loss of OHCs does not contribute significantly to

hearing loss of Bsnd KO mice as OHCs are nonfunctional asshown by otoacoustic emissions. We similarly found degen-erative processes in the stria vascularis itself. The mechanismfor these degenerative processes remains unclear.

The normal endocochlear K+ concentration in inner-ear-specific Bsnd KO mice suggests that there is yet another, sofar unknown mechanism for basolateral Cl– exit in marginalcells of the stria vascularis. The normal steady state [K+],however, does not imply that the capacity for K+-secretionof the stria is normal. Indeed, when conditional Bsnd micewere crossed with a Cre line that totally deletes Bsnd in theinner ear, and additionally to some degree in the kidney, weobserved a collapse of Reissner’s membrane (343). This sug-gested that the K+-secretory capacity is indeed reduced inthe absence of barttin. This capacity may be just sufficientto keep endocochlear [K+] at normal levels when barttin isonly absent from the stria, but no longer with the changes inelectrolytes and hormones that are associated with renal saltloss in Bartter IV. These observations may explain why thehearing loss in adult Bartter IV patients often exceeds 60 dB(343).

How, then, does the loss of strial ClC-K/barttin chan-nels lead to the reduction in endocochlear potential? Workwith ion-selective microelectrodes (287) and Kir4.1 KO mice(241) strongly suggests that this voltage is predominantly aK+-diffusion potential that is generated through Kir4.1 K+

channels located in the intermediate cells (12) of the multi-layered strial epithelium (for reviews see (153,471)) (Fig. 4).This potential is high because the K+-concentration in thecleft between marginal and intermediate cells is kept lowdue to the avid K+-uptake by the NaK2Cl-cotransporter andNa,K-ATPase of marginal cells. Disruption of the basolat-eral ClC-K/barttin, Cl–-recycling pathway is expected to leadto an increase of [K+] in this cleft and hence to a decreasein this K+-diffusion potential. In addition, currents throughClC-K/barttin Cl– channels continuously depolarize the baso-lateral membrane of marginal cells during strial K+-secretion.The lack of this depolarizing current will also decrease theendocochlear potential. Mathematical modeling is needed formore quantitative predictions (154).

In addition to marginal cells of the stria vascularis, ClC-K/barttin channels are also expressed in basolateral mem-branes of potassium-secreting dark cells of the vestibular or-gan. Consistent with this localization, inner-ear-specific BsndKO mice also displayed slight vestibular symptoms (343).Contrasting with cochlear OHCs, no degeneration of vestibu-lar hair cells was observed.

Of note, some BSND mutations may entail congenitaldeafness that is associated only with mild renal symptoms(264) or nearly normal renal function (342). Japanese pa-tients with a G47R BSND mutation were congenitally deaf,but had only rather mild renal abnormalities (264). AnotherBSND mutation (I12T) was identified in several Pakistanifamilies with recessive autosomal, nonsyndromic deafness ofthe DFNB73 type (342). Patients homozygous for this mutantallele were congenitally deaf like patients with Bartter IV,




(A)

Reissner’s membraneScalamedia

K+

Striavascularis

Organ of Corti

+100 mV140 mmo/L [K

+]

etaidemretnIsllec lanigraMcells

Scala mediaendolymph

Basalcells

Fibrocytes

Bloodvessel

Stria vascularisintrastrial fluid

Ligamentum spiraleperilymph

K+

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2Cl–

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+80 mV150 mmol/L [K+]

+90 mV150 mmol/L [K+]

+100 mV1 mmol/L [K+]

0 mV5 mmol/L

[K+]

0 mV150 mmol/L

[K+]

0 mV150 mmol/L

[K+]

Intrastrialspace

Gap junctions

Tight junctions

NKCC1

Kir4.1

KCNQ1KCNE1

ClC-Kbbarttin

ClC-Kabarttin

Na/K ATPase

K+

(B)

Cl–

PostulatedCl– channel

Figure 4 ClC-K/barttin in the cochlea. (A) Model of potassium recycling in the cochlea of the inner ear. The endolymph in the cavity ofthe scala media displays a high K+ concentration of 140 mmol/L and a positive potential of +100 mV. Both the high [K+] and the potentialare established by the stria vascularis (light blue). Both parameters are important for the depolarizing K+ current through mechanosensitivechannels in the apical membrane of inner (red) and outer (green) hair cells. K+ leaves these sensory cells basolaterally into the perilymph,which displays the zero potential and low [K+] of normal extracellular space. While the perilymph is separated from the endolymph bytight junctions, potassium is transported back to the stria vascularis through a gap junction system. (B) The scheme represents a model ofhow potassium is secreted into the endolymph through the stria vascularis. In this multilayered epithelium, a layer of marginal cells, whichare connected by tight junctions and apically face the endolymph, and a layer of basal cells, also connected by tight junctions, isolate anintrastrial space with a low K+ concentration. K+ enters this space through Kir4.1 from intermediate cells, which can receive K+ througha system of gap junctions, and it is taken up by marginal cells that secrete it into the endolymph. Cl– exits the marginal cells throughbasolateral ClC-K/barttin channels. The presence of another, unidentified Cl– channel on the basolateral side is suggested by the normalendocochlear K+ concentration upon inner-ear-specific barttin deletion. [Models modified from, reference (343), with permission.]




but did not display any renal symptoms except for elevatedrenin levels and hypocalciuria. Some members of one fam-ily were compound heterozygotes for I12T and the loss-of-function E4X nonsense mutation. In addition to deafness andelevated plasma renin, these latter patients displayed nephro-calcinosis and borderline metabolic alkalosis. Detailed func-tional analysis of heterologously expressed ClC-Ka/Barttin orClC-Kb/Barttin channels revealed that the I12T mutation didnot affect their single-channel properties, but reduced whole-cell currents by decreasing the plasma membrane expression,an effect that was more pronounced for ClC-Ka (reduction to<20%) than for ClC-Kb (to <50%) (342). It appears thatthe kidney, but not the inner ear, can tolerate this reduc-tion in transport activity without overt pathology. In a waythis situation is similar to that with mutations in the K+-channel KCNQ1/KCNE1, where total loss of function leadsto deafness and cardiac arrhythmia (280, 370), whereas par-tial loss of function results only in cardiac arrhythmia in thedominant Romano-Ward variant of the LongQT syndrome(439). The relatively large tolerance of kidney function toreduced ClC-K transport activity as is evident from those pa-tients casts some doubts on the physiological impact of pos-sible regulatory mechanisms affecting ClC-K/barttin activity(28, 50, 96, 419, 424, 431) and on the renal impact of cer-tain polymorphisms in human CLCNKA and CLCNKB genes(51,171). On the other hand, the differential effect of the I12Tmutant on ClC-Ka and ClC-Kb may contribute to the selectiveimpairment of hearing (342) if strial transport depends moreon ClC-Ka (-K1) than on ClC-Kb, which in turn has a largeimpact on renal function. Although it is clear from humanand mouse genetics that both ClC-Ka and ClC-Kb contributeto the generation of the endocochlear potential, the relativecontribution of these isoforms could not be determined so far.

Polymorphisms in CLCNKA and CLCNKB mightcontribute to cardiovascular disease

As ClC-Ka and -Kb are involved in renal salt absorption, poly-morphisms in these genes might influence blood pressure. ACLCNKB polymorphism (T481S) that is present in 20% to40% of the population led to a drastic, approximately 20-foldincrease in currents when expressed together with barttin inXenopus oocytes (171) (but strangely not in mammalian cells(381)) and even gave currents without barttin (171). Sinceincreased basolateral Cl– channel activity in the TAL may in-crease renal salt reabsorption, it was investigated whether thisactivating polymorphism is linked to hypertension. An initialstudy indeed found a weak association of the T481S allelewith arterial hypertension (172), but this finding could not bereproduced in several other cohorts (106, 195, 390, 442). Amore recent study, however, found an association with hyper-tension in a cohort from Ghana (381). BSND variants withpartial loss-of-function did not confer protection against hy-pertension in that population (380). In addition, several CLC-NKA polymorphisms (mostly noncoding single nucleotidepolymorphisms (SNPs)) were associated with salt-sensitive

hypertension in one study (19), and the activating CLCNKBT481S variant was associated with a protective effect againsthearing loss (118). It appears that the above association stud-ies should be interpreted cautiously until they are replicatedin more cohorts.

CLCNKA polymorphisms were also associated with heartfailure. A frequent polymorphism (R83G) in CLCNKA waspresent at an allele frequency of approximately 57% in Cau-casians with heart failure, compared to an approximately 50%frequency in healthy controls (51). This association was statis-tically significant in three independent cohorts. The presenceof a glycine at position 83 (in an extracellular loop betweenhelices B and C) was reported to reduce ClC-Ka/barttin cur-rents by approximately 50% compared to the R83 variant(51). The authors speculated that the increased propensity forheart failure with the G83 variant might be related to an in-crease in renin like that observed with total loss of ClC-Kbfunction in Bartter syndrome III. However, Clcnk1–/– micedisplay nephrogenic diabetes insipidus (249) but no salt loss.A functional link between the R83G variant and heart failureremains to be established.

Pharmacology of ClC-K/barttin channels

The pharmacology of ClC-K/barttin channels has been the fo-cus of intense research efforts (214, 216, 250, 318, 319, 478),leading to the identification of blockers that block ClC-K/barttin channels from the outside (215, 216). Some ofthese surprisingly block ClC-Ka/barttin with significantlyhigher affinity than the highly homologous ClC-Kb/barttin(215,319). ClC-K residues determining inhibitor affinity havebeen identified (319). Currents from ClC-K/barttin channelsare also potentiated by fenamates like niflumic acid (214),a substance better known for its inhibitory effect on Ca2+-activated Cl– channels, and the sites for this interaction havebeen mapped on the ClC-K protein (478).

Intracellular CLCs—Involved inVarious Physiological ProcessesThrough Their Role in Organellar IonHomeostasisThe members of the second and third branch of mammalianCLCs, ClC-3 through ClC-7, reside predominantly on com-partments of the endosomal/lysosomal pathway (175). Theirdifferential localizations partially overlap so that they seemto occupy all organelles from early endosomes to lysosomes(Fig. 5) as well as related organelles, such as synaptic vesi-cles and synaptic-like microvesicles (SLMVs). ClC-7 is alsofound in a specialized plasma membrane domain, the osteo-clast ruffled border. ClC-4 and -5 can reach the plasma mem-brane, probably in a recycling process, to some extent. ClC-4through ClC-7 have been shown to act as chloride/proton ex-changers, and this is also expected for ClC-3 because of thepresence of the “proton glutamate” and the close homolgy to




H+

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Figure 5 Localization of the intracellular CLC proteins to the endo-somal/lysosomal pathway. The scheme illustrates the proposed sub-cellular localizations of the members of the second and third CLC sub-families. While ClC-5 localizes to early compartments of the endocyticpathway, ClC-3 and -6 localize on late endosomes. The localizationof ClC-4 is less clear, and ClC-7/Ostm1 is the only lysosomal CLCprotein. The ATP-consuming proton pump acidifies the compartments,from the extracellular pH 7.4 to pH 4.5 in lysosomes. At least in earlyendosomal compartments, the shunt current is provided by the CLCproteins. For all intracellular CLCs except ClC-3, a Cl–/H+-exchangeactivity has been shown. For ClC-7, this activity has been shown toaccumulate chloride in lysosomes.

ClC-4 and -5 which are well-established Cl–/H+-exchangers.One cell physiological function of the intracellular CLCs isto support the acidification of the respective organelle by pro-viding the electrical shunt for the proton-pumping V-ATPase(105, 141, 175). This role seems to be less important for lateendosomal/lysosomal CLCs, as these organelles possess sig-nificant cation conductances. Recent data on ClC-5 and -7(291,448) clearly bolster the idea (174) that a fundamental roleof all intracellular Cl–/H+-exchangers is the proton gradient-driven luminal chloride accumulation. According to theirbroad tissue distribution and differential subcellular localiza-tion, ClC-3 through ClC-7 are involved in numerous physio-logical functions (Fig. 1). The phenotypes of their respectiveKO mouse models and the human diseases upon mutations intheir genes underline their physiological importance.

ClC-3: an intracellular CLC with manyproposed functionsClC-3 shares about 80% sequence identity with ClC-4 andClC-5, and together they constitute the second branch of

mammalian CLCs (Fig. 1). The ubiquitously expressed,intracellular ClC-3 is subject to many controversies inrespect to both its basic biophysical characteristics andits physiological functions in different organs and celltypes.

Various physiological currents have been assignedto ClC-3

Although ClC-3 was the first intracellular CLC to be cloned(37, 185), its biophysical properties remain controversial.Probably due to its weak surface expression, various mutu-ally incompatible biophysical properties have been assignedto ClC-3. At first it was reported to mediate slightly out-wardly rectifying Cl– currents, that were inhibited by proteinkinase C (185). The same group reported later that ClC-3currents are inactivated by physiological concentrations ofintracellular Ca2+ (184). ClC-3 was also proposed to consti-tute the swelling-activated Cl– channel (also referred to asvolume-regulated chloride channel, VRAC) (86, 433, 465).However, data from three independent ClC-3 KO mousemodels (83, 402, 467) provide unambiguous evidence thatswelling-activated anion currents are unaffected in hepato-cytes and acinar cells (402), in salivary acinar cells (17), andin cardiomyocytes (131) of these mice. Even the group thatoriginally put forward the hypothesis that ClC-3 is VRAC(86) reported that swelling-activated Cl– currents remainedintact in ClC-3-deficient cells (464). They, therefore, pos-tulated compensatory effects in the KO mouse (461, 462).However, this hypothesis requires that another closely relatedCLC also shows VRAC activity—which is clearly not thecase. Yet another current that was assigned to ClC-3 was thatof a Ca2+-dependent, CamKII-activated plasma membraneCl– channel (165) that may have a role in the modulation ofexcitatory synaptic transmission (443) and in the migration ofglioma cells (69). However, salivary acinar cells lacking ClC-3 displayed normal Ca2+-activated Cl– currents (17) and thesecurrents differ fundamentally from those mediated by ClC-4and -5. Moreover, the single-channel currents recorded frominside-out patches of hippocampal neurons that were ascribedto ClC-3 (443) strongly resemble those of the ubiquitously ex-pressed ClC-2 (412, 449) which is prominently expressed inthose neurons (345, 387).

The Weinman lab has reported different currents for het-erologously expressed ClC-3 (209, 210). They were stronglyoutwardly rectifying and displayed a Cl–>I– conductancesequence that is typical for CLCs (209) and that dif-fers from VRAC currents. Mutating the “gating glutamate”eliminated the rectification (210) as in ClC-4 through -7(119,206,275,400). The homology to the established Cl–/H+-exchangers ClC-4 through -7 (206, 275, 322, 362) and thepresence of the “proton glutamate” strongly argues for ClC-3being an exchanger as well.

The Lamb lab reported ClC-3 currents that resembledthose observed by the Weinman lab (248). When they ex-pressed ClC-3 in HEK cells, they observed strongly outwardly




rectifying currents that were converted into almost ohmic cur-rents by mutating the “gating glutamate,” like observed be-fore for ClC-3 by Li et al. (210) and for ClC-4 and -5 (119).The difference between the measured chloride reversal po-tential and that calculated by the Nernst equation suggestedthat ClC-3 is a Cl–/H+-exchanger like the other intracellularCLCs (248). However, due to the strong outward rectification,currents near the reversal potential are low and are expectedto be contaminated by background currents of the expressionsystem. Lately, Lamb and colleagues postulated that acidicpH uncoupled this chloride current from proton transport(246).

Obviously not all functions assigned to ClC-3 can be true.Errors or misinterpretations happen easily due to the low cellsurface expression of ClC-3 that also lead to reports of nega-tive results (119, 322, 454).

Tissue distribution and subcellular localization

ClC-3 is expressed broadly and has been detected in brain,retina, adrenal gland, pancreas, epididymis, kidney, liver,skeletal muscle, and heart (37, 185, 242, 402).

Like the other CLCs of the second and third homol-ogy branch, ClC-3 resides predominantly on intracellular or-ganelles. ClC-3 endogenously expressed in various tissues(242,262,298,402,445), as well as heterologously expressedClC-3 (146, 210, 404, 453, 477), has been localized to com-partments of the endosomal pathway. Epitope-tagged ClC-3colocalized with cotransfected ClC-4 or ClC-5 and with mark-ers of both early and late endosomes (404). In the same studyoverexpressed ClC-3 was shown to coimmunoprecipitate withoverexpressed ClC-4 and ClC-5, but not with ClC-6 or ClC-7.So far, heterodimerization of endogenous ClC-3 with anotherCLC has not been reported. Enlarged intracellular, ClC-3-positive compartments that are formed upon ClC-3 overex-pression also stained positive for late endosomal and lyso-somal markers (210). Subcellular fractionation showed thatin ClC-7-deficient brain, ClC-3 is shifted partially into lyso-somal fractions, indicating that it localizes at least partiallyto late endosomes (327). At least when overexpressed, ClC-3may be trafficked via the plasma membrane to its intracellulardestination (477). Most studies ascribing plasma membranelocalization to ClC-3 (86, 165, 169, 184, 185, 263, 443) havebeen performed without knockout controls or with heterolo-gously overexpressed ClC-3. Internalization from the plasmamembrane requires the interaction of clathrin with an amino-terminal leucine-rich stretch of ClC-3 (477). This interac-tion seems to be independent of the adaptor protein AP-2because AP-2 binding to ClC-3 was not abolished by muta-tion of this stretch (477). Another study has confirmed theClC-3/clathrin interaction without detecting AP-2 as a furtherinteractor (393).

In neurons, ClC-3 additionally localizes to synaptic vesi-cles (137, 357, 402), and in PC12 cells, chromaffin cells andpancreatic β cells to SLMVs (242, 357). Although no di-rect binding of AP-3 to the cytosolic amino- or carboxy-

terminal region of ClC-3 was detected (393, 477), transportof ClC-3 to synaptic vesicles and SLMVs of PC12 cells de-pends on this adaptor protein (357, 375). Reports claimingthat ClC-3 would additionally be present on large dense-corevesicles (LDCVs) (18, 78, 208) are questionable (177) andin KO-controlled studies ClC-3 was not detected on LDCVs(242).

There is a splice variant of ClC-3, called ClC-3B, in whichthe carboxy-terminal part starting within CBS2 is replaced bya different sequence. This leaves the CBS consensus intactbut adds a PDZ-binding motif (294). ClC-3B binds to thePDZ domains of EBP50/NHERF-1 (294), of PDZK1 and ofthe Golgi-localized protein GOPC (127). These interactionsseem to determine the subcellular localization of heterolo-gously expressed ClC-3B, but a physiological role of thissplice variant remains to be uncovered.

Degeneration of hippocampus and retinain knockout mice

Three groups have independently generated Clcn3–/– mice(83, 402, 467). All three ClC-3 KO mouse lines displaysevere degeneration of the hippocampus and retina (Fig. 6).Agreeing with its localization to synaptic vesicles, ClC-3is expressed prominently in the inner and outer plexiformlayers (IPL and OPL) of the retina (402). However, thestructures that are most vulnerable to degeneration uponClC-3 deficiency are the outer nuclear layer (ONL) and theouter segments (OS), that is, the photoreceptors (402, 467).

WT

KO WT KO

RPE

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Figure 6 Neuronal degeneration in ClC-3 KO mice. (A) Nissl-stained frontal brain sections show severe neuronal cell loss that leadsto a complete loss of the hippocampus in adult ClC-3 KO mice. (B)Retinal sections of P28 WT and ClC-3 KO mice reveal a complete de-generation of photoreceptors, with a loss of the photoreceptor outersegment (OS) as well as the nuclei that form the outer nuclear layer(ONL). RPE, retinal pigment epithelium; IS, photoreceptor inner seg-ments; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, innerplexiform layer; GCL, ganglion cell layer. [Images adapted from, ref-erence (402), with permission.]




Postnatal retinal degeneration progresses until the miceare completely blind and their electroretinograms show noresponse to applied flashes already a few weeks after birth.

In the brain, neuronal cell loss is most prominent in thehippocampus, which is totally lost after a few months. How-ever, neurodegeneration is by no means restricted to the hip-pocampus. Only subtle differences are found between thethree ClC-3 constitutive KO mouse model lines, which mightbe explained by differences in the genetic background. In thefirst mouse (402), neuron loss began in the CA1 region ofthe hippocampus by P12 and then progressed rapidly over thenext weeks. In contrast, in the second mouse line neurode-generation started in the dentate gyrus and spread to CA3 andthen CA1 over months (83). Features of the lysosomal storagedisease neuronal ceroid lipofuscinosis (NCL) were observedin the third line (467). These include the typical neuronalaccumulation of the mitochondrial ATP synthase subunit c.However, there were only minimal NCL features in the firstClC-3 KO model (402) when compared to the ClC-7 KOmouse with its severe NCL-like lysosomal storage disease(183).

The third ClC-3 KO mouse line was reported to dis-play an initial upregulation of GABAA receptors followedby a loss of GABA-synthesizing neurons of the dentate gyrus(83). These mice also displayed an increased sensitivity toGABAA receptor-affecting drugs, suggesting an alterationof inhibitory GABAergic signaling. A more recent study re-ported a reduction of the frequency and amplitude of minia-ture inhibitory postsynaptic currents (mIPSCs) of CA1 neu-rons from P18 to P25 KO mice (341). However, this is instriking contrast to earlier data from CA1 neurons of P13to P15 mice from the first knockout line which showed un-changed mIPSCs (402). It remains unclear whether this dis-crepancy is due to the genetic background of the lines orto the advanced age of the mice displaying weakened mIP-SCs.

Despite this severe neurodegeneration, ClC-3 KO miceare viable and can reach more than 1 year of age. ClC-3is broadly expressed and may have important physiologicalfunctions in other organs as well. For example, exocytosisof LDCVs is diminished in chromaffin cells from KO mice(242). Glucose-induced release of insulin has been reportedto be reduced upon loss of ClC-3, although the changes inplasma glucose level upon glucose load were unaltered (78,208). Also reduced basal insulin levels have been reportedfor ClC-3 KO mice (242). This might be explained by theobserved concomitant increased leptin level (242), since leptindirectly or indirectly reduces insulin levels (23, 272). Ourlaboratory found no alteration in serum leptin and insulinconcentrations in β-cell-specific ClC-3 KO mice (Maritzenand Jentsch, unpublished data). Clcn3–/– mice have furthernonneuronal phenotypes, but the mechanisms leading to thesephenotypes may be hard to define in this multisystem disorder.For example, these mice accumulate less body fat and remainsmaller than their WT littermates (83,402), a fact which mayimpact insulin secretion.

Role of ClC-3 in the physiology of vesicularorganelles

As ClC-3 localizes to endosomes and related intracellularcompartments, it is likely to play a role in the luminal acid-ification and/or chloride accumulation of the respective or-ganelles (174). Indeed, the steady-state luminal pH of anendosome-enriched fraction from liver of ClC-3 KO mice wasreported to be slightly elevated compared to wildtype (467).Using pH- and chloride-sensitive dyes, both acidification andthe increase of luminal chloride were reported to be dimin-ished in early and late endosomes of cultured hepatocytesfrom ClC-3 KO mice (146).

Synaptic vesicles from ClC-3-deficient mice acidify lessefficiently in vitro (402). However, acidification is not abol-ished and pH gradient-dependent uptake of the neurotransmit-ter dopamine was found unaffected. Also mIPSCs in acutehippocampal slice preparations, which depend on both thesynaptic content of GABA and on the postsynaptic recep-tor density, were not different from WT control mice (402).The reduction in glutamate uptake was ascribed to a selectiveloss of glutamatergic synaptic vesicles, which was reflectedby the reduced level of the vesicular glutamate transporterVGLUT1 (402). Recently the idea that the electrical shuntfor synaptic vesicle acidification is predominantly providedby ClC-3 has been questioned by a report that attributed thisfunction to VGLUT1 (364). The authors even reported thatClC-3 were present on less than 0.05% of the synaptic vesiclesas estimated by calibrated immunoblotting signals. Our labo-ratory, however, could not reproduce this quantification in anequivalent approach (Weinert and Jentsch, unpublished data)and ClC-3 has been detected in mass spectrometry analysisof immuno-purified synaptic vesicles (137), confirming theoriginal identification of ClC-3 as a synaptic vesicle protein(402). Chloride conductance by VGLUT1 had been describedearlier (24). Takamori and colleagues showed that chloride-dependent acidification was abolished in synaptic vesicles ofVGLUT1 KO mice (364). The authors hypothesized that theearlier effect observed for ClC-3-deficient synaptic vesicles(402) resulted indirectly from the concomitant reduction ofVGLUT1 in these mice. A further study has corroboratedthe notion that Cl– transport through ClC-3 is not requiredfor the acidification of VGLUT1-positive, excitatory synap-tic vesicles (341). Crude synaptic vesicle preparations fromClcn3–/– mice, which contain mainly excitatory synaptic vesi-cles, showed no defect in Cl–-dependent, V-ATPase-mediatedacidification. However, it was not reported whether thoseexperiments were performed before reduction of VGLUT1levels in these mice. Nevertheless, after depletion with anti-VGLUT1 antibodies, the remaining population, enriched inVGAT-positive inhibitory vesicles, displayed a difference be-tween genotypes. Vesicles from ClC-3-deficient mice stillacidified, but to a lesser extent than those from WT mice.This finding agrees with the first study on synaptic vesicleacidification upon ClC-3 disruption (402) inasmuch synapticvesicle preparations from KO mice contained less VGLUT1,




possibly due to an (unexplained) loss of VGLUT1-positivevesicles (402). An acidification defect of inhibitory synap-tic vesicles is also consistent with the reported reduction inmIPSC amplitude and frequency (341), which, however, con-tradicts with earlier findings (402). Immunodepletion of theinhibitory synaptic vesicle population from WT mice with anantibody against ClC-3 left a vesicle population that acidifiedto a similar extent as the inhibitory population from Clcn3–/–

mice (341). However, the antibody used is not specific forClC-3 (177, 242) and the results have to be considered withcare. The same antibody was then used to further immuno-deplete synaptic vesicle populations from rat that had beenenriched in inhibitory synaptic vesicles. The resulting vesiclepopulation was claimed to represent a rat model for ClC-3knockout vesicles and it also showed less acidification thanthe vesicle population before ClC-3 immunodepletion (341).However, the nature of the remaining ClC-3-negative vesi-cles is unclear and they may differ in many respects fromClC-3-positive vesicles. Although ClC-3 may contribute tothe acidification of inhibitory synaptic vesicles, there are stillmany open questions. Is ClC-3 involved in neurotransmitterloading, and if so, how does it influence VGAT transport ac-tivity? Another question concerns the function of ClC-3 onexcitatory synaptic vesicles. Possibly ClC-3 is indeed not in-volved in their acidification. How then does ClC-3 deficiencylead to a loss VGLUT1, a neurotransmitter transporter whosemode of operation is also still controversial? Even if ClC-3plays a crucial role in inhibitory synapse function, why is thehippocampus so vulnerable to degeneration in the KO mouse,whereas the cerebellum with its many GABAergic interneu-rons remains virtually unaffected?

The mechanism that leads to the attenuation of LDCV ex-ocytosis in endocrine cells of ClC-3-deficient mice (78, 208,242) has remained equally elusive (177). Priming of thesegranular compartments requires acidification and possiblychloride influx as it can be blocked by DIDS (18). ClC-3 hasbeen proposed to provide the required chloride conductance(18, 78, 208). However, a localization of ClC-3 to LDCVshas not been demonstrated convincingly by either study (177)and ClC-3 does not copurify with markers of LDCVs, such asinsulin of isolated islets or INS-1 cells. It rather localizes toendosomes and SLMVs in KO-controlled experiments (242).This suggests that the effect on LDCV exocytosis upon lossof ClC-3 is rather indirect, possibly due to a trafficking defectin the endosomal system.

It was also suspected that ClC-3 provides the countercur-rent for the activity of the NADPH oxidase in neutrophilsand vascular smooth muscle cells (262, 269, 270). Therebyit may be involved in ROS signaling by endosomes (262).This function is in agreement with the strong voltage depen-dence of ClC-3. Due to their strong outward rectification,intracellular CLCs would be virtually inactive at luminal pos-itive voltages. This has been an enigma in the field, becauseendosomal/lysosomal compartments are generally believedto be inside-positive due to the activity of the electrogenicproton pump (note, however, that model calculations pre-

dict an inside-negative voltage in the presence of an H+-ATPase, 2Cl–/H+-exchanger, and a proton leak (448)). Asthe NADPH oxidase rather transports negative charge intothe lumen, this problem would not occur for the combina-tion of ClC-3 with the NADPH oxidase. ClC-3 has beenreported to be directly involved in phagocytosis and the res-piratory burst in neutrophils (269, 270). Through reactiveoxygen species (ROS) signaling, it may play an indirect rolein their chemotaxis and shape changes (432). ROS result inthe formation of H2O2, which in turn has been reported toincrease the swelling-activated chloride current (428). Theinvolvement of ClC-3 in endosomal ROS production has re-cently been proposed as an explanation for the upregulationof the swelling-activated chloride current upon ClC-3 over-expression (247). This opens the possibility that further rolesassigned to ClC-3, such as in cell cycle regulation and cellmigration (62,144,182,230,239,335,408,438,463,466), arejust very indirectly linked to the activity of ClC-3. Clearly, itis not known precisely to which organelles ClC-3 localizesand what function it exactly exerts in these. Gaining more in-sight into the answers to these questions will help understandwhy ClC-3 deficiency leads, probably by indirect ways, to thevarious phenotypes such as neurodegeneration and reducedendocrine secretion.

ClC-4: a mysterious chloride/proton exchangerThe second homology group of CLC chloride channels andtransporters consists of ClC-3, ClC-4, and ClC-5, whichshare close to 80% sequence identity (Fig. 1). The least isknown about ClC-4, but it is thought to localize like ClC-3and ClC-5 on vesicles and compartments within the endo-somal/lysosomal pathway (174). Although the conventionalClC-4 KO mouse model did not display an obvious phenotype(344), several physiological functions have been proposed forClC-4. However, these are often controversial and remain tobe confirmed.

ClC-4 is a broadly expressed chloride/protonexchanger

In humans, ClC-4 expression is most prominent in muscle andbrain (423) whereas ClC-4 expression in rat is highest in liverand brain and additionally found in heart, skeletal muscle,spleen, and kidney (176). In mouse, ClC-4 was shown to beexpressed in brain, intestine, and kidney (265, 266). In thewild Mediterranean mouse Mus spretus, the Clcn4 gene mapsto the X chromosome as it does in rat and humans, whereasin the inbred laboratory mouse strain Mus musculus, it hasbeen translocated to chromosome 7 (347). This provides apossible explanation for the divergent expression pattern ofClC-4 between rat and mouse (282).

ClC-4 resides predominantly on intracellular vesicles, buta minor portion of ClC-4 reaches the plasma membrane,at least when expressed heterologously. This localizationmade ClC-4 easily accessible for biophysical analysis, which




(B)

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Figure 7 Basic electrophysiological properties of ClC-4. (A) Two-electrode voltage-clamptraces of ClC-4 expressed in Xenopus oocytes show strongly outwardly rectifying chloridecurrents which a rapidly activated at positive voltages. (B) Neutralizing the “gating gluta-mate” in ClC-4 converts the Cl–/H+-exchanger into a pure Cl– conductor which results insignificant currents also in the negative voltage range. [Traces taken from, reference (119),with permission.]

showed that ClC-4 yields strongly outwardly rectifying cur-rents (Fig. 7) that decrease with acidic extracellular pH (119).ClC-4 currents closely resemble those of ClC-5 in their recti-fication, pH sensitivity, and SCN–>NO3

–>Cl–>Br–>I– con-ductance sequence (119,149). ClC-4 can be inhibited by Zn2+

when expressed in Xenopus oocytes (301). However, inhibi-tion by Zn2+, which ClC-4 shares with ClC-2 (64), can hardlybe considered specific.

ClC-4 currents, like those of other intracellular CLCs,do not reflect a pure Cl– conductance but rather a voltage-dependent electrogenic exchange of Cl– and H+ (322, 362).The stoichiometry of Cl–/H+-exchange has not yet been de-termined for ClC-4, but it seems safe to assume that it is2:1 like in the bacterial EcClC-1 and in ClC-5 and ClC-7(2, 206, 481). Similar to those transporters, neutralizing the“gating glutamate” (2) converts the ClC-4 Cl–/H+-exchangerinto a pure Cl– conductor. It also drastically changes its volt-age dependence from strongly outwardly rectifying to nearlylinear (Fig. 7) (119,322,362). Like observed with other CLCexchangers (281, 472) ClC-4 anion transport can be partiallyuncoupled from H+-transport by polyatomic anions like NO3

–

and SCN–, without losing, however, its strong rectification(8, 119). The “slippage mode” underlying uncoupled aniontransport has been investigated in detail recently (8). Severalstudies have addressed the biophysical properties of ClC-4(8, 119, 149, 300, 301, 322, 362, 426).

ClC-4 resides on intracellular compartments

There is currently no consensus on the subcellular localiza-tion of ClC-4. It appears that the antibodies used in severalstudies have not been validated by staining ClC-4 KO tissueas negative control, or by using antibodies against a differentClC-4 epitope as positive control. Part of the difficulty mayarise from the fact that ClC-3, -4, and -5 are about 80% iden-tical at the amino acid level. Based on immunohistochemistrydata, Bear and colleagues reported that ClC-4 colocalizes notonly with the chloride channel CFTR in the apical brush bor-

der membrane of the intestinal epithelium (265), but also tosubapical vesicles of the proximal renal tubule epitheliumwhere it colocalized with endocytosed FITC-dextran (266).Another group found ClC-4 overexpressed in HEK 293 cellsin intracellular compartments where it colocalized with ClC-3and partially with ClC-5 (404). Another group also expressedClC-4 heterologously in HEK 293 cells and skeletal mus-cle fibers and reported ClC-4 colocalization with SERCA2,a marker for the endoplasmic reticulum (299), a finding thatwas corroborated by Western blots of subcellular fractions.These authors also identified an N-terminal motif necessaryfor targeting ClC-4 to the ER. Nonetheless, further studiesusing specific antibodies and appropriate controls are neededto unambiguously determine the subcellular localization ofClC-4.

Physiology of ClC-4

Reports on the possible physiological functions of ClC-4 arenot less controversial. Bear and colleagues first suggestedthat ClC-4 might mediate intestinal Cl– secretion (265), butsuggested later that it facilitates endosomal acidification andhas a role in endocytosis similar to ClC-5 (266). They latertested this hypothesis by analyzing ClC-4-null mice they hadgenerated by breeding Mus spretus with Mus musculus (267).Due to the different chromosomal localization of the Clcn4gene, backcrossing results in male hybrids that lack ClC-4(6). These mice display no immediately apparent abnormalphenotype but they are infertile. Bear and colleagues reporteda more alkaline endosomal pH in ClC-4-null than in WTfibroblasts as well as reduced receptor-mediated uptake oftransferrin (267). On the other hand, conventional disruptionof ClC-4 in mice did not lead to proteinuria or impaired prox-imal tubular endocytosis (344). It was also suggested that inhepatocytes, ClC-4 is important for copper incorporation intoceruloplasmin: It was proposed that ClC-4 shunts currents ofCu2+-ATPases in the secretory pathway (441). However, our




own unpublished data showed no change in ceruloplasminoxidase activity in liver lysates of Clnc4–/– mice.

ClC-4 may form heteromers with ClC-3 and ClC-5 (266,404), but whether this interaction occurs in vivo and is ofphysiological importance is not yet known. The possibilitythat ClC-4 and ClC-5 have partially overlapping functionshas been addressed for proximal tubular endocytosis (344).The additional disruption of ClC-4 did not impair further thereduced level endocytosis of ClC-5 KO mice (344).

ClC-5: a chloride/proton exchanger importantfor renal endocytosisWithin the second branch of CLC channels and transportersthe function of ClC-5 is best understood. ClC-5 is most highlyexpressed in renal and intestinal epithelia (400,425). It is alsofound in other tissues like brain and liver (400). Like ClC-3and ClC-4, ClC-5 resides predominantly on intracellular vesi-cles. It localizes predominantly on early and recycling endo-somes, but it may also be found to some degree in the brushborder membrane of proximal tubules. At least in proximaltubules, ClC-5 plays an important role in endocytosis. Theloss or dysfunction of ClC-5 leads to Dent’s disease (223),an X-linked disorder that is characterized by hypercalciuria,nephrocalcinosis, kidney stones, and renal failure.

Biophysical properties of ClC-5

Upon heterologous overexpression, a significant amount ofClC-5 is found at the plasma membrane (140,339,361). Thislocalization allowed biophysical studies which revealed thatClC-5, like its close homologue ClC-4, mediates strongly out-wardly rectifying currents (119, 400) with an anion conduc-tance sequence of SCN–>NO3

–>Cl–>Br–>I– (119). ClC-4and ClC-5 are now known to operate as voltage-dependentCl−/H+ exchangers (322, 362). The strong outward rectifica-tion of ClC-5 precluded a determination of the coupling ratioby measuring reversal potentials (322,362). However, quanti-tative comparison of currents with proton fluxes across Xeno-pus oocyte membranes using a pH-sensitive dye allowed theconclusion that it mediates Cl–/H+-exchanger with a 2:1 stoi-chiometry (481) like EcClC-1 (2) and other CLC anion/protonexchangers (206, 481).

Concatemers showed that each subunit of the homo-dimeric ClC-5 transporter performs Cl–/H+-exchange inde-pendently from the transport activity of the neighboring sub-unit (472). Mutating the “gating glutamate” uncoupled theCl− conductance from H+ countertransport and abolished therectification (119, 322, 362) and neutralizing the “proton glu-tamate” of ClC-5 abolished both Cl– and H+ transport (472).As observed earlier with EcClC-1 (281), anion transport ofClC-5 becomes partially uncoupled from H+ transport withNO3

– or SCN– (472). Comparison with the plant NO3–/H+-

exchanger AtClC-a (74) identified a Cl– coordinating serinein ClC-5 that is replaced by proline in AtClC-a. This exchangewas shown to affect NO3

– selectivity and NO3–/H+-coupling

(29, 321, 481). ClC-5 currents (like those of ClC-4) decrease

with acidic extracellular pH (119), an effect that was thor-oughly investigated in a recent study (320).

Like all other eukaryotic CLCs, ClC-5 displays two cy-tosolic CBS domains. It has been suggested that these domainsmight constitute binding modules for nucleotides that regulatethe activity of several CLC proteins such as ClC-2 (286,374).Structural and biochemical studies have shown that ClC-5binds ATP at the interface between CBS1 and CBS2 of eithersubunit (257, 450) and that ATP, as well as ADP and adeno-sine monophosphate (AMP) similarly potentiate ClC-5 cur-rents (482). X-ray scattering experiments suggested that ATPbinding induces a conformational change (451). Since notonly ATP, but also ADP and AMP similarly stimulate ClC-5transport, the physiological role of this effect remains unclear.

Tissue distribution, subcellular localization,and regulation of ClC-5

In the kidney, ClC-5 expression is highest in acid-transportingintercalating cells of the distal nephron and in proximaltubules (140,356). To a lesser extent ClC-5 is also expressed inthe TAL of Henle’s loop (79). In proximal tubule cells (PTCs),ClC-5 is expressed prominently below the brush-border in aregion densely packed with endocytic vesicles, where it colo-calizes with the V-type H+ -ATPase (proton pump) and withinternalized proteins early after uptake (140, 356). In PTCs,which endocytose low-molecular weight proteins that havepassed the glomerular filter, a portion of ClC-5 is also presentin the membrane of the brush-border (356). In intercalatedcells of the collecting duct and in intestinal epithelial tis-sue it similarly localizes to apical endosomes and colocalizeswith the proton pump in acid-secreting α-, but not in acid-reabsorbing β-intercalated cells (140, 425).

Several mechanisms have been proposed to regulate theexpression, localization and activity of ClC-5. On the tran-scriptional level, it was shown that the hepatocyte nucleartranscription factor 1 (HNF1), which is robustly expressed inproximal tubules, directly regulates the expression of ClC-5(407). Reduced expression levels of ClC-5 in HNF-1 KO kid-ney could be rescued upon transfection of HNF-1 in HNF-1KO PTCs (407).

On the protein level, it was suggested that ClC-5 isregulated by the ubiquitin system. The E3 ubiquitin ligaseWWP2 can interact with a PY-motif of ClC-5 that is locatedbetween the two CBS domains (323, 371). PY-motifs areknown to bind to WW-domains, protein interaction modulesfound in several HECT-domain ubiquitin ligases (113). Crys-tallography has shown that the cytoplasmic region harboringthe PY-motif in ClC-5 is unstructured and accessible forprotein-protein interactions (257). ClC-5 point mutationsdestroying the PY-motif consensus sequence (e.g., Y762E)led to a roughly twofold increase in ClC-5 currents andincreased surface localization when expressed heterologouslyin Xenopus oocytes (371, 393). Dominant negative mutantsof the ubiquitin-protein ligase WWP2 increased ClC-5surface expression and currents when coexpressed with




WT ClC-5, but not with its PY-mutants suggesting thatClC-5 can be ubiquitinated in a PY-motif dependent manner,which then regulates the endocytic removal of ClC-5 fromthe plasma membrane. Also other WW-domain containingubiquitin ligases, that is, Nedd4 and Nedd4-2, were shownto interact with ClC-5 through its PY-motif (163). Theseauthors also directly observed ubiquitylation of ClC-5 whenthey incubated cells with the proteasome inhibitor MG-132.Surprisingly, this effect was only observed when cells wereincubated with albumin, a procedure thought to stimulateendocytosis. Knocking down Nedd4-2 with RNAi in opossumkidney (OK) cells (a cell culture model for PTCs) reducedalbumin endocytosis by about 25% (163), but it is unclearwhether this effect was mediated by reduced ubiquitylation ofClC-5. To test the physiological role of PY-motif-dependentubiquitylation of ClC-5, our laboratory generated knock-inmice in which this motif was disrupted by a point mutation(344). Surprisingly, the localization and abundance of ClC-5appeared unchanged in these mice, which neither showedlow-molecular weight proteinuria. Moreover, pulse-chaseexperiments to study endocytosis in vivo did not revealany impairment of either receptor-mediated or fluid-phaseendocytosis (344). By studying the PY-mutation in micelacking ClC-3 and -4, it was clearly shown that neither ClC-3nor ClC-4, with which ClC-5 might form heterodimers(266, 404), masked the effect of the ClC-5 PY-motif that hadbeen convincingly observed in vitro (344).

The C-terminal tail of ClC-5 may also interact with theactin depolymerizing protein cofilin (164) and the PDZ2-domain of NHERF-2, but not NHERF-1, PDZ-domain con-taining adaptor proteins (162). Because of its reported in-teraction with ClC-5, mutations in the gene encoding cofilinwere sought in patients with Dent’s disease, but none werefound (459). Silencing NHERF-2 in OK cells reduced cell-surface levels of ClC-5 and albumin uptake (162), whereassilencing NHERF-1 increased cell-surface levels of ClC-5 andalbumin uptake. Whether the effects on albumin uptake oc-curred through trafficking defects of ClC-5 remains unclear.However, neither NHERF-1 nor NHERF-2 mice displayedany low-molecular weight proteinuria (70). Yeast two-hybridscreens and coimmunoprecipitation assays suggested that thecarboxyterminus of ClC-5 may also interact with the kinesinfamily member 3B (KIF3B) (339). The heterotrimeric motorprotein containing KIF3B, kinesin-2, is known to facilitatetransport of late endosomes and Golgi-to-ER cargo (43,394),but it has also been reported recently to be involved in thetransport of cargo through recycling endosomes (368). Over-expression of KIF3B or siRNA-mediated knock-down in cellculture led to reciprocal changes in ClC-5 cell surface expres-sion and endocytosis (339). It is an open question whetherthese effects are due to a general impact of motor proteins onintracellular trafficking, or rather caused by directly interfer-ing with ClC-5. ClC-5 mutants lacking KIF3B binding willbe required to test the latter hypothesis.

Recently the N-terminus of ClC-5 was shown to bindthe adaptor protein AP-2 and clathrin in vitro. Although AP-2

binding to ClC-5 would fit well with the assumed recycling ofClC-5 via the plasma membrane, the disruption of its bindingmotif did not increase cell surface localization of heterolo-gously overexpressed ClC-5 (393).

Mutations in CLCN5 underlie Dent’s disease

ClC-5 was independently identified by linkage analysis as acandidate gene in patients with Dent’s disease (112) and byhomology cloning from rat brain (400). Mutations in CLCN5were identified in several forms of inherited kidney stonedisorders (223), which are now considered to be differentvariants of one syndrome, Dent’s disease. Dent’s disease is arare, X-linked recessively inherited disorder that is associatedwith low-molecular weight proteinuria, hyperphosphaturia,and hypercalciuria, as well as kidney stones and nephrocal-cinosis (458). Whereas the former symptoms like proteinuriaare constant symptoms, the latter ones are more variable andnot found in every patient carrying CLCN5 mutations. So farmore than 50 different mutations are known to cause Dent’sdisease. About half of these mutations result in early stopcodons leading to nonfunctional truncated forms of ClC-5.The majority of the missense mutations affect residues thatare located at or close to the interface of both subunits of thehomodimer (460), but the biological meaning of this observa-tion is not yet clear. Biophysical studies showed that almost allhuman missense mutations result in reduced or abolished ClC-5 currents (133, 134, 223, 224, 271, 459). When investigatedin heterologous expression, some of them showed changedsubcellular localization (133, 134, 228, 386) suggesting thatdefective targeting and trafficking of ClC-5 to endosomes area major determinant in the pathophysiology of Dent’s disease(444).

Not all patients with clinical symptoms of Dent’s dis-ease carry mutations in CLCN5, leading to the suggestionthat Dent’s disease is heterogeneous (159). OCRL1 wasidentified as a second independent gene that is mutated insome Dent’s disease patients (160), but there may be stillanother gene causing similar symptoms. Located also onthe X chromosome, OCRL1 encodes a phosphatidylinositol-4,5-bisphosphate-(PIP2)-5-phosphatase (403,474), which haspreviously been found to be mutated in the multisystem dis-ease Lowe syndrome (217,251). This disorder is characterizedby various symptoms such as congenital cataracts, intellectualdisabilities, and renal Fanconi syndrome that includes symp-toms observed in Dent’s disease. It is not yet entirely clearwhy certain mutations in OCRL1 only cause the renal symp-toms of Dent’s disease and not the full spectrum of symptomscharacteristic for Lowe syndrome, but recent work suggeststhat it might be related to the position of the mutations thatthereby may affect different OCRL1 splice variants (378).OCRL1 is a peripheral membrane protein that is localizedto the Golgi apparatus and early endosomes. It is a inositol5′-phosphatase that impinges on levels of phosphatidylinosi-tol 4,5-bisphosphate (452, 473). This lipid is enriched in theplasma membrane where it participates in nearly all events




that occur at, or involve the cell surface and plays a majorpart in transducing extracellular signals (81). Thus, mutationsin OCRL1 might influence a number of processes includingintracellular signaling and cytoskeletal dynamics, which willeventually result in an early endocytosis defect (98) similarto that observed with ClC-5 dysfunction.

Pathophysiology of Dent’s disease: indirect effectsof an impaired proximal tubular endocytosis

The subcellular localization of ClC-5 together with protonpumps on apical endosomes points to an involvement inearly endocytosis, consistent with the proteinuria observedin Dent’s disease. Indeed, two independent mouse models,the “Jentsch ClC-5 KO mouse” and the “Guggino ClC-5KO mouse” (324, 440) fully confirmed this hypothesis. Theyshowed a loss of low-molecular weight proteins into the urine,with particularly high levels of vitamin D-binding protein andretinal-binding protein (324). These proteins are also highlyelevated in the urine of patients with Dent’s disease. In thesemice, endocytosis by PTCs was studied in vivo (324). Ran-dom X-chromosomal inactivation of ClC-5 in heterozygousfemales (Clcn5+/– mice) enabled well-controlled studies withcells expressing or lacking ClC-5 side-by-side. Mice wereinjected with fluorescently labeled lactoglobulin (as markerfor receptor-mediated endocytosis) or with labeled dextran(marker for fluid-phase endocytosis) and kidneys were fixedby perfusion after a couple of minutes. These pulse-chase ex-periments revealed that the loss of ClC-5 drastically reducedapical fluid-phase and receptor-mediated endocytosis in a cell-autonomous manner (Fig. 8) (324). Moreover the expressionof the endocytic receptor megalin was decreased in PTCslacking ClC-5 suggesting a defect in recycling megalin backto the surface (324). Indeed, subsequent electron microscop-ical studies confirmed this hypothesis (60) and additionally

+/-ClC-5 Lactoglobulin

Clcn5 +/–

10 µm

Figure 8 Proximal tubule endocytosis defect in ClC-5 KO mice. Fluo-rescently labeled lactoglobulin (in blue) was injected into femaleClcn5+/– mice. Due to random X-chromosomal inactivation in females,proximal tubular cells (PTCs) either express (ClC-5 stained in red) orlack ClC-5 (indicated by arrows). Cells expressing ClC-5 accumulatedsubstantial amounts of lactoglobulin below the brush border whileneighboring ClC-5 KO cells had taken up much less protein indi-cating a reduced receptor-mediated endocytosis in a cell-autonomousmanner. [Images modified from, reference (324), with permission.]

revealed that also the coreceptor cubilin is drastically reducedat the brush border membrane of ClC-5 KO mice (60). Theimportance of megalin in renal endocytosis is underlined bythe megalin KO mouse, whose phenotype resembles in manyrespects that of Clcn5–/– mice (205).

The impairment of proximal tubular endocytosis mightbe explained by a reduced electrical shunt for endosomalproton pumps, leading to defective endosomal acidification(141), or by impaired endosomal Cl– accumulation (291).But what might be the mechanism leading to kidney stonesobserved in many, but not all patients with Dent’s disease?Both, patients (458) and ClC-5 KO mouse models (324, 440)lose inorganic phosphate into the urine. The small polypep-tide parathyroid hormone (PTH) regulates the reabsorptionof phosphate from the proximal tubule of the kidney by trig-gering the endocytosis of apical transport proteins like NaPi-2a, a sodium-coupled phosphate cotransporter (273), or theNa+/H+-exchanger NHE3 (73). PTH passes the glomerularfilter into the primary urine, and under normal circumstances,it is endocytosed by PTCs. The lack of ClC-5 impairs PTH en-docytosis and thereby entails a progressive increase in luminalPTH levels from the early S1 to the late S3 segments (Fig. 9).It was therefore suggested that the increased stimulation of lu-minal PTH receptors in more distal segments of the PT leadsto an enhanced internalization of NaPi-2a from the plasmamembrane, which in turn reduces phosphate reabsorption andleads to hyperphosphaturia (324). Also levels of NHE3 werefound to be reduced and localization was shifted from theapical membrane to intracellular vesicles in ClC-5-deficientmice (324).

But what about hypercalciuria, another prominent symp-tom of Dent’s disease, which is probably of even greaterimportance for the formation of kidney stones? PTCs are themain sites for enzymatic conversion of 25(OH)-VitD3 intothe active metabolite 1,25(OH)2-VitD3 by the mitochondrialenzyme 1α-25(OH)-VitD3-hydroxylase. Gunther et al. foundincreased mRNA levels of this enzyme in kidneys of ClC-5KO mice (141). Both increased stimulation of apical PTHreceptors and reduced endocytic uptake of VitD into PTCsmay be invoked for this upregulation. Assuming unchangedavailability of the precursor, an increase in 1α-25(OH)-VitD3-hydroxylase predicts elevated levels of the active hormone1,25(OH)2-VitD3. In fact, 1,25(OH)2-VitD3 concentrationsare slightly elevated in the serum of patients with Dent’sdisease (363). This hormone is known to increase the in-testinal absorption of calcium, which would then have to beexcreted by the kidney leading to hypercalciuria and kidneystones (324). However, there is one obstacle: the major up-take pathway of the precursor 25(OH)-VitD3 in PTCs is viaapical, megalin-dependent endocytosis (293) and ClC-5 KOmice lose 25(OH)-VitD3, and vitamin D-binding protein intothe urine (324). Although the ratio of 1,25(OH)2-VitD3 to25(OH)-VitD3 was increased in serum of ClC-5 KO mice,which can be explained by the increase in transcription of1α-25(OH)-VitD3-hydroxylase (141,244), the absolute serumconcentration of the precursor and the active form of vitamin




1,25-VitD

25-VitD

PTH

PTH

25-VitDPTH

1,25-VitD

25-VitD

PTH

25-VitD

VDRE

D-9kTRPV5

Megalin

PTH-R

1,25-VitD

VDR

RXR

( )

1,25-VitD

PTH

24-HYD 1α-HYD

PTH

25-VitDPTH

24-HYD 1α-HYD

Early proximal tubule Late proximal tubule Distal tubule

(C)(A) (B)

Decreasedin ClC-5 KO

Increased

Figure 9 Model for renal pathology caused by an impaired proximal tubular endocytosis as observed in ClC-5 KOmice and in patients with Dent’s disease. (A) Events in the early proximal tubule: After the small peptide PTH (parathyroidhormone) and various forms of vitamin D have passed the glomerular filter, loss of ClC-5 results in an impaired megalin-mediated endocytosis of PTH that would have been normally degraded in lysosomes. The uptake of 1,25(OH)2- and25(OH)-vitaminD3 together with their binding protein DBP, which requires binding to megalin, is also impaired (indicatedby red minus symbols). Under normal conditions 1,25-VitD reaches VitD receptors that regulate the transcription of nucleartarget genes (not shown) and 25-VitD is metabolized by mitochondrial enzymes: 1α-hydroxylase (1α-HYD) converts theprecursor 25(OH)-vitaminD3 to the active hormone 1,25(OH)2-vitaminD3 (1,25-VitD), whereas the 24-hydroxylase (24-HYD) inactivates 1,25-VitD. (B) Events in the late proximal tubule: The defect in ClC-5-dependent endocytosis results in anincreased luminal PTH concentration which leads to an enhanced stimulation of apical PTH receptors (indicated by greenplus symbols). The enhanced stimulation of PTH receptors together with the decreased uptake of 1,25(OH)2-vitaminD3enhances the transcription of the mitochondrial enzyme 1α-hydroxylase that converts the precursor 25(OH)-vitaminD3 intothe active metabolite 1,25(OH)2-vitaminD3, while decreasing transcription of the catabolizing enzyme 24-hydroxylase.However, 25(OH)-vitaminD3 megalin-dependent endocytosis to reach the enzyme is impaired, thus both 1,25(OH)2-and 25(OH)-vitaminD3 are lost into the urine. There is a balance between the changed transcription of enzymes thatcooperate to increase active vitamin D3, and the loss of the precursor and active form into the urine. Depending on theoutcome (increased or decreased concentration of active vitamin D3 in serum), this leads to different clinical phenotypes.(C) Events in the distal tubule: In contrast to proximal tubules, 1,25(OH)2-vitaminD3 enters distal tubular cells mainly bydiffusion of the free hormone over the membrane and binds to the vitamin D3 receptor which activates the transcription of1,25(OH)2-vitaminD3 dependent genes after heterodimerization with the retinoid X receptor (RXR). As the concentrationof 1,25(OH)2-vitaminD3 is increased in the lumen of ClC-5 KO kidneys, renal 1,25(OH)2-vitaminD3 target genes areselectively affected. VDR, vitamin D receptor; RXR, retinoid X receptor; PTH-R, parathyroid hormone receptor. [Modeladapted from, reference (244), with permission.]

D were reduced by about threefold and twofold, respectively(324). Consequently, there was no hypercalciuria in ClC-5null mice (324). Thus, it was speculated that the balance be-tween the reduced supply of the precursor and the increase ofthe hydroxylase will determine the appearance of hypercal-ciuria and kidney stones. The delicate balance between theseopposing processes might explain the variability of clinicalsymptoms of patients with Dent’s disease, as well as the dif-ference between the Jentsch mouse model and the ClC-5 KOfrom the Guggino lab (440). Guggino ClC-5-deficient micedeveloped renal tubular defects as observed for the Jentschmouse but additionally showed hypercalciuria and renal cal-cium deposits (440).

To better understand the malfunction in kidney of ClC-5KO mice, a gene expression profile was performed. Over-all, 720 genes involved in biological processes like lipidmetabolism, organ development, and organismal physiologi-cal processes were shown to be differently expressed (457).Additionally, Devuyst and colleagues found by RNA and pro-tein analysis increased cell proliferation and oxidative stressresponse. This was suggested to be due to an increased levelof type III carbonic anhydrase found in kidney lysates andurine of ClC-5 KO mice, in urine of megalin receptor KOmice and a Dent’s disease patient carrying a CLCN5 mutation(125). Whether these changes are direct consequences of, orsecondary to, the endocytosis defect remains elusive.




Dent’s disease illustrates impressively that impaired en-docytosis, even if occurring in a restricted set of cells, mayentail various and serious secondary complications. It alsobolsters the contention that proximal tubular endocytosis offiltered proteins does not primarily serve to conserve proteinsor amino acids, but is important for the recovery of vita-mins and hormones bound to various carrier proteins (293).This is not only apparent from the changes in PTH and vi-tamin D endocytosis and metabolism that ultimately lead tothe most severe symptoms of Dent’s disease, that is, kidneystones and nephrocalcinosis, but also from the upregulationof retinol target genes (244) that are expressed in the distaltubule that may be explained by the reduced endocytosis ofretinol-binding protein (324). A recent clinical report high-lights this aspect: Three Indian boys carrying CLCN5 muta-tions not only showed typical symptoms of Dent’s disease likehypercalciuria and low-molecular weight proteinuria, but alsorecurrent episodes of night blindness (376). As night blind-ness in these patients could be cured by giving vitamin A, itis most likely due to the urinary loss of vitamin A bound tothe retinol-binding protein.

Pathophysiology of ClC-5 KO in thyroidand intestine

Due to the restricted expression pattern of ClC-5, only a feworgans were investigated for a possible physiological roleof ClC-5. Although the liver expresses low levels of ClC-5no significant defects in endocytosis of asialofetuin could bedetected in the ClC-5 KO mouse (324). The expression levelof ClC-5 in the thyroid where endocytosis and subsequentintracellular processing of thyroglobulin is central to organfunction is rather modest with about 10% to 20% of the kidneylevel (243,421). The Jentsch ClC-5 KO mouse showed neitherdefects in megalin expression and function in transcytosis, norhistological abnormalities of the thyroid (243). The GugginoClC-5 KO mouse did not show an apical endocytosis defecteither, but displayed goiter that was attributed to impairediodide efflux by a downregulation of pendrin expression (421).The reason for these differences and the mechanisms leadingto a reduced expression of pendrin are not understood.

To test whether ClC-5 modulates the immune responseand thereby inducing susceptibility to ulcerative colitis in theintestine, the Guggino group induced colitis by dextran sul-fate sodium. Elevated colitis as well as immune dysregulationwas found in ClC-5 KO mice implying a role of ClC-5 inthe immunopathogenesis of ulcerative colitis (9). Along thesame line, our microarray analysis using intestine of ClC-5-deficient mice detected differences in genes implicated inthe immune system (ArrayExpress database: E-MEXP-495)(244), suggesting a role of ClC-5 in immune response. How-ever, although ClC-5 is highly expressed in the intestinal ep-ithelial tissue and in α-intercalated cells of the collecting duct,its precise physiological role in those cells needs further to beinvestigated.

ClC-5 is important for endosomal acidification

The defect in renal endocytosis, the expression of ClC-5 onapical endosomes and its colocalization with the proton pumpon vesicles of PTCs strongly suggested that the Cl– conduc-tance by ClC-5 is required for efficient acidification of endo-somes by providing an electrical shunt (324). This hypoth-esis was confirmed using isolated endosomal fractions fromkidney cortex of WT and KO animals. ATP-induced Cl–-dependent acidification was observed in endosomes from KOand WT animals, but the final level of acidification was indeedremarkably reduced in ClC-5 KO endosomes (141,291,324).Other groups have confirmed these findings. In endocyto-sis experiments with cultured PTCs, fluorescent sensors cou-pled to transferrin or α2-macroglobulin were used to mea-sure pH and Cl– concentrations in early/recycling endosomesand late endosomes, respectively (145). Luminal acidifica-tion and chloride accumulation were reduced in early, but notin late endosomes of cultured PTCs from ClC-5-null mice(145). Likewise, Guggino and colleagues showed a reducedendocytosis in primary PTC cultures from ClC-5 KO ani-mals compared to wildtype (444). Moreover Cl– depletionand treatment with bafilomycin A1, which inhibits the V-typeH+-ATPase, disturbed endocytosis in WT cells but did not im-pair endocytosis further in ClC-5 KO cells, thereby indicatingthat ClC-5 is important for Cl–- and proton pump-mediatedendocytosis (444).

Shortly after pinching off from the plasma membrane,the Cl– concentration inside endocytic vesicles might be ashigh as in the extracellular space (∼120 mmol/L), consider-ably higher than the cytoplasmic Cl– concentration in epithe-lial cells (∼30 mmol/L) or in neurons (∼10 mmol/L). It is,therefore, tempting to speculate that ClC-5 may directly acid-ify endosomes by exchanging endosomal Cl– for cytosolicH+ (362). Indeed, Smith and Lippiat proposed recently thatthis mechanism, which is independent from the H+-ATPase,accounts for a part of endosomal acidification (385). How-ever, there are some problems with this mechanism. A roughcalculation shows that with a 2:1 stoichiometry of Cl–/H+-exchange, lowering luminal [Cl–] from 120 to 30 mmol/Lwould raise luminal total [H+] by 45 mmol/L. When con-sidering a typical buffer capacity of 30 mmol/L H+/pH unit,this corresponds to a decrease in pH by 1.5 units to a lu-minal pH of 5.9 which is in the range for endosomes (butmore alkaline than lysosomes (∼4.75)). However, even whenone disregards the electrogenic nature of 2Cl–/H+-exchange,this calculated value can clearly not be reached because lu-minal [Cl–] must be higher than cytosolic [Cl–] to maintainthe pH gradient to the cytosol (pH ≈ 7.2). Considering thecharge transfer associated with 2Cl–/H+-exchange, it turnsout immediately that this mechanism is not feasible. Withoutan appropriate countercurrent, Cl– exit through the exchangercreates a lumen-positive potential, which not only would shutdown ClC-5 activity by voltage-dependent gating, but alsowould immediately lead to an equilibrium potential (in thiscase +28 mV) where no transport occurs. The H+-ATPase




ADP+Pi

2Cl–

H+

H+

ClC-5

Lumen

cytoplasm

ADP+Pi

Cl–

H+

ClC-5unc

Lumen

cytoplasm

ADP+Pi

Cl –

H+

Lumen

cytoplasm

? E211A

Knock-in(C) Wildtype(B) Classical model (A)

ATP ATPATP

Figure 10 Models for endosomal acidification. According to the classical model (A), Cl– channels provide theelectrical shunt for the proton-pumping V-ATPase in endosomes. But in reality, the Cl–/H+-exchanger ClC-5performs this role (B). Neutralizing its “gating glutamate” uncouples the Cl− conductance from H+ countertransport,which results in a pure Cl– conductor (C), going back to “classics” (A). [Model adapted from, reference (291), withpermission.]

would increase rather than decrease this voltage. The onlyavailable counterion could be Na+, with an initial lumen-to-cytosol gradient of approximately 140 to approximately 10mmol/L. Hence, if one postulates an endosomal Na+ channel,a total dissipation of this gradient would predict (based on thetransport of three charges per H+ by the 2Cl–/H+-exchanger)a maximum transfer of 130/3 = 43 mmol/L H+. Whereas thisvalue is consistent with the upper limit obtained in the afore-mentioned considerations on Cl– gradients, there is currentlyno evidence for a significant endosomal Na+-conductance.Moreover, the model predicts an osmotic shrinkage of endo-somes to less then a fourth of their initial size. Importantly, Cl–

concentration in endosomes was measured to be low shortlyafter pinching off from the plasma membrane, a finding thatwas tentatively attributed to the negative surface charges ofmembranes (145, 388), and endosomal [Cl–] increased ratherthan decreased during endosomal acidification.

ClC-5 may raise endosomal chloride concentration

It has been commonly accepted for decades that chlorideis required for sufficient acidification not only for vesiclesalong the endosomal/lysosomal pathway but also for synap-tic and secretory vesicles (254). Therefore, vesicular chloridechannels were long thought to electrically shunt the voltagecreated by the V-type H+-ATPase. In the absence of Cl– cur-rents, as observed in the ClC-5 KO, further proton pumpingwould be inhibited and hence acidification perturbed. How-ever, ClC-5 is a Cl–/H+-exchanger rather than a Cl– channeland it appears counterintuitive that such an exchanger shouldelectrically shunt proton pump currents because it mediatesa parallel H+-efflux during ATP-driven acidification. To ex-plore the biological consequences of coupling chloride fluxto proton gradients, we have generated ClC-5 knock-in micecarrying a single-point mutation in the “gating glutamate”that converts ClC-5 from a 2Cl–/H+-exchanger into a pure,uncoupled Cl– conductor (Fig. 10). Such a Cl– conductorshould efficiently support endosomal acidification as postu-

lated in the classical model of vesicular acidification. Thus,any potential phenotype of these mice, which we dubbedClC-5unc mice (unc for uncoupled), may not be attributed toimpaired endosomal acidification, but can be ascribed specif-ically to a loss of coupling of the Cl–/H+-exchange. In fact,ClC-5unc mice displayed normal acidification of renal endo-somes in vitro (Fig. 11) (291). Surprisingly however, ClC-5unc

mice displayed impaired proximal tubular endocytosis thatwas indistinguishable from that found in ClC-5-null mice.ClC-5unc mice also displayed hyperphosphaturia and hyper-calciuria (291). As early endosomal acidification was not im-paired, the similar phenotype of ClC-5 KO and ClC-5unc micemight be due to a reduced endosomal chloride accumulation.Mathematical modeling of a minimal vesicle containing justan H+ pump, an H+ leak, and either a 2Cl–/H+-exchanger ora Cl– channel predicts that stoichiometric 2Cl–/H+-exchangewould result in a higher vesicular Cl– concentration than apure Cl– conductance during active acidification (448). In-deed, using a dextran-coupled, ratioable Cl–-sensitive dye,lysosomes containing the WT ClC-7 Cl–/H+-exchanger dis-played higher luminal Cl– than their ClC-7 KO or ClC-7unc

counterparts (448). These findings suggest that endosomalchloride concentration may play a pivotal role in endocytosis.

Additionally, mathematically modeling the ATP-dependent acidification of the minimal vesicle predicts thatthe lumen becomes positive with a Cl– channel whereas it sur-prisingly reaches negative voltages with a Cl–/H+-exchanger(Fig. 12) (448). This might solve the dilemma that the stronglyoutwardly rectifying vesicular CLC proteins are thought to benearly inactive at lumen-positive voltages (322,362,400), butit conflicts with the common opinion that endosomes andlysosomes are inside-positive (122,296,396). Thus, consider-ing the effect of Cl–/H+-exchanger on vesicular voltage mightbe valuable for further conclusions. Furthermore, as a resultof the lumen-negative potential reached with the exchanger,a more acidic steady-state pH is reached when proton pumpcurrents are shunted by a 2Cl–/H+-exchanger rather than aCl– channel (448).




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Figure 11 Renal endosomal acidification of mice converting ClC-5 into a chloride conductor. (A) Averaged tracesof acridine orange fluorescence comparing ATP-driven acidification of endosomal fractions from renal cortex of WT(green) and ClC-5unc (red) (left panel) and WT and Clcn5– (black) mice (right panel). Acidification of WT and ClC-5unc vesicles occurred with similar efficiency but was severely reduced with endosomes from mice in which ClC-5 waslacking. (B) Fluorescently labeled dextran, a marker for fluid-phase endocytosis (green), was injected into femaleClcn5+/unc mice. Cells expressing ClC-5unc accumulated much less dextran than neighboring cells expressing the2Cl–/H+-exchanger ClC-5 (for details, see (291)). [Images taken from, reference (291), with permission.]

ClC-6: a neuronal chloride- /proton-exchangeron late endosomesClC-6 was cloned in parallel with ClC-7 (40). According totheir sequence identity of about 45%, ClC-6 and ClC-7 consti-tute the third subfamily of mammalian CLCs (Fig. 1). Like themembers of the second subfamily, ClC-3 through -5, they lo-calize to compartments of the endosomal/lysosomal pathway(197, 327). However, unlike members of the second branch,ClC-6 and ClC-7 were neither found to coimmunoprecipitatewith each other nor with any other CLC (404).

ClC-6 is a Cl–/H+-exchanger

It has been shown (186) that expression of ClC-6 can rescuethe growth phenotype of the yeast mutant gef1, in which thesingle yeast CLC is disrupted (136). However, due to its intra-cellular localization no ClC-6-specific currents were observedupon heterologous expression in Xenopus oocytes (40, 46) orCOS cells (45). This problem has recently been overcome byexpression of a ClC-6 fusion construct carrying an amino-terminal GFP tag (275). This construct reaches the plasmamembrane in Xenopus oocytes and in cultured mammalian

cells to a significant extent. As suggested by the presence ofa “proton glutamate,” ClC-6 was found to mediate Cl–/H+-countertransport (275) like members of the second mam-malian CLC subfamily (322, 362) and ClC-7 (135, 206, 448).Both ClC-6 (275) and ClC-7 (206) share basic biophysicalfeatures with the Cl–/H+-exchangers of the second CLC sub-family, such as the outwardly rectifying voltage dependenceand the deactivation by acidic external pH (pHo).

ClC-6 localizes to late endosomes of neurons

On the protein level, ClC-6 is virtually exclusively expressedin the nervous system (327) whereas ClC-6 mRNA hasbeen found in many more tissues, including brain, kidney,testis, lung, skeletal muscle, thymus, intestine, and pancreas(40, 95, 186). A physiological relevance of ClC-6 mRNAsplice variants identified by reverse transcription PCR (RT-PCR) (95) is questionable, because the expressed proteinswould be severely truncated. Whether the difference betweenthe ubiquitous RNA expression and the neuron-specific pro-tein expression of ClC-6 is due to translational control orto a difference in protein stability, possibly mediated by anunknown neuronal β-subunit, remains an open question.




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Figure 12 Minimal mathematical model for ATP-dependent vesicleacidification. Mathematical modeling for the ATP-dependent acidifica-tion of a minimal vesicle containing just an H+ pump, an H+ leak, andeither no Cl– conductance (−/−; black traces), a 2Cl–/H+-exchanger(+/+; green traces) or a Cl– channel (unc/unc; red traces). For details,see (448). (A) Simulations predict a more efficient vesicular acidificationwith the exchanger despite H+ efflux due to Cl–/H+ antiport comparedto the acidification predicted with a Cl– channel. There is no acidifi-cation without any Cl– conductance. (B) Upon acidification an inside-positive potential is predicted with a Cl– channel whereas the lumenbecomes negative with a Cl–/H+-exchanger. (C) The calculated in-travesicular Cl– concentration is higher with a Cl–/H+-exchanger thanwith a Cl– channel. The initial conditions were: pHi = 7.2; pHo =7.2; [cation+]i = 140 mmol/L; [cation+]o = 140 mmol/L; [Cl–]i =50 mmol/L; [Cl–]o = 50 mmol/L; U = 0 mV. [Graphs adapted from,reference (448), with permission.]

Like ClC-3, -4, -5, and -7, ClC-6 localizes to compart-ments of the endosomal/lysosomal pathway. Although anearly study showed ER localization of epitope-tagged ClC-6 upon overexpression (45), it is now accepted that nativeClC-6 resides predominantly on late endosomes (327). In KO-

controlled immunocytochemistry, ClC-6 colocalized partiallywith lamp-1 (lysosome-associated protein-1). In subcellularfractionation, it comigrated partially with the late endosomalClC-3 and with the late endosomal/lysosomal ClC-7 on a Per-coll gradient (327). Interestingly, ClC-6 was shifted partiallyinto lysosomal fractions in ClC-7 KO tissue (327). Whereasa late endosomal localization of endogenous ClC-6 was laterconfirmed in the human neuroblastoma cell line SH-SY5Y(167), heterologously expressed ClC-6 colocalized either ad-ditionally (404) or predominantly (167, 393) with markers ofearly and recycling endosomes. How exactly ClC-6 is tar-geted to its subcellular destination remains an open question.Endosomal sorting is determined by cytosolic amino- andcarboxy-termini (393). However, disruption of motifs that me-diate adaptor binding in vitro did not affect the early/recyclingendosome localization of ClC-6 (393). However, mutation ofan amino-terminal stretch of basic amino acids to alaninespartially shifts heterologously expressed ClC-6 from early en-dosomes to ClC-7-positive late endosomes/lysosomes (167).Interestingly, this basic amino acid stretch is also involvedin the incorporation of ClC-6 into detergent-resistant mem-branes (167).

(Patho-)physiology of ClC-6

In a large-scale association study the human CLCN6 locus,which encodes for ClC-6, has recently been linked to bloodpressure regulation (415). Furthermore, single nucleotidepolymorphisms in the intronic region of CLCN6 have beencorrelated with plasma levels of NT-proBNP (the N-terminalcleavage product of the B-type natriuretic peptide), a markerassociated with cardiac dysfunction (76). However, an ef-fect on the expression of ClC-6 has not been investigated andmechanistic insight as to how the neuronal ClC-6 protein mayaffect cardio-vascular functions is unclear. These associationstudies await replication in other cohorts.

More information on the physiological function of ClC-6has been gained from studying Clcn6–/– mice. These displaya normal life span, are of normal size and exhibit no other im-mediately apparent phenotype (327). However, after 4 weeksof age, lysosomal storage material becomes apparent both incentral as well as in peripheral neurons (327). Apart from lyso-somal markers, such as lamp-1 and cathepsin D, the depositscontain saposin D and the subunit c of the mitochondrial ATPsynthase (327). These latter proteins are typically present instorage material accumulated in NCL, a type of lysosomalstorage disease (304, 418). The neuropathologic phenotypeof ClC-6 KO mice (327) is much milder than that of ClC-3or of ClC-7 KO mice. Clcn6–/– mice are not blind and theydisplay little neuronal cell loss and microglial activation (327,328). Their phenotype resembles a milder, late-onset form ofNCL, Kuf’s disease. Out of 75 patients that were tested formutations in CLCN6, two carried heterozygous missense mu-tations (327). The relevance of this finding is unclear becauseno mutation was found on the other allele (327).




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Figure 13 Neuronal storage material in ClC-6 and ClC-7 KO mice. Electron micro-graphy shows the accumulation of electron-dense deposits (arrows) in ClC-6-deficient hip-pocampal neurons and in cortical neurons of ClC-7 KO mice. The localization of storagematerial is restricted to the initial axon segment in ClC-6-deficient neurons, whereas itis scattered through the entire soma of ClC-7 KO neurons. nuc, nucleus; scale bars, 2μm. [Images modified from, references (327) (left panel) and (183) (right panel) , withpermission.]

Another difference between the phenotypes of ClC-6 andClC-7 KO mice is the subcellular localization of the deposits(Fig. 13). Whereas in ClC-7-deficient neurons lysosomal stor-age material is found all over the somata (183), these depositsare restricted to proximal axons of ClC-6 knockout neurons(327). In about 20% of cortical neurons, this accumulationleads to a swelling of the initial axon segments (327). Animpairment of dorsal root ganglion neuronal function by thisstorage material may explain the reduced pain sensitivity ofClC-6 KO mice (327).

The molecular mechanism leading to the lysosomalpathology of ClC-6 KO mice remains elusive. The steady-state pH in lysosomes of cultured hippocampal neurons fromClcn6–/– mice is not different from wildtype (327). This doesnot exclude an alteration of late endosomal pH, which in turncould affect late endosomal/lysosomal enzymatic reactionsor traffic and eventually lead to lysosomal storage. However,recent studies on the early endosomal ClC-5 (291) and lyso-somal ClC-7 (448) indirectly suggest that also the Cl–/H+-exchanger ClC-6 may rather have a role in the luminal accu-mulation of chloride.

ClC-7/Ostm1: a Cl–/H+-exchanger importantfor lysosomal function and bone resorptionClC-7 and its β-subunit Ostm1 display a broad tissuedistribution and localize to lysosomes. In addition, theylocalize to the ruffled border of osteoclasts. ClC-7/Ostm1is engaged in diverse physiological processes includinglysosomal protein degradation and bone resorption and itsloss or dysfunction leads to several disease phenotypes (175).These phenotypes, together with recent work of the lastfew years that give insight into the molecular physiology ofClC-7/Ostm1, will be discussed in this article.

Basic biophysical properties of ClC-7

Based on homology, ClC-6 and ClC-7 form the third branchof mammalian CLCs (40) (Fig. 1). As in the case of ClC-6, theexclusive intracellular localization of ClC-7 has precluded abiophysical characterization for years. Like the other intracel-lular CLCs, ClC-7 displays a “proton glutamate,” suggestingthat it functions as a Cl–/H+-exchanger. This notion was sup-ported by a study showing chloride- /proton-exchange activityof native lysosomes (135), a compartment where ClC-7 lo-calizes as the only CLC (197). The Cl–/H+-exchange activitywas unambiguously attributed to ClC-7 in experiments us-ing isolated lysosomes from ClC-7 KO mouse tissue (448).Recently, the partial cell surface localization of ClC-7 mu-tants in endosomal sorting motifs (393) allowed for a muchmore detailed biophysical characterization of ClC-7/Ostm1in heterologous expression (206). This study revealed thatClC-7/Ostm1 shares basic biophysical features with otherCLC exchangers, such as a strong outward rectification, aCl–>I– conductance sequence, a decrease in transport activ-ity with acidic external pH and an exchange stoichiometryof 2Cl–:1H+. This work also revealed features on the trans-port mechanism of CLC exchangers that could not be stud-ied with other mammalian CLCs. The relatively slow activa-tion (steady state was not yet reached after 2 s of activatingdepolarization) and relaxation kinetics allowed tail currentanalyses of the “open transporter.” These currents revealedthat the transport process per se is almost linearly dependenton the voltage and that the outward rectification is nearlyexclusively due to voltage-dependent gating (206). Further-more, Leisle et al. (206) showed that Ostm1 is not only re-quired for the stability of ClC-7, as suggested previously (204)(see later), but it is also essential for the transport activity ofClC-7.




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Figure 14 Subcellular localization of ClC-7 and Ostm1 in mouse fibroblasts. Top panel: Costainingfor ClC-7 (green) and the lysosomal marker lamp-1 (red) revealed localization of ClC-7 on lysosomalcompartments in mouse fibroblasts. Lower panel: Immunofluorescence of ClC-7 (green) and its β-subunit Ostm1 (red) showed colocalization of the two subunits. [Images modified from, reference (448),with permission.]

ClC-7 localizes to lysosomes and to the ruffledborder in osteoclasts

ClC-7 mRNA seems ubiquitously expressed and was detectedin every sample of a large variety of murine tissues examinedby Northern blot (40). In situ hybridization revealed particu-larly high expression in brain, eye, spinal cord and in dorsalroot and trigeminal ganglia in mouse embryos (197), and inPurkinje cells, renal proximal tubules and Sertoli cells, and inpancreatic and tracheal epithelia of adult mice (186). This ex-pression pattern was consistent with that of β-galactosidaseunder the endogenous ClC-7 promoter in ClC-7 KO mice(197). X-Gal staining of these animals additionally showedexpression in osteoclasts and, at closer examination of ex-pression in the eye, expression in the lens, retinal pigmentepithelium and cells of the neuroretina (197). β-galactosidaseexpression was also reported for all regions of adult kidney(445). KO-controlled immunocytochemistry confirmed ex-pression in neuronal tissues (183), in microglia and in variouscortical nephron segments including proximal tubules (445).

ClC-7 colocalizes with the late endosomal/lysosomalmarker lamp-1 in mouse fibroblasts (197) (Fig. 14), hip-pocampal neurons (183), and PTCs (445). The lysosomal lo-calization has been confirmed by immunoelectron microscopyon cortical neurons (183) and by immunoblots of subcellularfractionations (197). ClC-7 belongs to the network of lyso-somal proteins whose expression is regulated by the tran-scription factor EB (TFEB) (359). Colabeling for ClC-7 withClC-3 and ClC-5, respectively, showed only limited overlap

in renal PTCs, showing that ClC-7-positive structures are dis-tinct from those positive for ClC-3 or ClC-5 (445). Subcellularfractionation demonstrated that ClC-7 is the only CLC resid-ing on lysosomes (327) and, like ClC-6, ClC-7 is unlikelyto form heteromers with any other CLC (404). Interestingly,ClC-3 and ClC-6 shift partially into lysosomal fractions ofmouse brain upon disruption of ClC-7 (327). ClC-7 is foundon late endosomes and lysosomes also upon heterologousexpression (204,404). The amino-terminus of ClC-7 exhibitsstretches of amino acid stretches that mediate binding to mem-brane traffic adaptor proteins of the adaptor protein complex(AP) and the Golgi-localized, γ-ear-containing, Arf-binding(GGA) family, respectively (393). The AP-binding motif(s)and a yet unidentified, carboxy-terminal motif are responsiblefor the sorting to late endosomes/lysosomes (393).

In bone-resorbing osteoclasts, ClC-7 does not only lo-calize to late endosomal/lysosomal compartments, but alsoresides in the membrane of the ruffled border (197). The pres-ence in this specialized plasma membrane domain, whichfaces the acidified resorption lacuna, is important for the roleof ClC-7 in bone physiology, which will be discussed later.

ClC-7 requires Ostm1 as a β-subunit

ClC-7 requires the small transmembrane protein Ostm1as a β-subunit both for its stability (204) and for its iontransport activity (206). A mutation in the Ostm1 gene, whichencodes Ostm1 (short for osteopetrosis-associated membrane




protein1), was shown to underlie the severe osteopetroticphenotype in the spontaneous mouse mutant grey-lethal(57). In an independent study, the same protein was foundas an interactor of RGS-Galpha-interacting protein (RGS-GAIP) and hence named GIPN (GAIP-interacting proteinN-terminus) (111). GIPN (Ostm1) was suggested to be an E3ubiquitin ligase with a RING finger domain (111). However,Ostm1 is a type I transmembrane protein with a signalpeptide, a highly glycosylated extracytosolic part and a rathershort cytosolic carboxy-terminal stretch following the singletransmembrane domain (204). This topology excludes thepreviously proposed function as a ubiquitin ligase becauseit puts the putative RING-finger domain outside the cytosol.It also makes a more recently proposed regulatory role inthe Wnt/β-catenin signaling pathway (107) hard to conceive.In that study, it was shown that overexpression of Ostm1enhances Wnt-stimulated transcription by increasing theβ-catenin/Lef1 interaction downstream of receptor activa-tion. Surprisingly, amino-terminal protein fragments, whichaccording to the Ostm1 topology are secreted from the cell(204), showed dominant-negative effects on this step of Wntsignaling (107). An explanation as to why overexpression offull-length Ostm1 and the truncation constructs had oppositeeffects on Wnt signaling would be hard to envisage.

Like ClC-7, Ostm1 is expressed ubiquitously(57, 111, 204). In osteoclasts, both genes are coregulatedby the transcription factor microphthalmia (252) and bothproteins are upregulated in microglia upon activation (234).Endogenous Ostm1 colocalizes perfectly with ClC-7 on lateendosomes/lysosomes (Fig. 14) and to the ruffled border ofosteoclasts (204). Both proteins can be coimmunoprecipitated(204, 234). Protein levels, but not mRNA levels, of ClC-7and Ostm1 are strongly reduced in KO tissues lacking therespective other protein (204). ClC-7 is reduced to about 5%of WT level in the grey-lethal mouse. It was hypothesized thatthe highly glycosylated amino-terminus of Ostm1 normallyprotects ClC-7, which lacks consensus sites for N-linkedglycosylation, from the activity of lysosomal proteases. Thereduction of Ostm1 in the ClC-7 KO mouse can be explainedby impaired ER export followed by ER-associated degrada-tion. Whereas ClC-7 alone traffics to lysosomes when heterol-ogously expressed, both ER export (204) as well as lysosomaltargeting (393) of Ostm1 require ClC-7. However, it has beensuggested recently that ClC-7 also depends on Ostm1 for itsdelivery to lysosomes of microglia (234). The transmembranepart of Ostm1 is required and sufficient for Ostm1 to becotrafficked with ClC-7 to lysosomes, as the other parts canbe substituted by the equivalent parts of the plasma membraneprotein CD4 (206). Coexpression of the closest homologueof ClC-7, ClC-6, does not support ER export of Ostm1 (204).

ClC-7 currents require the coexpression of Ostm1 con-structs containing at least the transmembrane and extracy-tosolic amino-terminal part (206). While the transmembranepart might be solely required for physical interaction andcotrafficking, the glycosylated amino-terminal part of Ostm1may modulate ClC-7/Ostm1 transport activity (206). Because

ClC-7/Ostm1 currents are decreased by acidic external pHand require Ostm1 (206), it is obvious that acid-activated cur-rents previously ascribed to ClC-7 (84, 181, 295) are ratherendogenous currents of the respective expression system. Inretrospect, the lack of complementation of the gef1 mutant byClC-7 (186) might have been due to the lack of Ostm1.

Because ClC-7 and Ostm1 only function together as acomplex, ClC-7 KO mice and Ostm1-deficient grey-lethalmice develop virtually identical phenotypes, including severeosteopetrosis, a short life span of only a few weeks, accumu-lation of lysosomal storage material and neurodegeneration,retinal degeneration and gray fur in an agouti background, allof which will be discussed later.

ClC-7/Ostm1 is involved in bone resorption byosteoclasts

ClC-7/Ostm1 is present on the ruffled border of osteoclasts(197, 204). This specialized domain of the osteoclast plasmamembrane faces the resorption lacuna, the sealed space be-tween the osteoclast and the bone (409). Build-up of the ruf-fled border involves massive lysosomal exocytosis, resultingin the presence of lysosomal membrane proteins, includingthe V-ATPase, in this domain. Active acidification of the re-sorption lacuna by the V-ATPase (34) requires an electri-cal shunt, similar to the luminal acidification in the endo-somal/lysosomal pathway. This countercurrent is thought tobe carried by chloride transport through ClC-7/Ostm1. In-deed, acidification of the resorption lacunae of ivory-attachedosteoclasts from ClC-7 KO mice was nearly abolished as re-vealed by staining with the pH-sensitive dye acridine orange(197). Probably as a consequence of impaired acidification,ClC-7 KO mice show impaired degradation of calcified bonematerial (197). In a later study, an acidification defect wasalso shown for a crude microsome preparation from a mix-ture of bone cells from ClC-7 KO mice (278). However, theproportion of vesicles stemming from osteoclasts and in par-ticular the resorption lacuna might be small. Surprisingly theassay was performed in the presence of high potassium andthe potassium ionophore valinomycin, a procedure that in-troduces an electrical shunt that should render acidificationindependent from a shunt by ClC-7. Apart from the impairedacidification of the lacuna, ClC-7-deficient osteoclasts dis-play underdeveloped ruffled borders (197), hinting toward arole of ClC-7 in lysosomal exocytosis. However, Clcn7–/– os-teoclasts are able to degrade decalcified bone to some extentin vitro (278).

Impaired bone resorption leads to dense, fragile bones ina disease called osteopetrosis (414). Of course, not only thelack of an electrical shunt, but also a disruption of the protonpump itself may diminish the acidification of the resorptionlacuna. Indeed, mutations in the a3 subunit of the V-ATPase(288) have been found to cause osteopetrosis in mice(211,373) and humans (116,199). Both these mice, as well asthe ClC-7 KO (Fig. 15) and the grey-lethal (Ostm1–/–) micedevelop a severe osteopetrosis accompanied by secondaryeffects, such as a lack of tooth eruption and extramedullary




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Figure 15 Bone phenotype of ClC-7 mouse models. Microcom-puted tomography images of tibiae show increasing severity of os-teopetrosis from Clcn7unc/unc to Clcn7–/– mice, which even lack thebone marrow cavity. [Images adapted from, reference (448), with per-mission.]

blood production due to obliteration of bone marrow cavities(139,197,204). ClC-7-deficient mice accumulate abnormallylarge osteoclasts (279). The osteopetrosis of ClC-7 KOmice was rescued by osteoclast-specific expression ofClC-7 under the tartrate-resistant acid phosphatase (TRAP)promoter (183). This experiment demonstrates that osteo-petrosis is a cell-autonomous consequence of a loss of ClC-7in osteoclasts. Surprisingly, a recent report states that TRAPpromoter-driven expression of Ostm1 in grey-lethal mice didnot rescue osteopetrosis (310). This result may suggest thatOstm1 has other functions in addition to its role as a ClC-7β-subunit. However, it was not ascertained in that report(310) that expression levels of Ostm1 were at least as high asin the wildtype.

Most humans suffering malignant infantile osteopetro-sis have mutations in the TCIRG1 gene, which encodesthe a3 subunit of the V-ATPase. Another approximately10% of these patients carry mutations in CLCN7. By nowabout 50 different human CLCN7 mutations are known(30, 49, 65, 117, 197, 207, 306, 317, 434, 435, 476). All hu-man OSTM1 mutations (57, 240, 305, 336, 337, 389) lead toan early stop codon that will result in the synthesis of anextracytosolic fragment. Whereas some human CLCN7 mu-tations lead to an early stop codon or to a frame shift, mostare missense mutations. Some of these latter mutations (e.g.,the dominant mutation G215R) interfere with the subcellulartargeting of ClC-7/Ostm1 and result in an ER retention of themutated transporter (206,369), whereas other missense muta-tions strongly reduce the ion transport activity (206). Anothersubset of osteopetrosis-causing mutants is targeted togetherwith Ostm1 to their normal subcellular localization and retainsion transport activity when studied in the background of theplasma membrane-targeted mutant (206). Interestingly, someof those mutants yield currents with accelerated activationand relaxation kinetics. It is unclear whether such changesin kinetics are pathogenic. Rather surprisingly, other disease-causing mutants give WT-like currents. In these cases, reducedprotein stability in native tissues, as shown for the R762Q mu-tation (197), might be responsible for their pathogenic effect.

A few studies investigated the properties of cultured os-teoclasts derived from patients with osteopetrosis. Osteoclasts

from patients carrying dominant mutations show an impaireddegradation of the inorganic as well as of the organic phaseof bone in culture (61, 152). A microsome preparation fromosteoclasts of patients heterozygous for the dominant G215Rmutation was reported to display strongly impaired acidifica-tion. However, those experiments were again performed in thepresence of potassium and valinomycin (151). The pathogeniceffect of the G215R mutation may be ascribed predominantlyto a partial ER retention of the mutant (206, 369).

Osteopetrosis represents a phenotype opposite to os-teoporosis, a disorder particularly common among elderlywomen. It was hence proposed (197) that inhibition of ClC-7/Ostm1 transport activity might be used to treat osteoporo-sis. This concept is bolstered by the finding that polymor-phisms in ClC-7 are associated with bone mineral densityvariations in human (89, 198, 316), suggesting a direct corre-lation between ClC-7/Ostm1 activity and bone homeostasis.The recently achieved cell surface expression, which greatlyfacilitates measurements of ClC-7/Ostm1 transport activity(206, 393), should allow the establishment of screening as-says.

The function of ClC-7/Ostm1 in lysosomalphysiology

Like Clcn2–/– and Clcn3–/– mice (38,402), Clcn7–/– and grey-lethal mice display retinal degeneration (197, 204). The de-generation becomes apparent at about 2 weeks after birth.Hardly any photoreceptors are left when ClC-7- or Ostm1-deficient mice reach the age of 4 weeks (197,204). Blindnessoften accompanies osteopetrosis in humans. In most cases,this results from a compression of the optical nerve by theosteopetrotic narrowing of the optical canal (401). Althoughthis narrowing occurs also in ClC-7 KO mice, the fact thatthe number of Clcn7–/– ganglion cells is not significantly re-duced at 4 weeks of age argues against retinal degenerationbeing secondary to osteopetrosis (197). Constriction of theoptical canal as a cause for retinal degeneration was excludedby the observation that this degeneration remained when theosteopetrosis of Clcn7–/– mice is rescued by ClC-7 expressionunder the TRAP promoter (183).

A progressive neuronal degeneration is observed in thecentral nervous system of Clcn7–/– and grey-lethal mice(183,204). This is most evident in the CA3 region of the hip-pocampus. Central nervous system symptoms have also beendescribed for human patients with mutations in CLCN7 (117)or OSTM1 (53, 305). In both Clcn7–/– and grey-lethal mice,neurodegeneration is accompanied by microglial activationand astrogliosis (183, 328). ClC-7 was also disrupted in aneuron-specific and regionally confined manner using mice inwhich Clcn7 exons were flanked by loxP sites (445). Becausethese survived much longer than constitutive Clcn7–/– mice,their neurodegeneration could be followed over a much longertime span. Perhaps not surprisingly, these mice revealedthat neurodegeneration occurred only in the areas whereClC-7 has been eliminated from neurons. Reactive microglia




lamp-1 VillinClC-7

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Figure 16 Enlarged late endosomal/lysosomal compartments in ClC-7-deficient PTCs. Kidney sectionsfrom mice with a chimeric ClC-7 deletion in renal proximal tubules were immunostained for ClC-7 (green),the late endosomal/lysosomal protein lamp-1 (red) and the proximal tubule marker villin (blue). Lamp-1-positive vesicles are drastically enlarged in ClC-7-deficient cells (KO) compared to their appearance inClC-7-expressing cells (WT). [Images modified from, reference (445), with permission.]

accumulated in only those areas in which ClC-7 had beendisrupted. As ClC-7 had been eliminated only in neurons,these results indicated that neurodegeneration is causedby a cell-intrinsic defect in neurons and that ClC-7 dis-ruption in immunoreactive microglia (as in Clcn7–/– mice)has no noticeable effect (445). In mice lacking, the lateendosomal/lysosomal ClC-7 or Ostm1, neurons accumulateelectron-dense lysosomal storage material (183, 204). Unlikedeposits in Clcn6–/– mice (327), storage material is scatteredthroughout the somata (Fig. 13). The lysosomal storagematerial of ClC-7 KO mice is autofluorescent and stainspositive for the subunit c of the mitochondrial ATP synthase(183). This is a hallmark of NCL, which is also oftenassociated with visual impairment (130). The similarities ofClC-7/Ostm1-deficient mice with typical NCL models havebeen highlighted recently in a detailed comparison of brainsfrom mice lacking ClC-7 or Ostm1 (328). ClC-7 KO andgrey-lethal mice show widespread atrophy of white mattertracts. A similar atrophy was observed with human patientswith OSTM1 mutations (53). It is consistent with the reducedlevel of sphingomyelin, sulfatide, and galactosylceramide inbrain from grey-lethal mice (329). Myelination defects havealso been reported for the peripheral nervous system in skinbiopsies of patients with OSTM1 mutations (10).

Lysosomal storage can be also found in renal PTCs ofClcn7–/– and grey-lethal mice (183, 204). In both neuronsand PTCs, the staining of the late endosomal/lysosomalmarker protein lamp-1 is altered. Whereas it appearsincreased and more diffuse in ClC-7-deficient neurons(183,445), lamp-1-positive structures are drastically enlargedin ClC-7/Ostm1-deficient PTCs (Fig. 16) (445). Endocytosisper se is unaffected and endocytosed cargo is delivered tothese enlarged vesicles. However, proteolytic degradation ofendocytosed protein (taken up by fluid-phase as well as byreceptor-mediated endocytosis) is slowed in PTCs lackingClC-7. This was elegantly shown by in vivo pulse-chaseexperiments. Mice carrying a chimeric ClC-7 deletion inproximal tubules allowed for the direct comparison betweencells expressing or lacking ClC-7 within the same tubule(445). Consistent with reduced lysosomal protein degrada-

tion, LC3-II, a marker for autophagic structures, accumulatedin brain and kidney of ClC-7 KO mice (445). It is not clearwhether this results from a reduced clearance of autophagicmaterial by lysosomes or from increased autophagy (446).Intriguingly, the enlargement of late endosomes/lysosomesin ClC-7-deficient PTCs is unlikely due to an accumulationof the endocytic cargo that is drastically reduced by theadditional disruption of ClC-5 (445).

In analogy to the roles of ClC-3, -4, and -5 in the acidi-fication of their respective compartments (141, 145, 146, 266,267,402), an elevated lysosomal pH was proposed to explainthe lysosomal phenotype upon loss of ClC-7/Ostm1. How-ever, already acridine orange-stained compartments in ClC-7KO osteoclasts indicated the presence of acidified lysosomes(197). Ratiometric measurements with a pH-sensitive dyethat was chased into lysosomes showed that ClC-7/Ostm1is dispensable for the maintenance of lysosomal pH. Nei-ther cultured neurons nor fibroblasts from Clcn7–/– or grey-lethal mice displayed an altered steady-state lysosomal pH(183,204). Similar results were obtained with primary alveo-lar macrophages from Clcn7–/– mice (395). Seemingly contra-dicting this body of data, reduced LysoTracker fluorescence inHeLa cells which were partially ClC-7-depleted by treatmentwith a single siRNA has been interpreted to indicate reducedlysosomal acidification (135). However, other parameters thatdetermine the averaged fluorescence intensity, such as the sizeand number of acidic compartments, have not been quantified.This makes a quantitative statement about lysosomal pH un-reliable (82). It is further disconcerting that treatment withscrambled control siRNA changed the LysoTracker fluores-cence, too, albeit into the opposite direction. Recently, it hasbeen shown that microglia activation leads to an upregulationof lysosomal ClC-7/Ostm1 and to a further acidification oflysosomes (234). It remains to be tested whether ClC-7 playsa direct causative role in the regulation of lysosomal pH inthis particular cell line. Not only ClC-7, but also even chlorideas a shunting ion is dispensable for lysosomal acidification bythe V-ATPase in vitro (448) and in living RAW macrophagesafter transient disruption of the pH gradient (395). Chlorideinflux can support acidification, but the main countercharge




Steady-state pHdetermined by voltage V

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Figure 17 Lysosomal transport characterization. carbonyl cyanide 3-chlorophenylhydrazone (CCCP) -induced alkaliniza-tion assay of lysosomes that were preloaded with the ratiometric pH indicator Oregon Green-dextran. The protononophorerapidly dissipated lysosomal pH in Clcn7+/+ (green), Clcn7unc/unc (red), and Clcn7–/– (black) fibroblasts, demonstrating thepresence of countercurrents. Lysosomes of Clcn7unc/unc mice reached a more alkaline pH than Clcn7–/– lysosomes, sug-gesting that the uncoupled transporter (ClC-7unc) mediates a Cl– conductance. However, Clcn7unc/unc lysosomes reacheda less alkaline steady-state pH than WT lysosomes. Since the steady-state pH is determined by voltage, which depends onthe Cl– diffusion potential, the difference in lysosomal pH suggests higher lysosomal chloride concentration in WT than inClcn7unc/unc lysosomes. Alkalinization of Clcn7–/– lysosomes by protonophore treatment argues for lysosomal conductancesin addition to ClC-7 most likely by a so far unknown cation conductance. Mathematical modeling of such an experiment(on the lower right) confirms the plausibility of the conclusions (for details, see (448)). The simulated vesicle contains either aCl–/H+-exchange activity, or a Cl– conductance or none of these, corresponding to the three genotypes (same color code asin the experimental data). In addition, the vesicle contains a cation conductance and a large proton conductance simulatingCCCP. The initial conditions were: pHi = 4.75; pHo = 6.4; [cation+]i = 100 mmol/L; [cation+]o = 10 mmol/L; [Cl–]i =120 or 80 mmol/L as indicated; [Cl–]o = 20 mmol/L. With the same initial [Cl–], the luminal pH of vesicles containing aCl–/H+-exchange activity or a Cl– conductance reaches the same value. Lower initial luminal [Cl–] in vesicles with a Cl–conductance may explain the different final pH values. [Graphs taken from, reference (448), with permission.]

for proton pumping seems to be provided by the lysosomalefflux of monovalent cations (395). The difference to endo-somes that need ClC-5 for acidification (141, 145, 291) maybe explained by the lower cation conductance on endosomescompared to lysosomes (422). Run-down of transport activ-ity during the long organelle isolation protocol might explainthe absence of a significant lysosomal cation conductance inbiophysical experiments demonstrating the Cl–/H+-exchangeactivity of lysosomes (135).

It has been speculated that the Cl–/H+-exchange activityof ClC-7/Ostm1 serves to accumulate chloride into acidiclysosomes (174), in analogy to nitrate accumulation intoArabidopsis vacuoles by AtClCa (74). This hypothesis hasbeen addressed by the generation of mice (Clcn7unc/unc) inwhich the “gating glutamate” of ClC-7 was replaced by ala-nine (448). This mutation uncouples ClC-7/Ostm1-mediatedchloride transport from proton countertransport (206, 448).Lysosomes of fibroblasts from these mice showed normalV-ATPase-mediated acidification in vitro and displayed noaltered steady-state pH (448). However, the difference inlysosomal pH reached after alkalinization by protonophoretreatment in vivo suggests a lower steady-state chloride con-

centration in the lysosomal lumen (Fig. 17). A differencein the lysosomal chloride concentration was detected witha ratiometric Cl–-sensitive dye chased into lysosomes. BothClC-7 KO fibroblasts and those expressing uncoupled ClC-7exhibited significantly reduced luminal chloride levels (448).Build-up of storage material, neuronal cell loss, microgliosis,and retinal degeneration are as pronounced as in ClC-7 KOmice (448). Hence, it was proposed that the severe lysoso-mal pathology of both Clcn7–/– and Clcn7unc/unc mice is dueto reduced luminal chloride. Intriguingly, Clcn7unc/unc micecarrying the uncoupling ClC-7 mutation present milder os-teopetrosis than Clcn7–/– mice (Fig. 15) (448). To explainthe intermediate severity of osteopetrosis, one can argue thatuncoupled ClC-7/Ostm1 can partially fulfill the role of ClC-7/Ostm1 in acidifying the resorption lacuna. It may providethe electrical shunt for proton pumping, while chloride ac-cumulation per se is not needed for bone resorption. Theremaining impairment in osteoclast function could then beexplained by a reduced exocytic delivery of lysosomes for thebuild-up of the ruffled border, thus indirectly by a lysosomaldefect. One further difference between mice lacking ClC-7or Ostm1 and mice expressing the uncoupled ClC-7 mutant




regards their hair pigmentation. In an agouti background, inwhich WT mice have brown hair, Clcn7–/– and grey-lethal(hence the name) mice are gray (139, 197). This suggestsa role of ClC-7/Ostm1 in the physiology of melanosomes,lysosome-related pigmentation organelles of specialized cells(338). The interaction between ClC-7 and the sorting adaptorAP-3 (393) would be consistent with a melanosomal local-ization of ClC-7, but how ClC-7/Ostm1 participates in hairpigmentation is unknown. The observation that mice withthe uncoupled ClC-7 mutation have brown fur like WT miceshows that the role of ClC-7/Ostm1 in pigmentation doesnot require Cl–/H+-exchange activity (448). Whether chlo-ride transport by ClC-7/Ostm1 is actually involved remainsto be investigated.

The obvious question is what function luminal chlorideserves and how its impairment leads to lysosomal dysfunc-tion. The chloride gradient could mediate secondary transportof metabolites or luminal chloride could regulate degradativeenzyme activity directly as shown for cathepsin C (63). Onthe one hand, activity of the lysosomal enzyme TPP I wasnot altered in cultured fibroblasts and neurons of ClC-7 KOmice (183), on the other hand do these cells not accumulatestorage material, either. In contrast, PTCs, which do displaydeposits, exhibit a slowed degradation of endocytosed protein(445). The finding that these cells also display enlarged lamp-1-positive structures when endocytosis is inhibited by theadditional ClC-5 knockout argues against the accumulationof cargo as the primary cause for the lysosomal phenotype.The late endosomal marker lysobisphosphatidic acid (LBPA)is also present on these enlarged structures, which indicatesan alteration in endosomal-lysosomal trafficking (445). Inanalogy, the importance of chloride accumulation in early en-dosomes by ClC-5 (291) can also rather be expected to liein the regulation of trafficking. A trafficking defect due toClC-7/Ostm1 dysfunction could also underlie the underde-velopment of the ruffled border in osteoclasts. No direct rolefor chloride has been ascribed to membrane traffic steps sofar. But chloride might also indirectly regulate fusion and fis-sion of organelles by its impact on other ions. For example, amodulation of endosomal calcium channels by luminal chlo-ride has been described (355) and calcium is discussed as aregulator of late endosomal/lysosomal trafficking (231).

ConclusionMore than 20 years ago, the first CLC was identified molec-ularly (179). Since then, enormous knowledge about all bio-logical aspects of this chloride transporter family has beengained. The nine mammalian CLC family members have beencloned within only a few years, and in parallel the physio-logical importance of CLCs became apparent from sponta-neous mouse mutants and human genetic disease. Alreadyin the first decade of research on CLCs, many laboratorieshave contributed to elucidating the transport mechanism andstructure-function relation of this anion transporter family by

numerous mutagenesis and electrophysiological studies. Inthe second decade, the generation of KO mice and the crystalstructures of bacterial CLCs and of various CBS domains hadstrong impact in this area of research. An important findingwas that some CLCs, such as the intracellular mammalianmembers, are Cl–/H+-exchangers rather than Cl– channels.This is of great relevance with respect to their physiologicalroles.

Genetically modified mouse models for the different CLCchannels and exchangers have significantly helped to uncoverthe amazingly broad physiological roles of this protein fam-ily. These mouse models display a wide spectrum of pheno-types. For human patients with some of the correspondingdiseases, responsible mutations have been found in the genesof the respective CLC or its β-subunit. These diseases includemyotonia, Bartter syndrome (with or without accompanyingdeafness), Dent’s disease, and osteopetrosis with neurodegen-eration. The plasma membrane CLCs are involved in regulat-ing electrical excitability by stabilizing the plasma membranevoltage, in the ion homeostasis of extracellular space and intransepithelial transport. A role common to all intracellularCLCs seems to be supporting vesicular acidification and/orluminal chloride accumulation. Due to their differential tis-sue expression and subcellular localization, they are involvedin different physiological processes. Finally, we would liketo mention the important insights into CLC functions gleanedfrom model organisms such as the yeast S. cerevisiae, the plantA. thaliana, and the worm C. elegans, which was beyond thescope of this review.

Despite the tremendous progress in our understanding ofthe physiological roles of CLCs, there are still many openquestions. To gain further insight, it will be of importance toidentify further regulatory mechanisms for CLC transporters,such as signaling and posttranslational modifications. Furtherinteractors, not only proteins but also lipids, will certainly bediscovered. So far, only two obligate β-subunits have beendescribed, but we expect more to follow. More specific pointsthat are unclear include the role of ClC-3 on synaptic vesicles,the mechanisms leading to retinal degeneration in ClC-2, -3,and -7 KO mice and to lysosomal storage material in ClC-6and -7 KO mice. Of great interest is the general role of in-tracellular CLCs in vesicular ion homeostasis. Recent worksuggests that luminal chloride accumulation is a crucial cellbiological function of the second and third CLC subfami-lies. This raises the question how intravesicular chloride isinvolved in the physiology of subcellular organelles.

AcknowledgementsWe thank the past and present members of our laboratoryfor their important contributions to understanding the bio-logy of CLC proteins. Our work has been supported in partby the Deutsche Forschungsgemeinschaft, the Bundesminis-terium fur Bildung und Forschung, the European Union, thePrix Louis-Jeantet de Medecine, and the Ernst-Jung Preis furMedizin.




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