Canal Calcio T

Molecular Physiology of Low-Voltage-ActivatedT-type Calcium Channels

EDWARD PEREZ-REYES

Department of Pharmacology, University of Virginia, Charlottesville, Virginia

I. Introduction 118II. Electrophysiology of Native T-type Currents 120

A. Low-theshold calcium spikes 120B. Neuronal 122C. Heart 125D. Kidney 127E. Smooth muscle 128F. Skeletal muscle 129G. Sperm 129H. Endocrine tissues 130I. Cell lines 131J. Single-channel recordings 131

III. Regulation 134A. Hormonal inhibition 134B. Hormonal stimulation 135C. Guanine nucleotides 136D. Protein kinases 136E. Voltage 137

IV. Molecular Cloning of T-type Channels 138A. Cloning 138B. Structure 140C. Distribution 141D. Electrophysiology of recombinant channels 141E. Auxiliary subunits? 146

V. Pharmacology 147A. Antihypertensives 147B. Antiepileptics 148C. Anesthetics 149D. Antipsychotics 149

VI. Conclusions 150A. Physiological roles 150B. Future directions 150

Perez-Reyes, Edward. Molecular Physiology of Low-Voltage-Activated T-type Calcium Channels. Physiol Rev 83:117–161, 2003; 10.1152/physrev.00018.2002.—T-type Ca2� channels were originally called low-voltage-activated(LVA) channels because they can be activated by small depolarizations of the plasma membrane. In many neuronsCa2� influx through LVA channels triggers low-threshold spikes, which in turn triggers a burst of action potentialsmediated by Na� channels. Burst firing is thought to play an important role in the synchronized activity of thethalamus observed in absence epilepsy, but may also underlie a wider range of thalamocortical dysrhythmias. Inaddition to a pacemaker role, Ca2� entry via T-type channels can directly regulate intracellular Ca2� concentrations,which is an important second messenger for a variety of cellular processes. Molecular cloning revealed the existenceof three T-type channel genes. The deduced amino acid sequence shows a similar four-repeat structure to that foundin high-voltage-activated (HVA) Ca2� channels, and Na� channels, indicating that they are evolutionarily related.Hence, the �1-subunits of T-type channels are now designated Cav3. Although mRNAs for all three Cav3 subtypes areexpressed in brain, they vary in terms of their peripheral expression, with Cav3.2 showing the widest expression. Theelectrophysiological activities of recombinant Cav3 channels are very similar to native T-type currents and can be

Physiol Rev

83: 117–161, 2003; 10.1152/physrev.00018.2002.

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differentiated from HVA channels by their activation at lower voltages, faster inactivation, slower deactivation, andsmaller conductance of Ba2�. The Cav3 subtypes can be differentiated by their kinetics and sensitivity to block byNi2�. The goal of this review is to provide a comprehensive description of T-type currents, their distribution,regulation, pharmacology, and cloning.

I. INTRODUCTION

Rises in intracellular calcium trigger a variety of pro-cesses including muscle contraction, chemotaxis, geneexpression, synaptic plasticity, and secretion of hor-mones and neurotransmitters. Although there are manychannels and pumps involved in controlling intracellularCa2� levels, voltage-gated Ca2� channels play a key rolein this process (50). Calcium is not only an importantsecond messenger, but its entry can also depolarize theplasma membrane, and thereby activate other voltage-gated ion channels. This property is especially importantfor neuronal T-type channels, which can generate low-threshold spikes that lead to burst firing and oscillatorybehavior (175, 377). This activity is especially prominentin the thalamus, where it plays an important role in sen-sory gating, sleep, and arousal (277). The term thalamo-

cortical dysrhythmias has been coined to describe patho-logical changes in these oscillations, and they have beenimplicated in a wide range of neurological disorders in-cluding absence epilepsy, the tremor associated with Par-kinson’s disease, tinnitus, neuropsychiatric disorders, andneurogenic pain (186, 242).

The ability of neurons to fire low-threshold Ca2�

spikes suggested the existence of low-voltage-activated(LVA) Ca2� channels. Since 1975 it has been recognizedthat there were at least two distinct types of Ca2� channel(reviewed in Ref. 153). Hagiwara, Ozawa, and Sand, usingtwo-microelectrode voltage-clamp recordings from star-fish eggs, found channels that were activated after smalldepolarizations of the membrane, LVA, and other chan-nels that required larger depolarizations of the membrane,high voltage activated (HVA) (154). LVA currents inacti-vated faster, more completely, and at more negative mem-brane potentials than HVA currents. Similarly, LVA andHVA currents were described in the marine pilewormNeanthes (123). Of historical note, the first recordings ofmammalian LVA channels were made by Moolenar andSpector in 1978 (290), who used the two-microelectrodevoltage-clamp technique on the mouse neuroblastomacell line N1E-115. However, these authors only observedone type of Ca2� channel, which in 10 mM Ca2� activatedat �55 mV and peaked at �20 mV. Subsequent studieshave shown that N1E-115 cells provide a convenient sys-tem to study the properties of native T-type currents (seesect. III). Using quinidine to block K� currents, Fishmanand Spector reported in 1981 (120) on the existence oftwo types of mammalian Ca2� channel. One type acti-vated at �50 mV, peaked at �20 mV, and inactivated

rapidly (� �15–30 ms). The second type peaked at 0 mVand inactivated slowly (� �2,000 ms). The existence ofLVA currents was firmly established by the work of manygroups, including those headed by Kostyuk (115, 420),Carbone and Lux (62), Armstrong (12), Bean (32), Feltz(54), and Tsien (303, 306). Whole cell patch-clamp record-ings showed that depolarizations in the �60 to �20 mVrange elicited rapidly inactivating currents, while higherdepolarizations elicited noninactivating currents. Plots ofthe current versus test potential (I-V) showed two com-ponents, with the LVA component appearing as a humpon the back of a larger HVA component. LVA currentsalso turned off, or deactivated, slower than HVA currents,producing slow tail currents (62). This property was stud-ied in great detail in GH3 cells, where slowly deactivating(SD) tail currents were found to activate at lower thresh-olds than fast deactivating (FD) currents (12). Tsien andcolleagues (306) extended the classification of dorsal rootganglion (DRG) channels and proposed that these chan-nels be called T type for transient, L type for long lasting,and N type for neither T nor L type. LVA currents werealso found to be resistant to rundown, unlike HVA cur-rents that required cAMP, ATP, and Mg2� in the pipettefor stability (115). T- and L-type channels were also de-scribed in cardiac myocytes from guinea pig ventricle anddog atrium and could be distinguished by their voltagedependence, kinetics, single-channel currents, pharma-cology, and conductance of Ca2� and Ba2� (32, 303). Theobservation that T-type currents inactivate at lower mem-brane potentials than L-type currents led to the develop-ment of an assay to separate these two components (32).The activity of both channels was recorded from a well-hyperpolarized potential such as �90 mV, then the activ-ity of L-type channels was recorded at a potential whereT-type channels are inactivated and L type are not (typi-cally �50 mV), then the L-type currents are subtractedfrom the T- plus L-type currents to isolate the T-typecurrent. Additional methods, such as tail current analysis,are required to isolate T-type currents in most other tis-sues due to the presence of HVA channels that also inac-tivate at �50 mV. Another possible contamination is volt-age-gated Na� currents that appear to conduct Ca2�

(ICa,TTX); however, these can be differentiated by theirsensitivity to tetrodotoxin (TTX) (162). Hallmark featuresof T-type channel electrophysiology are as follows: 1)they begin to open after small depolarizations of theplasma membrane (LVA); 2) their currents during a sus-tained pulse are transient; 3) they close slowly uponrepolarization of the membrane, generating a SD tail cur-

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rent; 4) they have a tiny, and equivalent, single-channelconductance of Ba2� and Ca2�; 5) they are relativelyinsensitive to dihydropyridines; and 6) their steady-stateinactivation occurs over a similar voltage range as activa-tion. In fact, T-type channels display a window current,i.e., there is a small range of voltages where T-type chan-nels can open, but do not inactivate completely. Thisproperty may be particularly important in controlling in-tracellular Ca2� levels (45). In conclusion, studies onnative channels have provided a toolkit for separatingCa2� channel subtypes, and these tools have proven use-ful in the characterization of both native and recombinantchannels.

T-type channels are expressed throughout the body,including nervous tissue, heart, kidney, smooth muscle,sperm, and many endocrine organs. These channels havebeen implicated in variety of physiological processes in-cluding neuronal firing, hormone secretion, smooth mus-cle contraction, myoblast fusion, and fertilization. In gen-eral, the electrophysiological properties of T-typecurrents recorded from various cell types are similar, butdifferences have been noted in how they inactivate and intheir pharmacology. This heterogeneity can be explainedin part by the existence of three T-type channels that areencoded on separate genes (90, 228, 324).

Voltage-gated Ca2� channels have been classifiedby their electrophysiological and pharmacologicalproperties, and more recently by their amino acid se-quence identity. There are at least 10 genes encoding�1-subunits of voltage-gated Ca2� channels (Fig. 1).Alignment of their deduced amino acid sequences sug-

gests that gene duplication and divergence of an ances-tral Ca2� channel gene gave rise to LVA and HVAsubfamilies. Further duplication of the HVA gene gaverise to Cav1 and Cav2 subfamilies. This duplicationmust have occurred well over 500 million years ago,since the nematode Caenorhabditis elegans containsone member of each subclass. Eventually the Cav1subfamily evolved into four genes, while both the Cav2and Cav3 subfamilies evolved into three. Cloning andexpression studies have established that the Cav3 fam-ily encodes LVA T-type channels (sect. IV), while theCav1 subfamily encodes L-type channels (although ex-pression of Cav1.4 has not been reported yet). Thesechannels play a critical role in excitation-contractioncoupling in cardiac, skeletal, and smooth muscle. Infact, the Cav1.1 gene has evolved beyond a Ca2� chan-nel, and its physiological role in skeletal muscle is as avoltage sensor, coupling membrane depolarization di-rectly to calcium release channels of the sarcoplasmicreticulum (SR) (340). In contrast, cardiac and smoothmuscle contraction requires influx of extracellular Ca2�

through Cav1.2 channels. In heart, this influx activates alarger release of Ca2� from intracellular pools via amechanism called calcium-induced calcium release(CICR) (37). Cav1.2 channels are an important thera-peutic target in the control of blood pressure and car-diac rhythm. The physiological roles of Cav1.3 channelshave been difficult to discern since pharmacologicaltools have not been described that can separate itsactivity from Cav1.2, and because these channels ap-pear to be coexpressed in a number of tissues. Incontrast, the activity of Cav2 family members can beselectively blocked by peptide toxins. Splice variationof the Cav2.1 gene results in channels that differ in theirsensitivity to the spider toxin �-Aga-IVA and thereforeencode both P- and Q-type channels (55). The Cav2.2gene encodes N-type channels, which are characterizedby their irreversible and potent block by the snail toxin�-conotoxin-GVIA. Studies with these toxins indicatethat Cav2.1 and Cav2.2 channels mediate the Ca2� influxinto presynaptic terminals that trigger neurotransmitterrelease. In contrast to these channels, it has been dif-ficult to determine which native Ca2� current is carriedby Cav2.3 channels. Recombinant Cav2.3 channels re-semble T-type channels in their sensitivity to nickel andtheir voltage dependence of inactivation, leading to theearly suggestion that they might encode T-type chan-nels (376). However, Cav2.3 channels require strongerdepolarizations for channel opening, i.e., are HVA chan-nels, and they deactivate 10-fold faster than T-typechannels (228). Studies with SNX-482, a tarantula toxinthat selectively blocks recombinant Cav2.3 channels,suggests that these channels mediate R-type currents insome tissues (299).

FIG. 1. Evolutionary tree of voltage-gated Ca2� channels. Tree isbased on an alignment of the putative membrane-spanning regions andpore loops of the human channels. Alignment of the full-length se-quences yields a similar pattern, although all the branch points areshifted to the left due to inclusion of nonconserved sequences. Low-voltage-activated (LVA) channels appear to have diverged from an an-cestral Ca2� channel before the bifurcation of the high-voltage-activated(HVA) channel family into Cav1 and Cav2 subfamilies. (Adapted fromPerez-Reyes E, Cribbs LL, Daud A, Yang J, Lacerda AE, Barclay J,Williamson MP, Fox M, Rees M, and Lee J-H. Molecular characterizationof T-type calcium channels. In: Low Voltage-activated T-type Calcium

Channels, edited by Tsien RW, Clozel J-P, and Nargeot J. Chester, UK:Adis International, 1998, p. 290–305.)

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Electrophysiological studies of recombinant chan-nels show that Cav3.1 (formerly �1G) and Cav3.2 (�1H)have similar activation and inactivation kinetics, but canbe differentiated by their recovery from inactivation andsensitivity to block by nickel (209, 230, 351). The kineticproperties of these �1-subunits closely resemble nativeT-type channels. In contrast, recombinant Cav1 and Cav2channels require auxiliary subunits for normal gating be-havior. This suggests that native T-type channels may beformed by a single �1-subunit. Cav3.3 (�1I) channels alsogenerate LVA currents; however, these currents activateand inactivate much more slowly than T-type channels.Native currents with these properties have only beendescribed in a limited number of studies (19, 99, 177, 316,398). Although there is no drug that is highly selective(�10-fold) for T-type over other ion channels, block ofT-type channels appears to contribute to the therapeuticusefulness of antihypertensives, antiepileptics, anesthet-ics, and possibly antipsychotics. Therefore, T-type chan-nels provide a novel target for drug development.

II. ELECTROPHYSIOLOGY OF NATIVE

T-TYPE CURRENTS

A. Low-Threshold Calcium Spikes

Low-threshold calcium spikes (LTS) have been de-scribed in slices and in isolated neurons from a variety ofbrain nuclei such as inferior olive (243, 244), thalamicrelay (96, 183), medial pontine reticular formation (146),lateral habenula (437), septum (10), deep cerebellar nu-clei (241), CA1-CA3 of the hippocampus (163, 307), asso-ciation cortex (122), paraventricular (399) and preopticnuclei of the hypothalamus (385), dorsal raphe (60), glo-bus pallidus (296), and the subthalamic nucleus (40).Typically the spike is crowned by a burst of action poten-tials (Fig. 2). Addition of TTX to block voltage-gated Na�

channels abolishes the fast action potentials, effectivelyisolating the slowly activating and inactivating LTS. Nu-merous studies have shown that the LTS is mediated by a

FIG. 2. Low-threshold Ca2� spikes generate burst firing. A: many neurons can generate two distinct patterns of actionpotential firing in response to a depolarizing stimulus. Regular, or tonic, firing is elicited when the neuron is depolarizedfrom a resting membrane potential near �55 mV. In contrast, when the membrane potential is below �70 mV, the samedepolarizing stimulus triggers a high-frequency burst of action potentials. [From Huguenard JR. Low-voltage-activated(T-type) calcium-channel genes identified. Trends Neurosci 21: 451–452, 1998, with permission from Elsevier Science.]B: a representative example of currents that generate burst firing. Depolarization of the plasma membrane by hyper-polarization-activated current (Ih) leads to activation of T-type currents (IT), and a second phase of depolarization calledthe low-threshold Ca2� spike. Riding on top of the low-threshold Ca2� spike are a burst of Na� spikes mediated by fastvoltage-gated Na� channels. High-threshold Ca2� and K� currents can also be activated by the low-threshold calciumspikes. Ca2� entry during the burst leads to activation of Ca2�-activated K� currents, which in combination withvoltage-gated K� channels repolarize the membrane. (From Bal T and McCormick DA. Synchronized oscillations in theinferior olive are controlled by the hyperpolarization-activated cation current Ih. J Neurophysiol 77: 3145–3156, 1997.)C: thalamic neurons of transgenic mice lacking expression of Cav3.1 do not fire bursts. Current-clamp recordings arefrom neurons held at �60, �70, or �80 mV, then depolarized or hyperpolarized by current injections of varyingmagnitudes as indicated below the set of traces. When the resting membrane potential is �60 mV, a depolarizing stimulustriggers tonic firing in both wild-type (�/�) and transgenic (�/�) animals. When the membrane potential (Vm) is loweredto �70 mV, a hyperpolarizing stimulus triggers burst firing in neurons from wild-type, but not transgenic animals. Whenthe resting membrane potential is �80 mV, a depolarizing stimulus only triggers burst firing in neurons from wild-typeanimals. (From Kim D, Song I, Keum S, Lee T, Jeong MJ, Kim SS, McEnery MW, and Shin HS. Lack of the burst firingof thalamocortical relay neurons and resistance to absence seizures in mice lacking �1G T-type Ca2� channels. Neuron

31: 35–45, 2001, with permission from Elsevier Science.)



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Ca2� conductance; it is not observed in Ca2�-free externalsolutions, and it can be blocked by divalent cations suchas Co2� and Ni2�. Definitive proof that thalamic LTS aremediated by T-type channels was provided by studies ontransgenic mice, where knockout of the Cav3.1 gene abol-ished these spikes and burst firing (Fig. 2C) (206).

With a few notable exceptions (7, 191), LTS cannotbe triggered by depolarization of the neuron from theresting membrane potential, which is typically between�60 and �65 mV. However, LTS can be observed after ahyperpolarizing pulse is delivered. This process has beencalled “deinactivation” and is due to recovery of channelsfrom inactivation. Table 1 lists a number of studies wherethe voltage and time dependence for recovery of thesespikes has been examined in detail. LTS began to appearafter the neuronal membrane was hyperpolarized below�69 mV, and full-amplitude spikes were observed whenthe membrane was below �73 mV. The rate of recoverywas measured by varying the duration of a hyperpolariz-ing pulse, then testing for the presence of an LTS uponreturn to the resting membrane potential. These studiesfound that the LTS deinactivated relatively quickly, withhalf of it recovering in �100 ms and full recovery after 200ms. Therefore, one physiological role of the LTS is as apacemaker. Although increases in intracellular concentra-tions of Ca2� act as a second messenger for a variety ofprocesses, in this case the important property is thatinflux of the divalent cation leads to direct depolarizationof the membrane. The amplitude of this depolarizationwas typically 25 mV (Table 1), which raised the mem-brane potential to approximately �40 mV. Since thethreshold of many voltage-gated Na� channels is around�55 mV, the LTS can trigger a burst of action potentials.

These Na�-dependent action potentials are brief (1–2 ms)and of high amplitude (�80 mV), and in some thalamicneurons of very high frequency (�400 Hz). High-thresholdCa2� channels will also begin to open at potentials morepositive than �40 mV (385). This will lead to more influxof Ca2� and, if present, activation of Ca2�-dependent K�

channels, which can lead to an afterhyperpolarization(AHP) (40, 244). Low-threshold channels can recoverfrom inactivation during the AHP and could begin to fireagain as the membrane potential returns to its restinglevel. Hyperpolarizations can also activate Ih channels,which allow the influx of both Na� and K�, thereby depo-larizing the cell and directly triggering another LTS (Fig. 2).In this scenario Ih are the primary pacemaker current (254).Clearly there are a variety of ionic conductances that medi-ate pacemaker activity, and it is an oversimplification toimplicate a single channel as the mediator of a pacemakercurrent. This is especially true in the heart (61).

Another way that low-threshold channels are in-volved in generating oscillations includes reciprocal con-nections with an inhibitory interneuron. Due to its impor-tant role in controlling brain activity, the best studied ofthese circuits involves the GABAergic neurons of thethalamic reticular nucleus that regulate the activity ofthalamic relay neurons (377). Interestingly, the firing pat-terns of thalamic relay neurons vary with the state ofconsciousness (277, 378). In the awake state and duringrapid-eye-movement sleep, they fire in tonic mode: theresting membrane potential is relatively depolarized, theLTS is inactivated, and excitatory inputs faithfully triggeraction potentials. In deep sleep, or during thalamocorticaldysrhythmias, these neurons fire in an oscillatory modecalled burst firing; the resting membrane potential is hy-

TABLE 1. Properties of neuronal low-threshold Ca2� spikes

Brain Region

RestingMembrane

Potential, mVThreshold,

mVAmplitude,

mV

Deinact VThreshold,

mV

Deinact VMidpt,

mV

Deinact TimeMidpt,

ms

Deinact TimeMax,ms

ReferenceNos.

Subthalamic nucleus �50 20 �78 80 120 40Septal nucleus �60 14 �63 �77 70 175 10Hippocampus, CA1–CA3 �60 �63 11 307Thalamic nuclei �64 �54 23 �55 �65 60 170 183Lateral thalamic nuclei �60 �65 20 �69 50 96Lateral geniculate nuclei 30 �75 �66 457Lateral geniculate nuclei �60 �68 35 �64 �72 456Lateral habenular nucleus �62 �65 30 �65 �68 437Paraventricular nucleus �66 �60 40 200 300 399Medial preoptic nucleus �60 34 �73 �77 120 400 385Dorsal raphe nucleus �67 �60 �70 400 800 60Cerebellar nuclei �57 24 �72 �78 241Cerebellar nuclei �58 �65 20 �72 �77 200 400 2Inferior olivary nucleus �64 �65 35 �70 �75 40 75 243, 244Hypoglossal motoneurons ��60 �65 10 �83 200 400 421

The properties of low-threshold Ca2� spikes (LTS) were measured using current-clamp recordings. Threshold was defined by the voltagewhere a depolarizing pulse triggers a LTS. The amplitude of the LTS is shown and does not include the fast Na�-dependent action potential. Thevoltage and time dependence for deinactivation (recovery) of the LTS spike is shown. Deinact., deinactivation; midpt, midpoint; CA1–CA3,hippocampal pyramidal neurons.



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perpolarized, allowing full-amplitude low-threshold spik-ing (Fig. 2A). Such firing is thought to underlie spike-wavedischarges observed during absence seizures (176). Sim-ilar patterns of brain activity have been detected in avariety of neurological disorders by magnetoencephalog-raphy and have been termed thalamocortical dysrhyth-mias (242).

B. Neuronal

1. Isolated neurons

LVA T-type currents have been characterized usingwhole cell voltage-clamp recording of neurons isolatedfrom a variety of brain regions. Very large T-type currents(�1 nA) have been recorded from cholinergic neurons ofthe basal forebrain (6), from relay neurons of the ventro-basal thalamus (222), and from sensory neurons of dorsalroot ganglia (436). Prominent T-type currents have beenfound in neurons from many brain regions (Table 2). I-Vrelationships are typically measured by holding the cell at�90 mV, and then currents are elicited by depolarizingpulses that increase in 10-mV increments. T-type currentsbegin to activate when the membrane is depolarizedabove �60 mV. At threshold potentials, the currents ac-tivate relatively slowly, requiring many milliseconds toreach peak, and also inactivate relatively slowly. At higherpotentials both activation and inactivation are faster. Thisproduces a stereotypical pattern in the family of currenttraces where successive records cross each other, remi-niscent of voltage-gated Na� channels. In general, HVAchannels do not produce this criss-crossing pattern be-cause inactivation rates are much slower than activationrates (334). I-V curves commonly show two components,with the LVA currents peaking at �30 mV while the HVAcurrents peak around 0 mV. In many neurons, T-typecurrents can be measured in isolation with test pulses to�30 mV and below. However, at higher potentials bothLVA and HVA currents are activated, complicating esti-mates of the LVA activation curve. Both I-V curves showa peak then decrease at more positive potentials, which isdue to a reduction of the electrochemical gradient forcalcium ions as the test potential approaches the theoret-ical reversal potential (�100 mV; see Fig. 7 for example).Therefore, the fraction of channels activated by a testpulse continues to increase beyond the peak of the I-Vcurve. This explains why estimates of the midpoint ofchannel activation (V0.5) derived by normalizing the cur-rent at each test potential to the maximum current ob-served (I/Imax) produces values that are more negative(�50 mV) than estimates that take into account changesin driving force (�41 mV). To circumvent this problem,activation curves have also been estimated by measuringthe amplitude of slowly deactivating tail currents at aconstant voltage. Most of these studies used a test pulse

of constant duration (isochronal), which assumes that therates of channel activation and inactivation are voltageindependent. However, this is not the case, so this methodcan underestimate channel activation at negative poten-tials where channels open slowly. It can also underesti-mate activation if channels inactivate during the testpulse. This problem can be overcome by varying theduration of the activating pulse, so that the tail current iselicited at the peak of the current (288). So far thismethod has only been applied to recombinant channels.Another experimental variable that affects the position ofthe activation curve is the concentration and choice ofcharge carrier. Millimolar concentrations of positivelycharged divalent cations can bind to surface charges onboth the channel and the membrane, thereby reducing theeffective transmembrane voltage gradient (164). An effectcalled surface charge screening. Increasing external[Ca2�] from 2 to 10 mM shifts the apparent gating ofT-type channels by �10 mV (128).

A hallmark of T-type currents is that they are tran-sient; currents reach a peak then decay. In solutionswhere K� channels are blocked, this decay is due tochannel inactivation. The inactivation rate is measured byfitting the current trace with an exponential function andis typically reported with a � in milliseconds. Plots ofinactivation rate versus test potential reveal two phases:inactivation is slow at �60, gets faster between �50 and�30, then is constant above �30 mV. Activation rateshave a similar voltage dependence, leading to the notionthat channels must activate before inactivating, and thatthe inactivation process is essentially voltage-indepen-dent. Although most T-type currents inactivate relativelyrapidly (�30 ms), there are also reports of slowly inacti-vating currents (Table 2). This variability was interpretedas evidence for multiple T-type channel genes (179). Thishypothesis was supported by the cloning of T-type chan-nels that differed in this property; Cav3.1 and 3.2 displayfast inactivation, while Cav3.3 displays slowly inactivatingcurrents. Notably, Cav3.3 mRNA is expressed in the samebrain regions as the slow currents (177, 228, 393). Exclud-ing these cases, the average inactivation � from 22 studiesis 20 ms.

The voltage dependence of inactivation is also mea-sured at “steady-state” by holding the membrane at vary-ing potentials (prepulse), then assaying for available T-type channels with a test pulse to �30 mV (h�). Toapproximate steady-state, many studies have used a pre-pulse duration of 1 s, which is considerably longer thanthe time required to inactivate open channels. However,increasing the prepulse duration to 10 s results in a�10-mV shift in the apparent h� curve (53, 389). Despitethe use of different prepulses and concentration of diva-lent cations, there is good agreement between the 34studies shown in Table 2, and the average midpoint of theh� curve is �77 mV (Table 2). Somewhat surprisingly this



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TABLE 2. Properties of T-type currents in isolated neurons

TissueDivalent,

mMThreshold,

mVI–V Peak,

mVImax,pA

V0.5,mV

h�,mV

�inact,ms �recov, ms Nickel, �M

ReferenceNos.

Basal forebrainStriatum 5 Ca �60 �40 �90 �53M �88 288 170Accumbens 5 Ca �64 �33 �51 �43C �80 397 80Septum 2.5 Ca �54 �1,320 �40M �49 16 5 6Septum 2 Ba �70 �40 �500 �59M �84 147

Amygdala 10 Ca �60 �20 �20 �38C �70 300 188Cerebral cortex

Temporal 2 Ca �60 �30 �47 �86 400 50 (28%) 354Piriform 5 Ca �60 �30 �250 �44T �86 22 257

HippocampusDentate granule 2 Ca �50 �30 �224 �65 25 458L–Minterneurons

2 Ca �60 �30 �100 �48M �84 12 100 400 126

CA1 pyramidal 10 Ca �60 �30 �150 �46C �79 15 430 (63%) 3897,000

CA1, 3 pyramidal 2 Ba �57 �100 �76 25 820 400Thalamus

Ventrobasal 3 Ca �60 �25 �350 �52T �84 20 251 85Ventrobasal 3 Ca �60 �30 �280 �59T �81 30 350 200 (66%) 179Ventrobasal 5 Ca �70 �1,400 �96 15 353 222LGN 10 Ca �48 �15 �186 �67 36 299 500 (83%) 387LGN 5 Ca �60 �23 �200 �45M �64 20 615 50 (100%) 159LGN-interneuron 2 Ca �70 �110 �55M �81 20 218 318LGN-relay 2 Ca �70 �289 �64M �86 20 206 318Reticular 3 Ca �50 �10 �131 �49T �78 80 570 200 (54%) 179Reticular 2 Ca �70 �50 �128 �64T �85 25 275 50 (38%) 409Lateral habenula 1 Ca �65 �35 �150 �62T �81 58W 507 177

HypothalamusHypothalamus 10 Ca �65 �30 �93 40 940 600 4Hypothalamus 10 Ca �60 �30 �150 �46C �79 15 215 389Hypothalamus 5 Ca �50 �20 �400 20 50 (50%) 293Supraoptic 2 Ca �64 �60 �250 �65 200 110Supraoptic 2 Ca �60 �30 �400 42 50 (84%) 119Paraventricular 10 Ca �59 �33 �402 �67 12 100 (67%) 253

Midbrain and ponsArea postrema 5 Ca �65 �10 �36 �72 10 (100%) 157Cochlear nucleus 2 Ca �75 �40 �400 �59M �89 500 (50%) 101

CerebellumPurkinje 10 Ca �60 �20 �400 �40C �79 23 3,300 110 189

Dorsal rootganglionDRG 2.5 Ca �65 �35 �2,000 �82 100 (95%) 436DRG 10 Ba �60 �25 �41C �60 25 330 295

Nodose ganglion 5 Ca �60 �20 �400 �48 30 400 54Nodose 10 Ca �55 �15 �400 �29C �44 30 226

Pelvic ganglion 10 Ca �50 �25 �500 �38T �57 10 231Olfactory 2.6 Ca �57 �36 �50 �82 429

Olfactory 3 Ca �70 �40 �60 �45C �66 100 (71%) 200, 201Retina

Rod bipolar 10 Ca �60 �30 �35 �36T �59 30 1,000 (80%) 316Cone bipolar 10 Ca �60 �20 �66 �35T �60 55 1,000 (85%) 316

Oligodendrocytes 20 Ba �50 �25 �150 �63 10 47

The divalent cation concentration used to measure the currents is shown. Properties of the current-voltage relationship are described in termsof the voltage threshold where currents become detectable, the voltage where they peak (I–V peak), and the maximum amplitude observed (Imax).Methods used to calculate midpoints of the activation curves (V0.5) are denoted with the following superscripts: C, chord conductance; T, tailcurrents; M, I/Imax. The midpoint of the steady-state inactivation curve is reported (h�). Kinetics of inactivation (�inact) can vary with test potentials;therefore, the values shown represent the voltage-independent rate, which was typically measured at �10 mV. WCurrents decayed in a biexponentialmanner; to simplify comparisons, a weighted tau was calculated according to the following equation �W � A1�1 � A2�2, where A represents thefraction of current decaying with the respective time constant. The values shown for recovery from inactivation represent the voltage-independentrate, which was typically measured at �90 mV. Tail current kinetics continuously vary with repolarization potential, so when possible the valuesshown represent those obtained at �90 mV. In some cases recovery from inactivation was found to be biphasic, so the values represent the tau ofthe fast component, the fraction of channels that recover fast (in parentheses), and the tau of the slow component. Nickel responses represent eitherthe IC50 value obtained or the concentration tested and the percent block observed (in parentheses). LGN, lateral geniculate nucleus; MDN,magnocellular dorsal nucleus; L–M, lacunosum-moleculare of hippocampus; DRG, dorsal root ganglion; inact., inactivation; recov, recovery.



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value is considerably more negative than the apparentthreshold of activation, indicating that channels can inac-tivate at potentials where they do not appear to open.Similar h� values have been obtained with the recombi-nant Cav3 channels (see Table 8), where this closed-stateinactivation has been studied in greater detail (127). Al-though most HVA channels inactivate at more positivepotentials, R-type and recombinant Cav2.3 channels inacti-vate over a similar voltage range as T-type channels (334).

The time course of recovery from inactivation is animportant property of T-type channels. LTS are oftentriggered after an inhibitory postsynaptic potential(IPSP), and this is due to fast recovery of T-type channelsduring the IPSP (deinactivation), followed by their open-ing as the membrane returns to its resting potential. Re-covery is often measured by varying the time between(interpulse) an inactivating pulse and a test pulse. In moststudies the interpulse voltage was �90 mV, and similarresults are obtained at more negative voltages. In con-trast, recovery is slower at potentials where channels canalso inactivate. There is considerable variability in thereported values for recovery, ranging from 100 to 3,300 ms(Table 2). Although most studies found that recoveryfollowed a monoexponential time course, others havefound evidence for a biexponential process. This discrep-ancy might be due to differences in the duration of theinactivating pulse, where long pulses allow channels toaccumulate in a second inactivated state from which theyrecover slowly. As observed in sensory neurons (53),recombinant channels recover with a monoexponentialtime course from short pulses and with a slower, biexpo-nential time course from long pulses, although this prop-erty is less pronounced for Cav3.3. Differences in recoverykinetics of T-type currents between different neurons canalso be due to which Cav3 isoform they express (209). Ofthe three isoforms, Cav3.1 channels recover the fastest(�120 ms from short pulses), and this time course issimilar to that observed for recovery of both native T-typecurrents (�300 ms; Table 2) and LTS (�150 ms; Table 1).

Early studies found that LVA channels were moresensitive to block by 50–100 �M nickel than HVA chan-nels (124, 152, 298). Subsequent studies found that manyneuronal T-type channels required �10-fold higher con-centrations for block, with many studies reporting IC50

values of �300 �M (Table 2). Studies on recombinantchannels provided an explanation for this diversity; onlyCav3.2 channels are highly nickel sensitive (IC50 �10 �M),while Cav3.1 and Cav3.2 are 20-fold less sensitive (230).Recombinant HVA channels also differ in their Ni2� sen-sitivity, with Cav2.3 being relatively sensitive (455). Al-though the mechanisms of block of recombinant HVA andLVA channels are complex and subject to multiple inter-pretations, a consistent finding is that block is greatest atpotentials near threshold. This voltage dependence wouldexaggerate block of LVA channels, making nickel appear

more selective than it is. In summary, block by 10–50 �MNi2� can be used to implicate Cav3.2 channels, but addi-tional evidence, such as a slowly deactivating tail current,is required to rule out Cav2.3 channels. Both these crite-ria, plus PCR, were recently applied to show the expres-sion of Cav3.2 in sympathetic ganglion neurons (231).

The temperature sensitivity of T-type currents hasbeen measured in thalamic neurons (85), DRG neurons(305), N1E-115 neuroblastoma cells (298), and GH3 cells(343). Heating from 22 to 37°C caused over twofold in-creases in current amplitudes and accelerated channelactivation and inactivation. In contrast, heating did notaffect the position of the I-V curve (305). The effects oftemperature have been found to be nonlinear, making itdifficult to calculate the effect of a 10°C increment (Q10)in temperature (298, 343). In the linear range, Q10 valueswith an average of 2.5 have been found for effects onamplitude, activation kinetics, and inactivation kinetics.

The pH sensitivity of T-type currents has been stud-ied in CA1 hippocampal neurons (405), ventrobasal tha-lamic neurons (365), and cardiac myocytes (83, 414).Acidification of the external solution decreased currentamplitudes, whereas alkalinization had the opposite ef-fect. In contrast, T-type currents are relatively insensitiveto changes in intracellular pH (405). The effect of externalpH is in part mediated by changes in the single-channelconductance, which when measured with 110 mM Ca2�

increased from 3.5 pS at pH 6 to 10.8 pS at pH 9 (414).Small changes in pH have also been shown to shift thevoltage dependence of activation and inactivation; chang-ing pH from 7.3 to 6.9 shifted both of these parameters by�3 mV, while increasing the pH to 7.7 had the oppositeeffect (365). Larger shifts in the voltage dependence ofactivation (�15 mV) have been observed using largershifts in pH (from 7.5 to 9.8) (83). The observed pKa

values were �7, indicating that T-type currents will beaffected by modest changes in extracellular pH (365, 414).

2. Slices

LVA currents have been characterized in slices pre-pared from numerous brain regions (Table 3). The moststriking difference between these data and that obtainedwith isolated neurons is that currents are much larger inslices. This difference has been attributed to loss of chan-nels that were localized on dendrites (97). In fact, T-typechannels appear to be preferentially localized to dendrites(77, 135, 195, 199, 328).

T-type currents recorded from slices also appear toactivate and inactivate at more negative potentials thanobserved in isolated neurons. The average threshold inslices was �70 mV, and peak currents were observed at�45 mV (Table 3), while in isolated neurons thresholdwas �60 and the peak occurred at �30 mV (Table 2). Thisdifference has been attributed to poor voltage clamp of



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neurons in slices (97). Consistent with this suggestion, thevoltage dependence of single T-type channels recordedfrom dendrites is similar to that observed in isolatedneurons (256).

In summary, T-type currents are found in neuronsthroughout the brain, with particularly large currentsfound in thalamic, septal, and sensory neurons. Theiractivation near the resting membrane potential and theirfast recovery from inactivation allow them to generateLTS. Their preferential localization in dendrites suggestsT-type channels play an important role in synaptic inte-gration.

C. Heart

Cardiac T-type currents have been the focus of manystudies due to their possible role as a pacemaker current.These studies have established that T-type current densi-ties are highest in conduction and pacemaker cells, andmuch reduced or nonexistent in ventricular myocytes.T-type currents are highest near birth and gradually de-cline but may reappear in pathological conditions. Cur-rents are larger in lower species and have a wider distri-bution. For example T-type currents are prominentthroughout the guinea pig and hamster heart but virtuallyabsent in adult ventricular myocytes of rat, cat, and dog.Although Cav3.2 was cloned from a human heart cDNAlibrary, T-type currents have not been detected in humanmyocytes (39, 235, 313).

Cardiac T-type currents were initially described indog atrium (32) and guinea pig ventricle (283, 303). Thesestudies showed that cardiac T-type currents resembledthose recorded from neurons, but differed from L-typechannels in terms of their kinetics (transient), pharmacol-ogy (dihydropyridine insensitive), conductance of diva-lent cations (Ca2� � Ba2�), lack of regulation by �-adren-ergic agonists, and smaller single-channel conductance ofBa2�. Subsequent work has established their presence in

myocytes isolated from dog Purkinje (367), rabbit sino-atrial node (152), cat atrium and latent pacemaker cells(462), hamster ventricle (362), and rat atrium (446). Ingeneral, the currents were small, displaying a peak cur-rent density below �3 pA/pF. In contrast, prominent T-type currents (��10 pA/pF) have been recorded frommyocytes isolated from shark (271), chick (202), finch(48), and mollusks (452).

Calcium channels were originally classified by theirinactivation kinetics: rapidly inactivating or transientchannels were called T type, while slowly inactivating, orlong-lasting channels were called L type (303, 306). Itshould be noted that this only holds for currents carriedby Ba2�, as cardiac L-type currents carried by Ca2� caninactivate at a similar rate as T-type currents. This is dueto calcium-induced inactivation of the L-type channel,which appears to be mediated by calmodulin tethered tothe carboxy terminus of the channel (111). This inhibitionis triggered with every heartbeat as Ca2� entry triggers alarger release of Ca2� from internal stores (37). In con-trast, Ca2� currents through T-type channels inactivate abit slower than Ba2� currents (116, 410), which excludesa similar regulation by calmodulin. Another major differ-ence between these channels is that L-type channels con-duct Ba2� threefold better than Ca2�, whereas T-typechannels conduct both equally well (discussed further insect. IIJ).

A proposed physiological role for cardiac T-typechannels is as a pacemaker current during diastolic de-polarization. These studies have been hampered by thelack of a selective blocker, relying heavily on the ability of40 �M nickel to block T- but not L-type currents (152,298). Current-clamp studies of spontaneous action poten-tials showed that nickel slowed the late phase of depolar-ization, and hence slowed the firing of rabbit sinoatrialnodal cells (SAN). Similar results have been obtained by anumber of groups using rabbit SAN (98, 353) or cat latentpacemaker cells (462). Tetramethrin (0.1 �M) was re-

TABLE 3. Properties of T-type currents in brain slices

Brain RegionDivalent,

mMThreshold,

mV

I–V

Peak,mV

Imax,pA

V0.5,mV

h�,mV

�inact,ms

�recov,ms Nickel, �M

ReferenceNos.

Cerebellum 0.5 �60 �33 �690 �51M �86 14 100 (19%) 292Thalamus

LGN 1 �65 �53 �1,500 �61M �77 26 100 200 (53%) 93LGN 2 �70 �45 �1,812 �61M �84 17 151Laterodorsal, IT,f 2 Ca �80 �55 �670 �86 32 398Laterodorsal, IT,s 2 Ca �80 �65 �630 �98 55, 69 25 (50%) 398

Hypothalamus MDN 2 �83 �50 �1,051 �70M �82 12 100 500 (100%) 302Brain stem respiratory 1.5 �65 �40 �550 �59M �81 200 (50%) 107Dorsal horn 2 Ba �54 �20 �1,000 �45M �86 14 200 (91%) 347

The voltage dependence of activation was estimated by normalizing the current observed at each test potential to the maximum ( M ) (I/Imax),fit with a Boltzmann function, then reported as the midpoint voltage (V0.5). A single concentration of nickel was tested, and the percent block isshown in parentheses. LGN, lateral geniculate nucleus; MDN, magnocellular dorsal nucleus.



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ported to produce selective block as well, and to producesimilar effects on pacemaker cycle (152); however, sub-sequent studies failed to confirm this selectivity (165).

Similarly, a number of studies have found lower sen-sitivity and selectivity for nickel (Table 4). Two possibleexplanations for these disparate results are 1) that heartexpresses two isoforms of the T-type channel, Cav3.1 andCav3.2, and these isoforms differ in their sensitivity tonickel, and 2) that isoform expression is age and speciesdependent. Cloned Cav3.2 channels are blocked at 20-foldlower concentrations than Cav3.1 (or Cav3.3), displayingan IC50 of �10 �M (230). There is a strong correlationbetween nickel sensitivity and Cav3.2 expression, suggest-ing that native Cav3.2 channels are as sensitive as thecloned channel (323). This suggests that nickel (�100�M) can be used to implicate Cav3.2 channel expression.Expression in atrium appears to be species specific:Cav3.1 appears to predominate in rat and mice (91, 236),while in guinea pigs it is Cav3.2 (323). In fact, carefulmeasurement of the nickel dose response in guinea pigatrium revealed a biphasic response, with 80% of thechannels being blocked with an apparent IC50 of 23 �M,while 20% of the channels were inhibited with an IC50 of1,350 �M. Rabbit and cat SAN cells appear to predomi-

nantly express Cav3.2 channels. In contrast, in situ hybrid-ization of mouse heart suggests that Cav3.1 channels pre-dominate, although both isoforms were readily detectedand both were enriched in SAN relative to atrium (49). Itshould be noted that distinct in situ probes might differ intheir ability to detect their cognate sequence, makingsuch comparisons difficult. PCR amplification of mouseheart detected the expression of a splice variant of Cav3.1that differs in the III-IV loop (91).

Expression of T-type currents in developing hearthas also been studied. T-type currents are readily detect-able in neonatal hearts, increase slightly to a peak be-tween postnatal days 4 and 8, then decline slowly to asteady-state (236, 246). In other studies T-type currentswere only detected in neonatal rats, disappearing by post-natal day 21 (236). In contrast to L type, the biophysicalproperties of T-type currents are unchanged during neo-natal development. Changes in nickel sensitivity have notbeen investigated but may be informative. No differencein T-type current density was detected in SAN myocytesfrom newborn (3–10 days old) and adult (41–48 days old)rabbits (329). Expression of Cav3.2 mRNA is higher infetal human heart than adult, whereas Cav1.2 shows theopposite pattern (331).

TABLE 4. Properties of T-type currents in peripheral tissues

TissueDivalent,

mMThreshold,

mVI–V Peak,

mV pA/pFImax,pA

V0.5,mV

h�,mV

�inact,ms

�deact,ms �recov, ms Nickel, �M

ReferenceNos.

HeartAtrium 5 Ca �50 �30 �0.3 �24 20 32Atrium 5.4 Ca �50 �30 �1.0 �75 �40M �57 15 197 50 (66%) 444Atrium 2 Ca �50 �30 �2.6 �42C �68 13 1.3 160 236Atrium 1.8 Ca �50 �30 �350 �24T �59 23 323Atrium 2.5 Ca �60 �40 �115 �38C �59 10 116SAN 2.5 Ca �47 �10 �2.1 �88 �23C �75 3.2 144 40 (100%) 152Latent pacemaker 2.7 Ca �50 �20 �3.3 �90 �31C �57 40 (54%) 462Purkinje 2 Ca �60 �30 �1.7 �391 �48C �68 10 100 100 (90%) 165Purkinje 5 Ca �40 �25 �2.9 �700 �47M �37 4 83 (67%) 50 (47%) 410

444Ventricle 5.4 Ca �50 �10 �100 11 100 (100%) 129Ventricle 20 Ba �50 �30 �5.8 �858 �43C �77 13 362

Skeletal muscle 1.8 Ca �55 �42C �63 13 4510 Ca �45 �15 �3.6 �400 �24C �41 16 224 (65%) 5.4 38

5,160Spermatocytes 10 Ca �60 �20 �6.5 �218 �47C �64 7 1.6 118 200 (75%) 350Adrenal

Glomerulosa 20 Ba �45 �30 �7T �56 7 82Fasciculata 10 Ca �50 �15 �8.2 �272 �17G �50 18 1.7 400 (50%) 20 287

4,800Pituitary

Pars intermedia 10 Ca �50 �15 �150 5T 1.8 84Lactotropes 5 Ca �48 �12 �90 �28M �66 15 100 (80%) 240Melanotropes 11 Ba �50 �20 �30 �61 19 204Gonadotropes 20 Ca �40 �20 �75 �27C �61 15 3 262

Pancreas�-Cell 10 Ca �40 �16T 21 2.5 320�-Cell 15 Ba �60 �30 �35 �14M 100 (100%) 25

Definitions are as in Table 2. Methods used to calculate activation curves are denoted with the following superscripts: C, chord conductance;T, tail currents; G, constant-field equation of Goldman-Hodgkin-Katz; M, I/Imax. In some cases recovery from inactivation was found to be biphasic,so the values represent the tau of the fast component, the fraction of channels that recover fast (in parenthesis), and the tau of the slow component.SAN, sinoatrial node.



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Cardiac hypertrophy appears to trigger a return tothe neonatal pattern of gene expression (388), leading toreexpression of T-type channels in rat and cat ventricularmyocytes (173, 268, 308). RNase protection assays indi-cate that Cav3.1 transcripts are upregulated in a rat modelwhere hypertrophy is induced by infarction (173). Al-though levels of Cav3.2 transcripts were not examined,the sensitivity of reexpressed currents to nickel blocksuggests a contribution of Cav3.2 channels as well (173,268). T-type currents are also reexpressed in dedifferen-tiated rat ventricular myocytes (114). T-type currentshave been found to be increased twofold in cardiomyo-pathic Syrian hamsters relative to other hamster strains(46, 362). Studies using 8-mo-old animals found that thevoltage dependence of activation and inactivation wasshifted to more negative potentials in ventricular myo-cytes from cardiomyopathic animals (362). In contrast,studies using 1-day-old hamsters found that only inactiva-tion was shifted (46). The relevance of these observationsto human pathologies is unclear as T-type currents havenot been detected in myocytes isolated from normalatrium (235) or diseased ventricle (39, 337).

Calcium influx through voltage-gated channels acti-vates intracellular Ca2� release channels, leading to largerelevations in intracellular Ca2� and hence muscle con-traction. The role of L-type channels in this process is wellestablished. T-type channels are also capable of triggeringthis CICR, albeit much more weakly in both cardiac (370,461) and skeletal muscle (133). T-type current-inducedcontraction developed more slowly, suggesting that thesechannels are not localized near SR stores as are L-typechannels. This observation coupled with the lower ex-pression of T-type channels in working myocytes indi-cates that they do not play a major role in excitation-contraction coupling. Consistent with this notion,mibefradil has little effect on cardiac inotropy at doses

that lower blood pressure (356). In contrast, T-type chan-nels may be localized near SR in atrial pacemaker cells ofthe cat (180). These authors suggested a novel pacemak-ing mechanism where Ca2� influx through the T-typechannel triggers subsarcolemmal Ca2� sparks, which inturn activate Na�/Ca2� exchange currents that depolarizethe membrane to threshold.

A role for T-type channels in triggering atrial natri-uretic peptide (ANP) secretion has been inferred from theeffects of mibefradil (236, 434). Evidence that mibefradilacted on T-type channels was provided by the observationthat L-type blockers were much less potent blockers, orstimulated ANP secretion.

D. Kidney

Relatively little is known about the physiologicalroles of T-type channels in kidney. T-type currents haveonly been recorded from smooth muscle cells (SMC)isolated from rat interlobular and arcuate arteries (143).Heterogeneity in Ca2� currents was observed, with ap-proximately one-third of the freshly isolated myocytesdisplaying only T-type currents (“T-rich”), one-third only Ltype, and the rest a mixture. Currents in T-rich cells weresome of the largest ever recorded for cardiovascularsmooth muscle (�156 pA in 2.5 mM Ca2�; Table 5), whichallowed their recording in physiological Ca2� solutions.Consistent with their assignment as T-type channels, cur-rents activated and inactivated at voltages 20 mV morenegative than observed for L-type currents. In contrast tomost T-type currents, these SMC currents activated (I-Vpeak �10 mV) and inactivated (h� � �50 mV) at voltagesthat were slightly more positive, which might be charac-terized as mid-voltage activated. They also inactivatedabout twofold slower during a pulse (�inact � 46 ms at �10

TABLE 5. Properties of LVA currents in smooth muscle

TissueDivalent,

mMThreshold,

mVI–V Peak,

mV Imax, pAh�,mV

�inact,ms Nickel, �M

ReferenceNos.

ArteriesCoronary 10 Ba �60 �10 �125 �65 14 130, 131Coronary 20 Ba �40 �30 �250 �55 332Aortic 20 Ca �60 �30 �140 �80 20 600 3Aortic 1.5 Ca �60 �30 ��100 �65 339Aortic 20 Ba �40 �12 24 339Mesenteric 10 Ba �40 �30 �63 372Rabbit ear 110 Ba �30 �10 �20 �55 34Rat tail 20 Ba �50 �38 46 428

VeinsPortal 5 Ca �45 �10 �263 �50 13 246

OrgansLung 10 Ca �50 �10 �200 448Uterus 1.8 Ca �60 �30 �300 �70 453Bladder 1.8 Ca �56 �10 �200 �60 100 (100%) 382Colon 2 Ca �60 �10 �385 �58 20 30 (50%) 445

Definitions are as in Table 2.



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mV). Atypical currents with similar properties have beenreported for SMC isolated from rat portal vein (315) andfrom the terminal branches of guinea pig mesenteric ar-teries (291).

Quite surprisingly, the human tissue that expressesthe most mRNA for Cav3.2 is the kidney (90, 438). Incontrast, both Cav3.1 and Cav3.3 are predominantly ex-pressed in brain (228, 288, 289, 324). Skøtt and co-workers(11, 156) have studied the distribution Cav3.1 and 3.2 inkidney and concluded that Cav3.1 was in the tubules,while Cav3.2 was in renal smooth muscle (11, 156). RT-PCR indicated that mRNAs for Cav3.2, and to a lesserextent Cav3.1, were expressed in rat (but not rabbit)glomerular afferent and efferent vessels, and vasa recta.The same study reported that K�-induced contraction ofperfused rabbit afferent arterioles could be blocked bylow concentrations of mibefradil (IC50 �10 nM) thatshould be selective for T-type channels. Nickel could alsoblock this response; however, the concentrations re-quired (1 mM) would be expected to block almost all Ca2�

channels. Similar results were obtained with rat isolatedperfused hydronephrotic kidneys: either 1 �M mibefradilor 100 �M nickel (a relatively selective dose) dilatedafferent and efferent arterioles constricted with ANG II(314). Selectivity of mibefradil for T-type channels wasdemonstrated by coadministration with the L-typeblocker nifedipine. In these experiments 1 �M nifedipinecaused a modest dilation of the efferent arteriole but didnot prevent the more pronounced dilation induced bymibefradil.

Cav3.1 was detected in dot blots of human fetal, butnot adult, kidney (288). RNase protection assays of ratkidney regions indicated that Cav3.1 was predominantlyexpressed in the inner medulla (11). RT-PCR of microdis-sected nephron segments indicated that Cav3.1 was ex-pressed in distal convoluted tubules, connecting tubules,and collecting ducts. This study also examined the distri-bution of Cav3.1 protein using a polyclonal antibodyraised against the first 22 amino acids of the deducedCav3.1 sequence (11). Immunoreactivity was detected inthe apical domains of the distal convoluted tubules and inprincipal cells of connecting tubules and inner medullarycollecting ducts. Preincubation of the antibody with pep-tide blocked the signal; however, Western blots were notpresented to demonstrate the selectivity of the antibody.These results suggested that T-type channels, along withendothelial calcium channels (ECaC), might be involvedin Ca2� reabsorption.

Calcium channel blockers are widely used in thetreatment of hypertension. It is generally accepted thatthey work by blocking L-type channels in vascular smoothmuscle, leading to vasodilation. Recent studies suggestthat part of their effect may be mediated by changes inrenal hemodynamics (172). In intact dogs, both nifedipineand mibefradil increased renal blood flow, whereas only

nifedipine affected glomerular filtration rate. Mibefradilhas also been reported to decrease renin secretion in ratswith renal artery clips, but had no effect on renin secre-tion from isolated juxtaglomerular cells (423). In conclu-sion, these studies suggest that T-type channels on affer-ent and efferent glomerular arterioles may be animportant therapeutic target (89). A possible advantage toa T-selective antihypertensive drug is that it may alsoprevent glomerular damage (192).

E. Smooth Muscle

In addition to L-type currents, SMCs isolated fromarteries, veins, and organs have also been reported tohave LVA currents. With the exception of the kidneyresults noted above, cardiovascular SMCs have tiny LVAcurrents. Therefore, most studies have had to use veryhigh concentrations of charge carrier, which shifts gatingto positive potentials, and currents have only been char-acterized in terms of their I-V relations and sensitivity toholding potential. These characteristics are not sufficientto establish an LVA current as T type, since HVA currentsactually represent a spectrum of mid- to high-voltage-activated currents. HVA currents also inactivate over awide range of potentials, with some R-type currents inac-tivating over the same voltage as T-type channels. Theseconcerns take on additional weight with the finding thatsome vascular SMCs express non-L-type HVA currents(291) such as P-type currents (155). Native P-type cur-rents can be mid-voltage activated (33). In addition, anickel- and mibefradil-sensitive LVA current has beendescribed in colonic myocytes that gates similar to T-typechannels but has different permeability properties (214).LVA channels can be classified as T type by their slow tailcurrents, which surprisingly have not been reported, andby their single-channel properties, which have been re-ported (34, 130, 450). Because T-type channels inactivateat the resting membrane potentials of most SMCs (�75 to�60 mV, reviewed in Ref. 168), it has been hard to discerntheir physiological role. Perhaps they contribute to basalintracellular Ca2� concentrations via their window cur-rent, or after transient hyperpolarizations induced byspontaneous transient outward currents (STOCs).

LVA currents have been found in the following arte-rial SMCs: coronary (130, 281, 332), aortic (3), mesenteric(150, 311, 372), rabbit ear (34), rat tail (327, 428), andmiddle cerebral arterioles (167). RT-PCR detected theexpression of both Cav3.1 and Cav3.2 in rat mesentericarterioles (150). Single-channel studies have establishedthe presence of 7.5-pS T-type channels in freshly isolatedguinea pig coronary SMCs (130). This study also reportedthat whole cell LVA currents could only be observed inhalf of the cells. In contrast, LVA currents were neverdetected in freshly isolated coronary SMCs from rabbits



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(269) or humans (332). LVA currents were found if thecells were cultured, and these currents appear to be Ttype based on their voltage dependence and 1:1 conduc-tance of Ca2� and Ba2� (332). Similar results have beenobtained with aortic SMCs; T-type currents are expressedin proliferating cultures, but neither in confluent cultures(3, 339) nor in freshly isolated cells (220). In fact, T-typecurrents were only detected in cells that were in G0 or G1

phases (220), leading to the hypothesis that they mightplay a role in proliferating SMCs (338). Consistent withthis notion is the finding that mibefradil could reduceneointima formation following balloon injury to rat ca-rotid arteries (355). However, it should be noted thatT-type currents were not observed in SMCs prepared frominjured rat aorta, although a transient downregulation ofL-type channels was documented (333).

LVA currents have also been detected in SMCs iso-lated from veins, such as saphenous (450), portal (246),and azygous (282). T-type single-channel currents wererecorded in saphenous vein SMCs (450). Isradipine (PN200–110) was found to block T-type currents of rat portalvein with an IC50 of 0.5 �M, while L-type currents wereblocked at 1 nM (246). The sensitivity of these T-typecurrents was greater than observed in other preparations(see sect. VA), suggesting that Cav3 isoforms may differ intheir pharmacology. Current-clamp recordings revealedthat portal vein SMCs could fire action potentials thatresembled those observed with cardiac myocytes (246).Isradipine (10 nM) inhibited the plateau phase with noeffect on the rising phase, suggesting that T-type channelsmight play a pacemaker role in these cells.

LVA currents have been described in SMCs frombronchi (185, 448), ileum (373), colon (445), bladder(382), and uterus (453). The results with pregnant humanuterine SMCs are notable because the T-type currents (�3pA/pF in 1.8 mM Ca2�) were larger than the L-type cur-rents (453). These currents activated and inactivated(h� � �70 mV) at potentials typical of most T-type cur-rents (Table 5). Current-clamp recordings indicated that adepolarizing pulse could trigger an action potential; how-ever, the threshold (�20 mV) was higher than observedwith neuronal LTS. Sizable (�200 pA) T-type currentswere also described for porcine bronchial smooth muscle,being detected in 30% of the cells, but absent from tra-cheal SMCs (448). LVA currents (�1.4 pA/pF) were foundto disappear when bladder SMCs were cultured (382).Concomitant with this loss was a shift in the threshold foraction potential generation from �25 to �15 mV, sugges-tive of a role for T-type channels.

F. Skeletal Muscle

Newborn rodent skeletal muscle was also used inearly studies to establish the existence of T-type channels

as separate from L-type channels (30, 81). Developmentalstudies of mice and rats have shown that the T-typecurrent is only found in embryonic and newborn muscleand disappears by 3 wk of age (29, 38, 142). Concomi-tantly, the slow L-type current increases. Studies on themuscular dysgenesis (mdg) mouse clearly establishedthat the T- and L-type channels were encoded on separategenes (30, 67, 212), and it is the L-type channel (Cav1.1)that plays a critical role in excitation-contraction coupling(395). Recent studies have shown that the T-type currentis important for myoblast fusion (45). In particular, it wasshown that T-type channels could generate sufficient win-dow currents to alter intracellular Ca2� concentrations,and trigger fusion. This hypothesis was further supportedby pharmacological experiments using low concentra-tions of Ni2� and amiloride, and by antisense oligonucle-otides directed against Cav3.2 (45). The selective expres-sion of Cav3.2 in skeletal muscle fibers has also beendemonstrated using single-cell PCR (38). The electrophys-iological properties of these currents (Table 4) and Ni2�

sensitivity closely resemble recombinant Cav3.2 currents,although differences have been noted in kinetics of acti-vation, inactivation, and recovery from inactivation (38).Early studies also noted kinetic differences as a functionof development (142), suggesting that these differencesare not due to rapid events such as phosphorylation, butrather slower events such as gene transcription of auxil-iary subunits (see sect. IVE).

G. Sperm

Calcium channels appear to play a role in mediatingthe egg-induced acrosome reaction that precedes fusion(for review, see Ref. 94). T-type channels have been im-plicated in this process, although other Ca2� conduc-tances may be involved (134, 435). One reason for thisuncertainty is that it is virtually impossible to patch clampmature sperm (335). Therefore, electrophysiological stud-ies have used spermatogenic cells, which are primaryspermatocytes in the pachytene stage. The only voltage-dependent Ca2� channels in these cells are T type, andtheir biophysical (350) and pharmacological properties(15) have been well characterized. PCR results indicatethat Cav3.2 is the predominant isoform expressed in hu-man testis (182, 375). Consistent with this result is thehigh sensitivity (IC50 � 34 �M) with which nickel blocksthe spermatocytic T-type current (15), although it shouldbe noted that Sertoli cells also express nickel-sensitiveT-type currents (225). Similar concentrations of nickelalso block the acrosome reaction (121, 375). Similarly, thefollowing compounds have been found to block sper-matocyte T-type channels and the acrosome reaction atsimilar concentrations: isradipine (PN 200–110), nifedi-pine, pimozide, amiloride, mibefradil, W-7, and trifluoper-



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azine (13, 15, 121, 247, 350, 375). Spermatocytic T-typechannels appear to be regulated by tyrosine and calmod-ulin-dependent protein kinases (14, 247).

H. Endocrine Tissues

1. Adrenal

T-type channels have been implicated in the secre-tion of aldosterone (76) and cortisol (109). Calcium cur-rents, and their regulation by secretagogues, have beenextensively studied in cells isolated from bovine adrenalzona glomerulosa and fasciculata. Glomerulosa cells ex-press a mixture of T- and L-type channels (82), whereasfasciculata cells predominantly express T-type channels(22, 287). Although these cells can be considered nonex-citable due to their lack of Na� channels, they are stillcapable of generating Ca2�-dependent action potentials(22). In contrast, adrenal chromaffin cells express HVACa2� and Na� currents but no LVA currents (16).

Serum K� is regulated by secretion of aldosteronefrom adrenal glomerulosa cells. Glomerulosa cells areexcellent K� sensors and depolarize after small increasesin serum K�, which in turn leads to the activation ofT-type channels, Ca2� influx, and stimulation of aldoste-rone synthesis and release. For example, increasing K�

from 2 to 5 mM causes the resting membrane potential ofisolated cells to depolarize from �97 to �78 mV (76).Aldosterone secretion is also stimulated by ANG II, aneffect mediated by stimulation of T-type channel activity(discussed in sect. IIIB).

Cortisol secretion from adrenal zona fasciculata cellsis regulated by ACTH, which depolarizes these cells byinhibiting a K� conductance (286). Depolarization wouldactivate T-type channels, leading to a rise in intracellularCa2�, and ultimately cortisol synthesis and secretion. Cor-tisol secretion can be inhibited by a variety of blockersincluding mibefradil, and this block occurs over the sameconcentration range as their block of the T-type channels(109, 141, 345). ACTH may also regulate the expression ofT-type channels (22).

Adrenal T-type channels are encoded by Cav3.2. Insitu hybridization of rat and bovine glands detected highlevels of Cav3.2 expression in the cortex, with only traceamounts of Cav3.1 and Cav3.3 (358). The biophysical prop-erties and sensitivity to Ni2� block of both glomerulosaand fasciculata cells are also consistent with their beingencoded by Cav3.2 (287, 358).

2. Pituitary

LVA Ca2� currents have been recorded from cells of theanterior and intermediate lobes of the pituitary gland includ-ing (84, 392, 439) lactotropes (240), corticotropes (261),gonadotropes (379), and thyrotropes (204). These cells gen-

erate spontaneous action potentials and Ca2� spikes, lead-ing to secretion of hormone (see Ref. 406 and referencestherein). Hormonal regulation of these LVA currents sup-ports the idea that these currents are involved in secretion(see sect. III). The voltage- and time-dependent properties ofthese currents are similar, but not identical to other nativeT-type currents (Tables 2 and 4). Notably the peak of the I-Vcurve is slightly depolarized. In addition, one study reportedthat the LVA current was fivefold larger in Ba2� than Ca2�

(379), a property typically ascribed to HVA currents. Assuggested previously, not all LVA currents are carried byT-type channels, and other criteria must be applied (19, 33,376). One such discriminating property is their tail deactiva-tion kinetics, and slowly deactivating T-type currents areclearly present in pituitary cells (84). Similarly, these chan-nels can be distinguished at the single-channel level by theirconductance for Ba2�, and again, pituitary T-type currentshave been clearly identified (203). In situ hybridization stud-ies suggest all three Cav3 isoforms are expressed, althoughCav3.2 was the predominant isoform detected (393). Consis-tent with this result, T-type currents were reported to benickel sensitive, with 80% of the current blocked by 100 �MNiCl2 (240). Currents recorded from GH3 cells are not nickelsensitive (IC50 � 777 �M), suggesting that this tumor-de-rived cell line most likely expresses Cav3.1 channels (160). Inconclusion, T-type channels appear to play a pacemaker rolein anterior pituitary, although they may also be involved instimulus-secretion coupling (258).

3. Pancreas

T-type channels appear to play a similar pacemaker rolein insulin secretion from pancreatic �-cells of the islets ofLangerhans. Increases in plasma glucose and its metabolismin �-cells leads to increases in cellular ATP and inhibition ofKATP channels (300). Closure of KATP channels initiates thepacemaker cycle by depolarizing the �-cell membrane toabout �55 mV. Riding on top of this slow plateau depolar-ization are rapid calcium-dependent spikes (reviewed in Ref.352). Insulin secretion can be blocked by a wide variety ofagents, indicating that these spikes activate L-, P-, and R-typecurrents (238, 258, 418).

T-type currents in pancreatic �-cells have been char-acterized at the whole cell (Table 4; Refs. 25, 320) andsingle-channel level (17, 348). Expression is species de-pendent, with little or no expression in rodents, butreadily detectable in humans (25, 426). Therefore, it isnotable that LVA currents were found in �-cells fromdiabetes-prone NOD mice (426). The expression of LVAcurrents in mouse cells has been reported to be stimu-lated by a 6-h treatment with cytokines (25 U/ml of inter-leukin-1� plus 300 U/ml of interferon-�; Ref. 427). How-ever, the I-V curve for the cytokine-induced currentpeaked between �10 and �20, while under similar re-cording conditions this group found that bona fide, slowly



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deactivating, T-type currents peaked between �20 and�10 mV (426). Human �-cells express both LVA and HVAcurrents, and in 20% of the cells tested, the LVA currentwas dominant (25). Voltage-clamp recordings indicatedthat 100 �M Ni2� could block the LVA current, whilecurrent-clamp recordings indicated that it slowed actionpotential frequency. Similar studies using the rat insuli-noma cell line INS-1 found that low concentrations ofnickel could completely block T-type currents (IC50 � 30�M) and partially block insulin secretion (42). Nickel-sensitive T-type currents (IC50 � 3 �M) have also beenrecorded from the mouse insulinoma cell line NIT-1 (426).Li and co-workers (463) also cloned a splice variant ofCav3.1 from an INS-1 cDNA library. The high sensitivity tonickel block suggests that Cav3.2 channels are also ex-pressed, and this conclusion is supported by Northernanalysis (438).

I. Cell Lines

T-type currents have been reported in a number ofcell lines such as 3T3 fibroblasts (73) and many lines oftumor origin, such as neoplastic B lymphocytes (128), therelated mouse neuroblastoma-derived lines N1E-115 (298,368), NG108–15 (196, 334), 140–3 (144), N18 (397), andND7–23 (213); human neuroblastoma lines IMR-32 (65)and SK-N-MC (18); rat pituitary lines GH3 (160, 270);human TT cells (also called h-MTC) and rat 6–23 (clone 6)cells from medullary thyroid tumors (43, 44); human Y79retinoblastoma cells (24); AT-1 from mouse atrium (351);rat smooth muscle lines A7r5 and A10 (272); and thepancreatic insulinoma lines INS-1 (rat; Ref. 42), HIT-T15(hamster; Ref. 259), and NIT-1 (mouse; Ref. 426). Many ofthese cell lines were reported to express almost exclu-

sively T-type currents, allowing characterization of thesechannels in the absence of contaminating HVA currents(Table 6). The study of T-type currents in cell lines hasadvanced our understanding of gating, permeation, andpharmacology (73, 160, 368). For example, Chen and Hess(73) developed models of gating using recordings from3T3 fibroblasts. They also showed that the T-type currentsof NG108–15 cells had different kinetics, leading them topredict the existence of multiple channel isoforms. Asimilar conclusion was reached from studies on TT cells,which also recovered from inactivation much moreslowly than observed previously (44). Differences in thepharmacology of T-type channels also suggested the ex-istence of distinct channel isoforms (160). The cloning ofmultiple Cav3 isoforms that differ in their kinetics, phar-macology, and recovery from inactivation supports thishypothesis (209, 228, 404).

J. Single-Channel Recordings

Single-channel studies provided conclusive evidencethat T-type channels were distinct from HVA Ca2� chan-nels (63, 303, 306). T-type channels were distinguished bytheir smaller currents, insensitivity to dihydropyridines,and voltage dependence of activation and inactivation. Inaddition to providing criteria for resolving T-type cur-rents, single-channel studies have provided insights intoboth hormonal regulation of activity and gating.

With isotonic Ba2� as the charge carrier (110 mM),the single-channel current at a test potential of 0 mV is�0.3 pA for T-type channels (Table 7) and �1.5 pA forL-type channels. Single-channel conductance is defined asthe slope of the line relating single-channel amplitudesversus test potential and is commonly reported in pico-

TABLE 6. Properties of T-type currents in cell lines

Cell LineDivalent,

mMThreshold,

mVI–V Peak,

mVImax,pA

V0.5,mV

h�,mV

�inact,ms

�deact,ms �recov, ms Nickel, �M

ReferenceNos.

HumanTT 10 Ca �40 �15 �211 �27T �51 16 2 1,900 5 (57%) 44IMR-32 10 Ca �50 �20 �80 15 200 (52%) 65Y79 20 Ba �50 �20 �87 �32C �40 20 24

RatC-cell 6-23 10 Ca �50 �15 �500 �31M �57 20 2 1,260 5 (22%) 43GH3 10 Ca �50 �20 �600 �33G �71 20 5 200 (80%) 777 160

1,000Mouse

MAb-7B 2.5 Ca �65 �35 �80 �82 16 128N1E-115 50 Ba �50 �20 �300 47 298NG108-15 10 Ba �60 �15 �140 �27C �53 20 2 334ND7-23 10 Ba �50 �30 �119 �67 18, 32 845 100 (60%) 2133T3 20 Ba �50 �20 �500 �65 10 2 100 73AT-1 2.5 �60 �35 �500 �48C �64 14 2.5 150 (73%) 351

1,200

Definitions are as in Table 2. Methods used to calculate activation curves are denoted with the following superscripts: C, chord conductance;T, tail currents; G, constant-field equation of Goldman-Hodgkin-Katz; M, I/Imax.



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seimens (pS). When recorded in Ba2�, the conductance ofT-type channels is 7–8 pS (Table 7), which is smaller thanthat observed for either N-type (13–15 pS) or brain L-typechannels (25–28 pS) (118, 125, 417). Somewhat surpris-ingly, all three families have a similar Ca2� conductance.For example, the L-type channel conductance dropsthreefold in Ca2� (149, 367). Recombinant Cav2.1 andCav2.2 channels also preferentially conduct Ba2� (�2-fold), whereas Cav2.3 conducts both ions equally well(56). In contrast, native T-type channels conduct Ca2� andBa2� (and Sr2�) equally well (64, 104, 367, 368). Theconductance plots differ in their relative position, withT-type channels gating at more negative ranges, and ex-trapolating to a reversal potential of approximately �40mV, while HVA currents appear to reverse at �60 mV (34,52, 118, 124, 194, 203, 213, 367, 417).

Estimates of permeability based on reversal poten-tials yield the opposite rank order, with Ca2� being morepermeable than Ba2� (128). Similar results have beenobtained using recombinant T-type (363) and native L-type channels (161) and suggest that Ca2� binds with highaffinity in the pore and permeates when displaced by asecond Ca2�. A very interesting property of all voltage-gated Ca2� channels is that they become highly perme-able to Na� in the absence of Ca2�. Apparently Na� arenot capable of displacing the bound Ca2�, and hence donot permeate. This block is unidirectional, since monova-lent cations can displace Ca2� bound in the pore when

they approach from the intracellular side of the channel(255, 363). The selectivity sequence for monovalentcations through T-type channels is Li�� Na� � K� �Rb� � Cs� (128, 255). In fact, T-type channels begin topass outward K� currents at physiological concentrationsof Ca2� and voltage (�28 mV; Ref. 128). HVA channelscan also pass outward currents, but this requires depolar-ization of the membrane beyond �70 mV (161). Our un-derstanding of Ca2� channel permeation is far from com-plete, and subject to multiple interpretations (275, 442).

Single-channel currents have also been studied atCa2� concentrations closer to the physiological levels,displaying a single-channel conductance of �2 pS in 5 mM(64) and 4.6 pS in 10 mM Ca2� (21). Whole cell studiesusing varying concentrations of Ca2� indicate that cur-rents increase sharply over the 1–10 mM range, thensaturate at higher concentrations. These studies have al-lowed estimates of the affinity of the open channel forCa2�. The average KD value from seven studies is 4.9 mM,with considerable variability (range 0.3�10 mM) (4, 22,54, 152, 160, 389, 429).

The transitions between open and closed states havebeen studied in detail in DRG neurons (64), cardiac myo-cytes (104), fibroblasts (73), and N1E-115 neuroblastomacells (368). T-type channels respond to depolarizingpulses by opening in bursts, that is, channels open andclose many times, become silent for a while, and at somevoltages may burst again. The fast opening and closings

TABLE 7. Single-channel properties

Source Divalent, mMCurrent at 0

mV, pAConductance,

pS SelectivityReference

Nos.

Rat DRG 40 Ca �0.2 5.2 Ca � Ba 64Chick DRG 110 Ba �0.4 8 124Mouse DRG 60 Ba �0.25 7.2 Ba � Sr 217Hippocampal CA3 pyramidal 95 Ba �0.3 7.8 118Septum and diagonal band 100 Ba �0.2 7.8 147Cerebellar Purkinje 110 Ba �0.3 9.0 52Pituitary menalotropes 110 Ba �0.4 8.1 203Hypoglossal motoneuron 110 Ba �0.3 7 417Retinal ganglion 96 Ba �0.36 8 194Cardiac ventricle 110 Ca �0.33 6.8 Ba � Ca 104Cardiac Purkinje 110 Ba �0.4 9.0 Ba � Ca 367Ear artery SMC 110 Ba �0.3 6.9 34Coronary artery SMC 110 Ba �0.38 7.5 130Saphenous vein 90 Ba �0.35 8 450Adrenal glomerulosa 110 Ba �0.25 8.7 Ba � Ca 27Pancreatic �-cells 100 Ba �0.3 6.4 348Neuroblastoma N1E-115 60 Ba �0.25 7.2 Ca � Ba � Sr 368Neuroblastoma ND7-23 110 Ba �0.25 7.9 213Neuroblastoma 140-3 110 Ba �0.3 6.84 144Low divalent

DRG 5 Ca 2 64Cardiac ventricle 10 Ca �0.16 4.7 21Coronary artery SMC 10 Ca �0.16 4.7 21Coronary artery SMC 10 Ba �0.2 4.6 130

Single-channel properties were measured from cell-attached patches using the indicated concentration of divalent cation as charge carrier. Theslope conductance of high-voltage-activated channels is similar to T-type channels when recorded in Ca2� solutions but can be distinguished by theircurrent amplitudes at a test potential of 0 mV. DRG, dorsal root ganglion; SMC, smooth muscle cell.



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can be modeled as transitions between a closed state C(C4 in Fig. 3) and an open state O. The transition rates outof the open state determine the mean open time, which istypically 0.5–2 ms (Table 7). Multiple bursts are seenduring depolarizing pulses just above the threshold forchannel opening. At more depolarized potentials a singleburst is observed, indicating that channels entered aninactivated state. Analysis of the time a channel spends inclosed states reveals a bimodal distribution; closingswithin a burst are short (�1 ms), whereas the time be-tween bursts is longer (�10 ms). Channels can also openpartially, leading to a subconductance state that has asmaller current (27, 64, 104). In contrast to other ionchannels (66), the gating behavior of this subconductancestate was found to be similar to the main conductancestate (64).

A distinguishing kinetic feature of T-type channels isthat whole cell current traces recorded during an I-Vprotocol produce a criss-crossing pattern, where succes-sive traces cross each other. This is largely due to slowactivation kinetics at threshold potentials (�60 to �40mV). At the single-channel level, T-type channels show along latency to the first opening, especially at thresholdpotentials, where 40 ms may pass before the first opening.This delay presumably reflects slow transitions betweenclosed states and can lead to a sigmoidal rise in the wholecell current. This rate is voltage dependent, such thatchannels open faster at more depolarized potentials (104).

In most sweeps (�70%) the channel does not open atall, resulting in a blank sweep (73, 104). This suggests thatchannels might inactivate without opening. Such closedstate inactivation has been observed in other channelsand is particularly prominent in Cav2 channels (321).Closed state inactivation also provides an explanation for

the whole cell observation that steady-state inactivationcan be observed at potentials more negative than channelopening. This inactivation is slow and voltage dependent(364). Inactivation from open (or the proximal closedstate C4) has similar kinetics and limits the channel to oneburst per sweep. Whole cell recordings suggest that thisrate is relatively voltage independent at potentials above�20 mV. Several models of T-type channel gating havebeen proposed that can simulate the observed activity.One reason these models differ is that they are based ondisparate results. Unresolved questions include the fol-lowing: Does the voltage dependence of the mean opentime explain the slowly deactivating tail currents ob-served at the whole cell level (64)? One study found thatopen times were shorter at more negative test potentials(64), another found no difference (104), while two foundthat they increased (217, 368). To complicate mattersmore, two studies have found evidence for a second typeof opening of longer duration (27, 147). Discrepancy inmean open time measurements has been attributed to thelow signal-to-noise ratio of channel openings (73). A re-lated question is: Does the burst duration vary with testpotential? Most studies report the burst duration at asingle voltage, although one study found that it increasedfrom 5 ms at �40 mV to 14 ms at �10 mV (104). Thesemodels might also differ because of T-type channel het-erogeneity, as evidenced by the distinct gating behavior ofthe three recombinant Cav3 channels (228). Recently, amodel has been proposed that can simulate the gatingbehavior of both recombinant Cav3.1 and Cav3.3 channels,although different rate constants for the transitions arerequired (127, 364). The model assumes that depolariza-tion leads to sequential movement of each of the four S4voltage sensors (see sect. IVB), culminating in a finalclosed state that precedes the open state. The open statecan either deactivate (return to C4) or inactivate (IO).Closed state inactivation is allosterically coupled to S4movement and becomes prominent after three S4 sensorshave moved (Fig. 3). Movement of the fourth voltagesensor does not affect the inactivation rate, so open chan-nels inactivate at the same rate as closed channels in C3and C4 states. The speed of voltage sensor movement andsubsequent transitions among closed states appears to beslower in Cav3.3 than Cav3.1, leading to slower activationkinetics. The channels also differ in their equilibriumbetween C4 and O states, with Cav3.3 favoring C4 whileCav3.1 favors O. Therefore, Cav3.1 channels inactivatemore from open states, whereas Cav3.3 channels inacti-vate more from closed states. The model accounts formost, but not all, of the observed properties of T-typechannels; in particular, it does not attempt to explainmultiple open states or ultraslow (�1 s) inactivation(127). In summary, voltage-gated Ca2� channels can bedistinguished by their single-channel properties, with T-

FIG. 3. Simplified gating scheme. The model represents transitionsof T-type channels between rested (R), closed (C), inactive (I), and openstates (O). Changes in the membrane potential are thought to inducemovement of the S4 voltage sensors, which leads to rearrangements inthe channel structure. Movement of three of the four S4 regions issufficient to induce closed states that inactivate relatively quickly. Theproximal closed state (C4) can also show closed state inactivation orlead to channel opening. Single T-type channels show a propensity toopen in bursts during a prolonged depolarization, and this representstransitions between C4 and O states. The model has been simplified fromthe version presented by Frazier et al. (127) by omission of C1, C2, I1, andI2. The effects of voltage suggest that S4 sensors must move back towardtheir original position before channels can recover from inactivation.



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type channels showing a smaller conductance in Ba2� andequal conductance of Ca2� and Ba2�.

III. REGULATION

Among the first criteria used to distinguish LVA fromHVA channels was their resistance to rundown in solu-tions that lack cAMP, ATP, and Mg2�, suggesting that LVAchannels were not phosphorylated by cAMP-dependentprotein kinase (115). Consistent with this hypothesis, hor-mones that stimulate adenylyl cyclase, such as the �-ad-renergic agonist isoproterenol, had no effect on T-typecurrents recorded from either canine atrial myocytes (32),rabbit sinoatrial nodal cells (152), canine Purkinje cells(166, 410), guinea pig ventricular myocytes (415), rabbitear artery (35), or guinea pig hippocampal CA3 neurons(117). Stimulation of L-type currents via cAMP-dependentpathways provided a positive control for most of thesestudies. Although some studies have reported a smallstimulation of T-type currents by isoproterenol (9, 283),this may be due to either incomplete separation of T- fromL-type currents (as evidenced by BAY K 8644 stimulation)or due to secondary regulation induced by changes ininternal Ca2� concentrations (411). In contrast to theapparent lack of protein kinase A (PKA) regulation, hor-monal regulation of T-type currents has been reported inmany systems. Both inhibition and stimulation have beenreported, and in some cases the same agonist did both.For example, the GABAB agonist baclofen has been re-ported to cause stimulation at 2 �M but inhibition at 100�M (361).

A. Hormonal Inhibition

Many hormones and neurotransmitters have beenreported to inhibit T-type currents, including dopamine,serotonin, somatostatin, opioids, ANP, and ANG II. Al-though most of these studies were performed on isolatedneurons, regulation has also been noted in secretory cellsof the pituitary and adrenal.

Dopamine inhibition of T-type currents has been de-scribed in DRG neurons (260), adrenal glomerulosa cells(312), pituitary melanotrophs (204), lactotrophs (240),and cells from the pars intermedia (309). Chick DRGcurrents were shown to be inhibited 60% by either 10 �Mdopamine or norepinephrine (260). Inhibition occurredwith no apparent effect on kinetics. Recovery upon wash-out was not observed in whole cell recordings. Similarinhibition was observed at the single-channel level, al-though in these experiments washout was observed. Sin-gle-channel current amplitude was not affected, suggest-ing that inhibition was due to a reduction in theprobability of opening (Po).

Dopamine (10 nM) inhibited T-type currents of rat

anterior pituitary lactotrophs by 25–40% (239, 240). Do-pamine shifted the h� curve from �63 to �77 mV (5 Ca)and accelerated current decay during the pulse. It had noeffect on voltage dependence of activation. Dopamine’seffects were reversible, mimicked by the dopamine ago-nist (D2 class) bromocryptine (10 nM), and prevented bythe D2 antagonist sulpiride (100 nM). Inhibitory regulationcould be abolished by omitting GTP from the intracellularsolution or by pretreatment with pertussis toxin. Inhibi-tory modulation could also be disrupted by inclusion ofantibodies that specifically recognize the �-subunit of Go,while antibodies against Gi subunits had no effect (239).Parallel studies showed that D2 receptors coupled to po-tassium channels via a different G protein, Gi-3�.

Dopamine inhibited rat adrenal glomerulosa T-typecurrent by 33% (103). Inhibition was mediated by the D1

receptor class as evidenced by 1) inhibition with the D1

agonist SKF 82958; 2) block by the D1 antagonist SCH23390; 3) but not by the D2 antagonist spiperone. Dopa-mine-mediated inhibition was blocked by agents that dis-rupt signaling by 1) G proteins [2 mM guanosine 5-O-(2-thiodiphosphate) (GDP�S)], 2) adenylyl cyclase (100 �M2,3-dideoxyadenosine), and 3) protein kinase activity(10 �M H-89). Consistent with an action through D1 re-ceptors, dopamine caused a stimulation of cAMP forma-tion. Somewhat surprisingly, bath application of 8-bromo-cAMP had no effect on basal T-type currents but couldblock the effects of dopamine. Addition of 100 nM G��subunits to the pipette solution did not affect T-typecurrents. However, G�� inhibition was observed afteraddition of 1 mM 8-bromo-cAMP to the bath. These re-sults suggested that G�� subunits might bind directly tothe T-type channel.

Somatostatin (10 nM) was found to inhibit T-typecurrents by 50% in rat somatotrophs (72). Somatostatincaused the currents to decay faster during a sustainedpulse and shifted the midpoint of the steady-state inacti-vation curve (h�) curve from �52 to �63 mV (chargecarrier was 5 mM Ca2�). This regulation was abolished bypertussis toxin. Neurotensin (200 nM) and substance P(200 nM) inhibited by 25% the T-type currents from ratcholinergic neurons (263). The �-opioid agonist Tyr-D-Ala-Gly-Met-Phe-Gly-ol (DAGO) was found to inhibit DRGcurrents by 50% (360). Enkephalin was found to inhibitT-type currents 20–40% in the neuroblastoma cell lineNG108–15 (196). In these cells there was no effect ofeither the phorbol ester 4�-phorbol 12-myristate 13-ace-tate (PMA), arachidonic acid (10 �M), or forskolin. Bra-dykinin (0.1 �M) inhibited 57% of the T-type current fromND7–23 cells, a cell line derived from DRG (213). Thisstudy also showed that (�)-baclofen (1 �M) could inhibitcurrents by 56%, whereas guanosine 5-O-(3-thiotriphos-phate) (GTP�S) inhibited by 20%. Pertussis toxin treat-ment did not block baclofen inhibition. Baclofen (20 �M)



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has also been reported to inhibit the T-type currents inhippocampal neurons by 53% (126).

Muscarinic inhibition of T-type currents from hengranulosa cells has also been reported (425). In contrastto most studies that report �40% inhibition, this studyreported 90% inhibition. Part of this difference may be dueto their use of the perforated patch technique, which isexpected to preserve the integrity of the intracellularmilieu. The effect of carbachol was blocked by atropine.In contrast, muscarinic agonists have been reported tohave no effect on LVA channels in cholinergic neurons (5).

ANP (3 nM) inhibited T-type currents of bovine ad-renal glomerulosa cells by 28% (274). Inhibition was de-pendent on the holding potential, with little block whenmembrane potential (VM) was �90 mV, but near completeblock when VM was �45 mV. This voltage dependence ofblock also caused a �10 mV shift of the h� curve to morenegative potentials. In contrast, ANP did not affect thevoltage dependence of activation as measured using tailcurrents (26). The effects of ANP were also studied at thesingle-channel level. Single T-type channels were identi-fied by their 8.3-pS conductance. Inhibition was accom-panied by a decrease in the number of active sweeps, withno change in the amplitude of single openings. The abilityof bath-applied ANP to modulate channels recorded in thecell-attached mode indicated that ANP induced the pro-duction of a soluble second messenger.

ANG II and the AT2 selective ligand CGP-42112 inhib-ited by 25% the T-type current in NG108–15 cells (58, 59).Inhibition was associated with a �5-mV shift in the I-V butno effect on the h� curve. Inclusion of 3 mM GDP�S in thepipette blocked the ANG II effect. In contrast, 3 mMGTP�S did not block the ANG II inhibition. This result issurprising since this concentration of GTP�S should havemaximally stimulated G protein signaling. Washout wasnot reported. A role for tyrosine phosphorylation in thesignaling pathway was suggested by the ability of 50 �Msodium orthovanadate to disrupt ANG II modulation ofthe T-type current.

B. Hormonal Stimulation

T-type currents can also be stimulated acutely by avariety of hormones. One of the first studies showed that10 �M serotonin could stimulate rat spinal motoneuronT-type currents by 66% in a slice preparation (36). Also ina slice preparation, dorsal horn neurons were found to bestimulated by substance P (1 �M), although some neuronswere inhibited (347). Cholinergic agonists also stimulateT-type currents, and this was demonstrated at the single-channel level. Carbachol and muscarine doubled the num-ber of openings of single T-type channels in adult guineapig hippocampal CA3 neurons (117). This stimulation wasblocked by 0.1 �M atropine. The modulation was likely

due to an increase in the probability of opening of eachchannel, as there was no change in the single-channelconductance, which was 7.1 pS in a solution containing 95mM Ba2�. Under similar conditions, carbachol inhibitedsingle L-type currents. Carbachol (50 �M) and serotonin(30 �M) were also shown to stimulate hippocampal T-type currents 54 and 61%, respectively (126). These effectswere blocked by the appropriate receptor antagonists andwere quickly reversible. Serotonin appeared to shift theI-V to the left (peak shifted from �25 to �30 mV), but thiseffect was not quantitated (126). Erythropoietin increasedT-type currents 20% in the human neuroblastoma cell lineSK-N-MC (18). It also stimulated increases in intracellularCa2� that was dependent on external Ca2� but not inter-nal stores. Norepinephrine acting via �-adrenoceptorswas reported to increase T-type currents in canine Pur-kinje cells by 69% (410).

Endothelin has been reported to stimulate T-typecurrents from both cardiac and smooth muscle myocytes(129, 181). In neonatal rat ventricular myocytes, endothe-lin-1 displayed an EC50 of 1.3 nM and a maximal stimula-tion of 54% (129). Heat inactivation of the endothelin, oromission of GTP from the pipette, occluded the effects.The stimulation induced by 10 nM endothelin wasblocked by 0.2 �M staurosporine or 20 �M H-7. Proteinkinase C (PKC) agonists stimulated the currents directly,and this could be blocked by H-7 as well. In guinea pigsmooth muscle myocytes, endothelin stimulated the ac-tivity of a 12-pS channel, that may or may not be carriedthrough T-type channels (181). Endothelin has been re-ported to have no effect on T-type currents recorded fromsmooth muscle cells from rat renal resistance arteries(143).

Acetylcholine stimulated T-type currents in NIH 3T3cells that had been transfected with recombinant m3 andm5 muscarinic receptors (322). Stimulation was modest(25%) and was accompanied by a leftward shift in the I-Vrelation. Parallel assays demonstrated that cAMP levelswere increased. T-type currents were also stimulated bytreatments designed to stimulate cAMP-mediated signal-ing, such as 500 �M 8-bromo-cAMP or 10 �M forskolin.Likewise, the PKA inhibitor 10 �M Rp-adenosine 3,5-cyclic monophosphothionate (RpcAMPS) inhibited basalactivity and occluded the response to acetylcholine.These results suggested that T-type channels might bedirectly phosphorylated by PKA. An apparent contradic-tion to this hypothesis was that T-type currents were notregulated in cells transfected with the m1 muscarinicreceptor, although these cells showed robust increases incAMP. The authors speculated that m1 receptors mightsimultaneously activate inhibitory PKC pathways that ob-scured the PKA-mediated stimulation. First they showedthat the PKC activator phorbol 12,13-dibutyrate (PDBu;0.5 �M) directly inhibited T-type currents as observed inother cells types. Next they showed that preincubation



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with the PKC inhibitor calphostin C (0.5 �M) allowedm1-mediated stimulation of the T-type current. Basal T-type currents were not regulated by acetylcholine in cellstransfected with either m2 or m4. However, inhibition ofT-type currents, as well as inhibition of cAMP formation,could be observed in these cells after stimulation of ad-enylyl cyclase by 10 �M forskolin. These results indicatethat hormonal regulation of T-type channels may dependon both basal phosphorylation levels as well as cross-talkbetween PKA and PKC pathways.

Barrett and colleagues (82) have extensively studiedthe stimulation of adrenal glomerulosa T-type Ca2� chan-nels by ANG II. Initial studies established the presence ofT-type channels in these cells through the use of tailcurrent analysis (82). Slowly deactivating currents gatedwith the voltage dependence expected of T-type channels,while fast-deactivating currents behaved like HVA, L-typechannels. ANG II increased the T-type tail currents by 50%(82). The effects of ANG II were also studied at thesingle-channel level (273). ANG II increased both thenumber of active sweeps and increased the number ofopenings per sweep, with no appreciable change in theamplitude of single openings. The ability of bath-appliedANG II to modulate channels recorded in the cell-attachedmode indicated that ANG II induced the production of asoluble second messenger. The effects of ANG II wereblocked by the ANG II receptor antagonist saralasin (1�M). ANG II also shifted the voltage dependence of acti-vation by 10 mV to more negative potentials, with nochange in the h� curve. Pretreatment of the cells withpertussis toxin abolished these effects, indicating thatANG II receptors couple through G proteins, possibly Gi

(251). The final step in this signal transduction pathwayappears to be phosphorylation of the T-type channel bycalmodulin-dependent protein kinase II (CaMKII; see be-low) (27, 252). However, not all studies have found anANG II stimulation of adrenal glomerulosa currents, withreports of either no effect (250) or frank inhibition (103,344).

C. Guanine Nucleotides

As noted above, T-type currents are regulated by Gprotein-coupled receptors. Therefore, this regulationshould require the presence of GTP and should be modi-fied by activators (GTP�S) and inhibitors (GDP�S) of Gproteins. Accordingly, no hormonal regulation was ob-served when GTP was omitted from the patch pipette(129, 239) or when it was replaced with GDP�S (59, 103).

The effects of GTP�S are difficult to interpret be-cause it can activate all G proteins. With the use ofphotoactivation of caged GTP�S in DRG neurons, lowconcentrations were observed to stimulate T-type cur-rents by 54%, while higher concentrations inhibited them

up to 87% (361). Inhibition was accompanied by an accel-eration of inactivation, such that the � decreased from 22to 18 ms. Neither cholera toxin pretreatment nor photore-lease of caged GDP�S had any effect. Pertussis toxinpretreatment blocked GTP�S inhibition, but not stimula-tion. GTP�S has also been shown to inhibit T-type cur-rents (40%) of rat nodose ganglion neurons (148). Thiseffect was blocked by pretreatment with pertussis toxin.

Somewhat surprisingly, G proteins do not appear tomediate the regulation of T-type currents by serotoninand nociceptin. Serotonin inhibition (25%) of T-type cur-rents has been observed in Xenopus sensory neurons(383, 384). Inhibition was not observed when the T-typechannels were recorded in the cell-attached patch config-uration, indicating that its effects were membrane delim-ited. Inhibitory regulation was not blocked by pertussistoxin pretreatment or inclusion in the pipette of GDP�S,GTP�S, or 5-guanylyl-imidodiphosphate (GMP-PNP). Theability of these manipulations to block the regulation ofHVA currents in the same neurons provided a convenientpositive control. It was concluded that G proteins are notinvolved in the serotonin inhibition of T-type channels. Asimilar conclusion was reached in a study on the nocicep-tin inhibition of DRG T-type currents (1). Nociceptin in-hibited currents with an IC50 of 0.1 �M, and in 22 of 77neurons it blocked 100% of the current at 1 �M. Inclusionin the pipette of GTP�S, GDP�S, or AlF3 did not alterresponse, leading the authors to conclude that a G proteinis not involved. It should be noted that nociceptin had noeffect on T-type currents from small nociceptive trigemi-nal ganglion neurons (51). The endogenous cannabinoidanandamide also blocks T-type currents independently ofG proteins and receptors (71).

The ability of pertussis toxin treatment to block reg-ulation indicates that Gi/Go proteins are involved. Supportfor this hypothesis comes from studies that used specificantibodies in the patch pipette to block regulation (239,251). Although the ��-subunits of G proteins have beensuggested to interact directly with T-type channels (103),more studies are required to establish this point.

D. Protein Kinases

With the notable exception provided by studies onfibroblasts (322), T-type currents appear not to be regu-lated by PKA. In contrast, T-type currents appear to beinhibited by members of the PKC family and stimulated bytyrosine kinases and CaMKII.

The effects of PKC have been evaluated by activatingthe kinase directly with compounds such as diacylglyc-erol 1-oleolyl-2-acetylglycerol (OAG), or various phorbolesters: PMA, 12-O-tetradecanoylphorbol-13-acetate (TPA),or PDBu. Although many studies have found no effect ofthese compounds (310), both stimulation and inhibition of



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activity have been reported. Studies on rat DRG neuronsfound that 10 nM PMA inhibited the T-type current by27%. Likewise, the inactive analog 4�-phorbol had noeffect. Of note, the PMA effect was not observed at roomtemperature, requiring preincubation at 29°C or higher(359). Similarly, 100 nM TPA was found to inhibit theT-type current by 26% in ventricular myocytes and Pur-kinje cells. A phorbol ester analog that does not activatePKC had no effect, while the protein kinase inhibitor H-8prevented TPA inhibition (411). In contrast, 50 �M H-7was not able to block the inhibition of hippocampal T-type currents by 10 �M TPA or 5 �M OAG (407). Inaddition, the TPA and OAG effects occurred faster thanwould be expected (200 ms), leading Toselli and Lux(407) to conclude that the compounds were blocking thechannel directly. PKC may in part mediate the inhibitoryeffects of arachidonic acid (459).

PKC-mediated stimulation of T-type currents has alsobeen reported (129). Currents in rat neonatal ventricularmyocytes were stimulated 30% by either 0.2 �M PDBu orPMA. The inactive analog 4�-PDBu had no effect. Stimu-lation was blocked by inclusion of 20 �M H-7 in thepipette.

The ability of CaMKII to stimulate adrenal T-typecurrents has been studied at both the whole cell andsingle-channel level, and recently with recombinant chan-nels. Inclusion of Ca-CaM in the pipette shifted the volt-age dependence of activation to more negative potentials(252). The CaMKII inhibitor KN-62 or the synthetic pep-tide inhibitor that corresponds to residues 290–309blocked this effect. Stimulation was also demonstrated atthe single-channel level in both cell-attached and excisedpatches (27). In cell-attached patches, manipulation ofintracellular Ca2� led to an increase in channel activitythat could be blocked by KN-62, but not the inactiveanalog KN-04. Channel activity could also be stimulatedwhen excised inside-out patches were exposed to cal-modulin and appropriate concentrations of Ca2�. Thisstimulation could be blocked with the autocamtide 2-re-lated inhibitory peptide (AIP). Stimulation could also bemimicked by the direct addition of a constitutively activemutant of CaMKII. Heat-inactivated kinase had no effect.As observed previously with ANG II (273), this regulationappeared to be due to an increase in active channels.These studies clearly establish that native bovine glo-merulosa T-type channels are regulated by CaMKII. Adre-nal cortical T-type channels are encoded by �1H, or Cav3.2(358). Recent studies have shown that calcium/calmodu-lin can stimulate the activity of cloned human Cav3.2channels when coexpressed with CaMKII�c in HEK-293cells (443). Stimulation could be blocked by CaMKII in-hibitors added to either the bath (3 �M KN-62) or to theintracellular pipette used for whole cell recording (AIP, 2�M). Replacement of pipette ATP with the nonhydrolyz-able analog 5-adenylyl-imidodiphosphate (AMP-PNP)

also blocked regulation. As observed with native chan-nels, stimulation was accompanied by an 11-mV hyperpo-larizing shift in the voltage dependence of activation, withno shift in the h� curve. In contrast, the activity of humanCav3.1 was not regulated under similar conditions. Thesestudies strongly suggest that the modulatory phosphory-lation site(s) reside directly on the �1-subunit of T-typechannels and that these sites are subtype specific, in thiscase only found on Cav3.2. Although CaMKII regulation ofT-type currents has not been reported in other systems, itmight have mediated the 30% stimulation observed incardiac myocytes (411).

E. Voltage

An intriguing property of many HVA Ca2� currents isthat their activity can be increased, or “facilitated,” bystrong depolarizing pulses. These prepulses can inducechannel conformations that are 1) more susceptible tophosphorylation, 2) have a lower affinity for divalentcations in the pore, or 3) have a lower affinity for Gprotein ��-subunits. The prepulse can also directly inducehigh activity gating modes (mode 2) of cardiac L-typechannels. Both native (below) and recombinant (140)T-type currents have also been reported to be facilitatedby prepulses.

The T-type currents of guinea pig coronary smoothmuscle myocytes were stimulated twofold by 200-ms pre-pulses to voltages above �30 mV (131). Studies where theprepulse potential was varied indicated that saturationoccurs at �10 mV. Use of longer prepulses (10 s) shiftedthis voltage dependency to the left, such that saturationoccurred at �60 mV. Changing the duration of the pre-pulse showed that the peak effect occurred between 160and 320 ms, which then decayed and was gone by 5 s. Thispotentiation was greater when the interpulse voltage was�100 compared with �80 mV, and at 36 versus 24°C.Potentiated currents inactivated faster (�inact 5.6 vs.14.5 ms).

Facilitation has also been observed in bone marrowcells, where T-type currents were stimulated twofold by a750-ms prepulse to �150 mV (330). Compared with theresults obtained in smooth muscle, stronger depolariza-tions were required; stimulation was not observed withprepulses lower than �30 mV, and saturated at �100 mV.Changing the duration of prepulse (�10-mV pulse)showed that half-maximal stimulation occurred at �250ms, and saturated by 1,000 ms. Changing the intervalbetween the prepulse and the test pulse showed thatpotentiation peaked after 1 s and decayed in a biexponen-tial manner: 9.4 and 43.5 s. Potentiation was not affectedby omission of ATP or GTP or bath application of non-specific protein kinase inhibitors (e.g., 100 �M H-7). Po-tentiated currents decayed slightly faster (�inact decreased



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from 17 to 13 ms; currents isolated by subtraction). Theseresults suggest that voltage directly affects channel activ-ity without the involvement of second messenger signals.The opposite conclusion was reached in studies of bull-frog atrial cells (8). Amphibian channels are stimulated by�-adrenergic agonists, and a prepulse enhances this stim-ulation. These effects were enhanced by GTP�S andATP�S, but blocked by either GDP�S or pertussis toxintreatment. From these studies it was concluded that Gproteins are involved and that phosphorylation of G pro-teins may play a role as well.

Facilitation of T-type currents from mouse sper-matogenic cells has also been reported (14). In this case,prepulses could only stimulate activity 50% above control.Prepulses to voltages greater than �30 mV were requiredto observe stimulation, and saturated around �60 mV.Short prepulses (�10 ms) were capable of stimulatingcurrents, and this effect wore off very slowly. Membrane-permeable inhibitors of tyrosine kinases (tyrphostins A47and A25) stimulated basal T-type currents but had noeffect on facilitated currents. This suggests that tyrosinekinases may tonically inhibit activity in this system andthat a prepulse somehow reverses their activity. Consis-tent with this hypothesis, inhibitors of tyrosine kinasephosphatases inhibited basal T-type currents and pre-vented voltage-induced potentiation.

In summary, T-type channels can be both stimulatedand inhibited by hormones and neurotransmitters that arecoupled to the Gq or Gi/Go families. Regulation appears tobe mediated by phosphorylation, with inhibition mediatedby PKC and stimulation by CaMKII. Additional studies arerequired to establish the location of the modulatory phos-phorylation site(s), whether G protein subunits are capa-ble of interacting directly with the channel, and the mech-anisms by which voltage regulates channel activity.

IV. MOLECULAR CLONING

OF T-TYPE CHANNELS

A. Cloning

Cloning of high voltage-gated Ca2� channels beganwith purification and microsequencing of the skeletalmuscle dihydropyridine receptor (396). Low-stringencyscreening of cDNA libraries with probes derived from the�1-subunit of skeletal muscle (�1S, or Cav1.1) led to thediscovery of cardiac (�1C, Cav1.2) and brain (�1D, Cav1.3)isoforms of the L-type family (Cav1; reviewed in Ref. 326).The more distantly related members of the Cav2 familywere cloned during the “brain era” (Fig. 4A) using evenlower stringency hybridization (374). PCR provided analternative approach, and primers based on highly con-served regions (IIIS5 and IVS5) were used successfully toisolate a fragment of Cav2.3 (357). Despite the efforts of

many groups, these approaches were not successful inisolating cDNAs encoding T-type channels.

Progress in the sequencing of human, yeast, and C.

elegans genomes provided a novel library that could bescreened with a computer, or in silico (Fig. 4B). Screeningwith the highly conserved P loop, or K� channel signaturesequence, allowed Ketchum et al. (205) to identify a novelK� channel sequence from Saccharomyces cerevisiae. Wedecided to try a similar approach using S5 segments. Thisled to the identification of two Ca2� channel-like proteinsthat were predicted from the C. elegans genome (325).These proteins were homologous to the skeletal muscleCa2� channel, and this similarity was noted in the Gen-Bank entry. We reasoned that there might be other se-quences in the GenBank that were similarly labeled andused a text-based search (Fig. 4B) to find sequences thatwere “similar to a calcium channel.” This led to the iden-tification of the human EST clone H06096 (324). Althoughthis clone is 1.4 kb long, only a part of the 5-sequence wasin the database. This fragment had a very low sequenceidentity to the S1 segment of other Ca2� channels, pre-cluding its direct identification by homology search. Thefull sequence of H06096 showed that it might encode aprotein with many of the hallmarks of voltage-gated chan-nels, notably the S4 voltage sensor sequence was wellconserved, and the pore loop sequence was highly homol-ogous to other Ca2� channels. Screening of the GenBankwith the H06096 sequence and the program BLAST iden-tified C54d2.5 as a C. elegans homolog. Similar to in vitrocloning where one “walks” through a cloning project us-ing probes to the ends, we screened the GenBank with the3-end of C54d2. This led to the identification of the ESTclone H19230. In vitro screening of a rat brain cDNAlibrary with H06096 allowed the cloning of Cav3.1 (324),whereas use of this clone to screen a human heart libraryled to the cloning of Cav3.2 (90). Screening of a rat brainlibrary with H19230 under low-stringency conditions al-lowed us to identify not only rat versions of Cav3.1 andCav3.2, but also Cav3.3 (228). A similar approach was usedby Monteil et al. (288) to identify C54d2.5 and the H06096and H19230 ESTs, which then allowed them to clone thefull-length human Cav3.1. Similar PCR approaches werealso used to clone a human Cav3.2 cDNA (438) and mouseand rat versions of Cav3.1 (211, 463). PCR approachesbased on the C. elegans sequence C27f2.5 led to thecloning of a distantly related channel gene whose func-tion remains unknown (227).

Sequencing of the human genome also played animportant role in the cloning of T-type channels. TheCav3.3 gene is located on chromosome 22, which was thefirst chromosome to be completely sequenced (105).BLAST search with rat Cav3.1 identified the coding re-gions of Cav3.3, which then allowed the design of PCRprimers to either clone its cDNA (228, 289) or to examine



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the splicing patterns of its gene (285). Proper identifica-tion of the 5- and 3-ends requires traditional cDNA clon-ing or specially designed PCR protocols. Recent studiesindicate that the human Cav3.3 mRNA contains an addi-tional exon that encodes 214 additional amino acids (140)than previously recognized (289). Studies comparing thefull-length to truncated Cav3.3 channels revealed differ-ences in channel expression and their ability to be mod-ulated by a strong prepulse (140).

Genetic mutations in various Ca2� channel subunitgenes have been associated with ataxic and epilepticphenotypes (187). As a first step in exploring such a link,we mapped the human and mouse genes for Cav3.1,CACNA1G (324), and Cav3.2, CACNA1H (90). The geneswere mapped using the Cambridge 4 radiation hybridpanel and fluorescent in situ hybridization. The locationsof the human genes are shown in Table 8. Mutations inhuman Cav3 genes have yet to be described.

All three T-type channel genes are alternativelyspliced, producing variants that differ in their intracellularloops (68, 284, 285, 463). Splicing produces Cav3.1 vari-ants that differ in at least two locations: 1) the II-III loop,where two variants are produced by insertion/deletion of

exon 16; and 2) the III-IV linker, where three (or actuallyfour, Ref. 229) variants are produced by a combination ofinsertion/deletion of exon 26 and use of an alternate5-splice site in exon 25 (288). These variants differ intheir electrophysiological properties (68) and may ac-count for the differences noted in the gating of T-typechannels between lateral geniculate (LGN) relay neuronsand interneurons (318). Splice variants of Cav3.3 havebeen detected by PCR that differ in the I-II loop andcarboxy terminus (285). Similarly, PCR has identifiedsplice variation in the III-IV linker of Cav3.2 in both rat(231) and human (182).

Cav3 cDNAs have been cloned from rat, mouse, andhuman sources (Table 8). Although all three have beencloned from rat and human, cloning from mouse is stillongoing. Comparisons of their nucleotide sequences re-veal a high level of conservation between species. Al-though species differences in the predicted amino acidsequence have been noted, some of these changes can beascribed to either frameshifts in the reading, cloning ar-tifacts, or differences in splicing. For example, it is likelythat a cloning artifact caused the deletion of three aminoacids from IIIS4 in a rat cDNA of Cav3.3 (279).

FIG. 4. A timeline of Ca2� channel cloning. A: graphillustrates the time of the first published reference to thecloning of each of the 25 subunits. Skeletal muscle Ca2�

channel subunits were purified, microsequenced, thencloned using degenerate oligonucleotide probes based onthe protein sequence. This initial period has been calledthe Muscle Era. Low-stringency screening of brain cDNAlibraries with the skeletal muscle probes led to the cloningof other �1- and �-subunits during the Brain Era. In thefinal epoch, or In Silico Era, subunits were first identifiedby database searching programs (BLAST) of Human andCaenorhabditis elegans genome sequencing projects, thencloned by standard in vitro methods. (Adapted from Ran-dall A and Benham C. Recent advances in the molecularunderstanding of voltage-gated Ca2� channels. Mol Cell

Neurosci 14: 255–272, 1999.) B: flow diagrams describingtwo ways of cloning in silico.



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B. Structure

Conservation of amino acid sequence (Figs. 1 and 5)and predicted secondary structure (Fig. 6) indicates thatT-type channels are evolutionarily related to the �-sub-units of K�, Na�, and HVA Ca2� channels (184). Althoughlittle is known about the structure of Na� and Ca2�

channels, recent advances with K� channels provide aframework to interpret their structures. The most signif-icant advance was the determination of the KcsA K�

channel structure by X-ray crystallography (102). Thischannel contains two transmembrane segments sepa-rated by a pore loop (Fig. 6). The second transmembranesegment forms the barrel walls of the pore, while theselectivity filter is formed by the pore loops that dippartially into the barrel. In addition to this fundamentalmotif, voltage-gated channels also contain four additionaltransmembrane segments, with one (S4) acting as a volt-age sensor. In K� channels, four proteins assemble toform one channel, while in Na� and Ca2� channels asingle protein contains four of these modules, or repeats.Each transmembrane segment can be identified by a num-ber to indicate the repeat it is in (traditionally a Romannumeral) followed by its position within a repeat (Fig. 6).For example, the third transmembrane segment of thethird repeat can be abbreviated IIIS3. Among voltage-gated ion channels, the most highly conserved sequencebelongs to the S4 segments, where every third residue ispositively charged. This region is thought to move out-

ward when the plasma membrane is depolarized. Little isknown about how this movement is transduced into chan-nel opening. The pore loops are also well conserved,especially within the members of a particular ion channelclass. In voltage-gated Ca2� channels, the pore loops allcontain the following signature sequence: T-x-E/D-x-W. InHVA channels, all four repeats have a glutamate residue inthe pore, leading to the abbreviation EEEE. In repeats IIIand IV of Cav3 channels, the glutamate has been replacedby an aspartic acid residue, or EEDD. The importance ofthese residues in Ca2� permeation has been extensivelyestablished in HVA channels (449), and recently withCav3.1 (391).

Site-directed mutagenesis studies on the S6 region ofCav3.1 suggest that these segments play a similar struc-tural role in these channels as in KcsA (266). This pro-vides a theoretical basis for the use of the KcsA crystalstructure to guide studies of drug binding pockets, whichin the case of Cav1.2 appear to be located within thechannel walls on the cytoplasmic side of the pore loops(174).

As shown in Figure 6, most of the channel is thoughtto be intracellular. The extracellular loops are predictedto be quite short, with the notable exception of the loopconnecting IS5 to the pore loop (98–103 amino acids).This extracellular loop contains six cysteine residues thatare conserved in all three Cav3 channels and a number ofpossible sites for glycosylation. Neuraminidase treatmentof cardiac myocytes stimulates T-type (but not L-type)

TABLE 8. Comparison of the three cloned T-type channels

Cav3.1 Cav3.2 Cav3.3Reference

Nos.

Alias �1G, CavT.1 �1H, CavT.2 �1l

Gene name CACNA1G CACNA1H CACNA1l 249Chromosomal location 17q22 16p13.3 22q12.3–13.2 90, 228, 324Database entries*

Human NP_061496, O43497 AAC67239, O95180 XP_001125, Q9UNE6Rat AAC67372, O54898 AAG35187, Q9EQ60 NP_064469, Q970Y8Mouse NP_033913, Q9WUB8 NP_67390

Tissue expression Brain, ovary, placenta, heart Kidney, liver, adrenal cortex,brain, heart

Brain 90, 228, 288,289, 324

Electrophysiology†Threshold, mV �70 �70 �70 209Peak of I–V, mV �30 �30 �30 209Activation kinetics, ms 1 2 7 209Inactivation kinetics, ms 11 16 69 209Deactivation kinetics, ms 3.0 2.2 1.1 209h�, mV �72 �72 �72 209Recovery from inactivation, ms 117 395 352 209Conductance, pS; Ba 7.5 9 11 228, 324, 438

PharmacologyNickel (IC50, �M) 250 12 216 230Mibefradil (IC50, �M) in 10 Ba2� 1.2 1.1 1.5 267Mibefradil (IC50, �M) in 2 Ca2� 0.27 0.14 267

* Accession numbers are for a representative GenBank entry followed by the SWISS-PROT entry. † Results for conductance were obtainedin 110 mM Ba2�, while all other electrophysiological measurements were made in 1.25 mM Ca2�. Activation and inactivation kinetics were measuredwith exponential fits to the currents elicited by test potentials to �10 mV. Deactivation kinetics were measured at �90 mV. Pharmacology wasmeasured in 10 mM BaCl2, or as noted, in 2 mM CaCl2 solutions.



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currents threefold, suggesting that glycosylation of thechannel inhibits its activity (116, 451). Despite the factthat the sequences of intracellular loops differ consider-ably across Na� and Ca2� channels, they have a similaroverall size: long loops connect repeats 1–2 and 2–3(range 207–375 amino acids for Cav3 channels), whilerepeats 3–4 are connected by short loops (55–62 aminoacids). Similarly, the amino termini are usually short(�100 amino acids), whereas the carboxy termini arequite long (433–493 amino acids). The sequence of theseloops is poorly conserved, with the exception of se-quences close to the end of the S6 segments (Fig. 5). InHVA channels these loops are important for binding aux-iliary subunits such as the �-subunit, binding to regulatoryproteins such as calmodulin or the G protein ��-complex,and are sites for protein phosphorylation (424). Althoughthe roles of these loops in T-type channels are presentlyunknown, it is likely they contain important regulatorysites as well.

C. Distribution

The expression patterns of these genes in peripheraltissues and brain have been studied using Northern blots,RNA dot blots, and in situ hybridization (Table 8). Cav3.1mRNA is primarily expressed in human brain, but also inovary, placenta, and heart (288, 324). A wider distributionwas noted in dot blots prepared from human fetal tissues,with strong expression noted in kidney and lung (288).Cav3.2 mRNA is primarily expressed in kidney and liver,but also in heart, brain, pancreas, placenta, lung, skeletalmuscle, and adrenal cortex (90, 438). Cav3.3 mRNA isalmost exclusively expressed in brain (228); the only pe-ripheral tissues that showed expression on dot blots wereadrenal (but see Ref. 358) and thyroid (289).

The distribution of all three Cav3 channel transcriptsin rat brain has been studied in exquisite detail using insitu hybridization (393), and similar patterns of expres-sion have been obtained using Northern blots of humanbrain mRNA (288, 289, 438). Their patterns of expressionare complementary in many regions, with most brainregions expressing more than one isoform. In fact, someneurons may express all three genes as suggested by thelabeling of olfactory granule cells and hippocampal pyra-midal neurons. Many brain regions showed heavy expres-sion of Cav3.1 mRNA, including thalamic relay nuclei,olfactory bulb, amygdala, cerebral cortex, hippocampus,hypothalamus, cerebellum, and brain stem. Two separatestudies have confirmed this pattern of mRNA expressionand extended the findings by showing a similar distribu-tion of Cav3.1 protein (88, 197). Cav3.2 mRNA expressionwas detected in olfactory bulb, striatum, cerebral cortex,hippocampus, and reticular thalamic nucleus. Cav3.3mRNA expression is high in olfactory bulb, striatum, ce-

rebral cortex, hippocampus, reticular nucleus, lateral ha-benula, and cerebellum. DRG neurons express bothCav3.2 and Cav3.3 mRNA (393). One study found that thisexpression was restricted to small and medium-sized neu-rons (393), while a second study reported that Cav3.3transcripts were equally abundant in large DRG neurons(454).

D. Electrophysiology of Recombinant Channels

Expression of recombinant Cav3 channels leads tothe appearance of robust currents in a variety of heterol-ogous expression systems. Currents mediated by thecloned �1-subunit are nearly identical to those recordedfrom native channels: they both open and inactivate atpotentials near the typical resting membrane potential,recover rapidly from inactivation, and close slowly pro-ducing prominent tail currents (Fig. 7).

The currents mediated by rat and human isoforms ofthe three Cav3 channels have been compared under avariety of experimental conditions (209, 218, 228, 279). Ingeneral these results agree quite well with each other,with the differences most likely due to recording condi-tions or sequence variability. For example, Cav3.1 andCav3.2 currents inactivate (�1.7-fold) faster with Ba2�

than Ca2� (209, 211). Similarly, recording conditions suchas choice of charge carrier or length of depolarizingpulses can affect estimates of both deactivation kinetics(211, 216, 430) and recovery from inactivation (430).Therefore, comparisons under one set of conditions arethe most relevant; Table 8 presents the results obtained in1.25 mM Ca2� (209), whereas Figure 7 results were ob-tained in 5 mM Ca2�.

All three Cav3 clones form LVA channels. Depolariza-tion of the membrane to �70 mV is sufficient to triggerchannel opening, and the I-V curves for all three channelspeak around �30 mV (Fig. 7B). Some studies have foundthat Cav3.3 currents activate at slightly more positivepotentials than either Cav3.1 or Cav3.2 (127, 289). Part ofthis difference can be ascribed to the methods used toestimate the voltage dependence of activation. Of note,not all the recombinant channels within a given prepara-tion gate at the same low potentials, with �40% of thechannels requiring much stronger depolarizations (�10 to�50 mV) for opening (288, 438). Activation curves con-structed using tail currents that detect both channelsshow a lower slope and higher midpoint of activation thanother methods.

The kinetics of the currents are very voltage depen-dent between �70 and �20 mV, producing a typical criss-crossing pattern of traces (Fig. 7A; Ref. 334). Therefore, itis useful to compare the kinetics of the three channels at�10 mV or above (Table 8). Cav3.1 and Cav3.2 both acti-vate relatively quickly at this potential (� � 1–2 ms) and



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FIG. 5. Alignment of the predicted amino acid sequence of human Cav3.1, 3.2, and 3.3 channels. Residues conservedin all three sequences are shown in the consensus sequence (CON). The approximate location of the membrane-spanningregions is indicated above the sequence. �-Subunits bind to the I-II loop of HVA channels through a sequence termed the�-interaction domain (AID). The consensus sequence of the AID is shown above the homologous region in the Cav3channels. The residues are colored according to the structural properties of the individual amino acids: red, positivelycharged; green, negatively charged; blue, polar; yellow, nonpolar.



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inactivate 10-fold slower (� � 11–16 ms). In stark con-trast, Cav3.3 currents activate and inactivate very muchslower. This difference is even greater when comparisons

are made using Xenopus oocytes (228), and it remains amystery why Cav3.3 channels behave so differently inamphibian eggs than in mammalian cells.

FIG. 5—Continued



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Recombinant Cav3 channels, like their native coun-terparts, close slowly producing prominent tail currents.The voltage protocol used to measure tail currents in-cludes a short pulse to open channels, followed by repo-larization to a voltage where they close (Fig. 7C). Al-though all three Cav3 channels show slow tail currentsupon repolarization to �90 mV, Cav3.1 are the slowest(� � 3 ms), while Cav3.3 are the fastest (� � 1 ms). Incontrast, HVA channels close 10-fold faster.

Despite large differences in the rate at which theyinactivate, the voltage dependence of steady-state inacti-vation of the three recombinant Cav3 channels is remark-

ably similar. The midpoint of the h� curves are �72 mV(Table 8). Native T-type currents inactivate over a similarrange. These studies indicate that T-type channels, asobserved with other channels, can reach inactivatedstates without passing through open states (364). In fact,during a depolarizing pulse up to 30% of Cav3.3, channelsinactivate without opening (127).

Cav3 channels recover rapidly from short-term inac-tivation. Cav3.1 channels recover fastest, displaying amonoexponential recovery with a time constant of �100ms (Fig. 7, Table 8). In contrast, Cav3.3 channels recoverthreefold more slowly, while Cav3.2 channels recover

FIG. 6. Structural models. A: hydrop-athy plots of the KcsA and Shaker K�

channels and repeat III of Cav3.1. Thehydropathy index varies from 3 to �1.Letters above the plots indicate the rela-tive position of the putative transmem-brane segments (S1–S6) and pore loops(P). B: transmembrane segments are alsotypically displayed in two dimensions assnake diagrams, wherein the protein isshown to snake its way in and out of themembrane. C: a model of the three-di-mensional structure of four-repeat ionchannels. The repeats are assumed to beoriented in a clockwise manner based onwork with Na� channels. The S5-P-S6segments are thought to form a similarstructure as the S1-P-S2 segments ofKcsA, with the S6 segment forming thewalls of the channel. The S4 segment ishighly charged, so it is likely to be sur-rounded by the other segments with wa-ter filling the cracks. The S1–S3 segmentsare shown to form the exterior of thechannel. The transmembrane segmentsare most likely composed of �-helices,but we have no clue to their orientation.The intra- and extracellular loops are notshown for clarity. D: a snake diagramwhere each amino acid is represented bya circle. Residues that are conserved inall three Cav3 channels are shown with asolid circle. (Adapted from Perez-ReyesE. Three for T: molecular analysis of thelow voltage-activated calcium channelfamily. Cell Mol Life Sci 56: 660–669,1999.)



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even more slowly (209). The channels also differ in theirrecovery from steady-state inactivation, and again, Cav3.2was found to recover the slowest (209). These resultssuggest the existence of multiple inactivated states, asnoted previously for T-type currents from sensory neu-rons (53). These results also suggest that recovery kinet-ics can be used to differentiate currents carried by nativeCav3.1 from Cav3.2 (351). Fast recovery from inactivationis a critical property of native T-type channels that allowsthem to participate in rebound burst depolarizations.

The ability of T-type channels to open at similarpotentials at which they inactivate suggests that theymight generate current under steady-state conditions.This is commonly referred to as a window current and is

operationally defined as the overlap region between theactivation and steady-state inactivation curves (Fig. 7, G

and H). All three Cav3 channels are predicted to generatewindow currents supported by �1% of the channels. Re-cording conditions and methods of analysis have a dra-matic effect on estimates of the window current, so theseestimates must be interpreted with caution. For example,analysis of Cav3.3 results obtained in Xenopus oocytesindicates that up to 2% of the channels can be open in thesteady-state. Evidence for window currents have alsobeen obtained in thalamacortical neurons, where theyplay an important role in signal amplification (441). Win-dow currents also appear to be important in determiningintracellular Ca2� concentrations (18, 69, 264). Expres-

FIG. 7. Electrophysiological properties of recombinant Cav3 channels. Recombinant channels were expressed inhuman embryonic kidney cells (293) and recorded with the ruptured patch method using 5 mM Ca2� as the chargecarrier. A: the current-voltage (I-V) relationship is typically measured using a series of step depolarizations where the testpotential (�80 to �10 mV) is increased 10 mV between runs. After each run the cell is held at �100 mV for at least 10 sto allow recovery. Recordings were made using a stably transfected cell line expressing Cav3.2. B: I-V curve is a plot ofthe current as a function of test potential. In this example the currents for each cell were normalized to the peak currentobserved, and then averaged. Typical I-V relations are shown for human Cav3.1 (‚), 3.2 (ƒ), and 3.3 (E; same symbols forall panels). The data were fit with an equation: G � Gmax (Vm � Vrev)/[1 � exp(V1/2 � Vm)/k], where G is conductance,Vm is the test potential, Vrev is the apparent reversal potential, V1/2 is the midpoint of activation, and k is the slope factor.C: tail currents are measured with a two-step protocol where the first step is designed to open channels (e.g., 2 ms to�60 mV), and the second step measures how fast they close at different repolarization potentials. Traces were obtainedwith Cav3.1 and Cav3.3 after repolarization to �90 mV. Deactivation kinetics are measured by fitting tail currents withexponential functions. D: deactivation kinetics vary with the repolarization potential. E: recovery from inactivation canbe measured using a three-step protocol where step 1 is a long depolarizing pulse used to inactivate channels, step 2 isa repolarizing step to voltages where channels recover, and step 3 is a test pulse to measure the fraction of channels thatrecovered. The duration of step 2 is increased in subsequent runs. Recovery is defined as the ratio of the peak currentin step 3 divided by the current in step 1. F: recovery time courses of the three human Cav3 channels. Data were fit withan exponential to calculate the rate of recovery. Cav3.1 channels recover fastest with a � of �100 ms, Cav3.3 areintermediate (250–300 ms), while Cav3.2 recover slowest (�400 ms). G: steady-state inactivation curves of the threehuman Cav3 channels estimated with 15-s prepulses. Smooth curves represent fits to the data with a Boltzmann equation.The foot of the activation curve is also shown. There is a small voltage range where these two curves overlap, leadingto the prediction that channels can produce steady-state (window) currents. H: fraction of channels open in thesteady-state can be estimated by multiplying the Boltzmann functions that describe activation and inactivation.



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sion of recombinant Cav3.1 or Cav3.2 was found to in-crease basal Ca2� in HEK-293 cells, and this increase wasblocked by appropriate concentrations of either mibe-fradil or nickel. Similarly, basal Ca2� is increased afterdifferentiation of the human prostatic cancer cell lineLNCaP, and this correlates with the expression of Cav3.2mRNA and currents (264). Hormonal regulation of theT-type window current provides cells with another mech-anism to regulate intracellular Ca2�. In adrenal glomeru-losa cells, ANG II increases T-type window currents byselectively shifting activation to more negative voltages(273, 443), an effect mediated by CaMKII phosphorylation(discussed in sect. IIID).

Another distinguishing property of T-type channels isthat they have a lower Ba2� conductance than HVA chan-nels. Therefore, studies of cloned T-type channels havefocused on the amplitude of single-channel currents inisotonic barium solutions. All three recombinant channelswere found to have small single-channel currents, corre-sponding to slope conductances in the 7–11 pS range(Table 8). In contrast, Cav1.2 channels display slope con-ductances of 20–30 pS, while Cav2 channels are in the13–16 pS range. Measurement of the single-channel am-plitude is complicated by the presence of subconductanceactivity (228). Failure to account for these subconduc-tance openings would result in lower estimates, and thismay explain why Cav3.2 was reported to have a conduc-tance of 5.3 pS (90). Subconductance activity has alsobeen observed with native T-type channels (64).

The effect of changing external pH has been studiedwith Cav3.1 (391) and Cav3.2 (95). In contrast to nativechannels, whose activity is affected by small deviationsfrom pH 7.2, the recombinant channels are largely unaf-fected by pH changes in the 8.2 to 6.9 range. Furtheracidification of the external solution to pH 5.5 decreasesthe currents measured during standard test pulses. Muta-tion of the aspartate in the dmain III pore loop to gluta-mate (EEDD to EEED) shifted the apparent pKa of Cav3.1from 6.0 to 7.0 (391). In addition,this mutation increasedthe sensitivity to Cd2� block. Therefore, each domaincontributes a negatively charged residue to form a high-affinity binding site for Ca2� (and Cd2�) in Cav3 channels(EEDD) as shown previously for Cav1 and Cav2 (bothEEEE) channels (75, 207, 319, 449). In addition to affect-ing permeation, shifting the pH from 8.2 to 5.5 produced adramatic 40-mV shift in the voltage dependence of activa-tion of Cav3.2, lowered its slope factor, and produced an11-mV shift in the h� curve (95). These results suggestedthat protonation of the channel affected the voltage de-pendence of transitions between deep closed states (R toC3 transitions in Fig. 3). Although it is unknown where onthe channel the affected residues lie, they may very wellbe in the pore. Consistent with this hypothesis, mutationsin the Cav3.1 pore also lowered its voltage dependenceand shifted activation to more positive potentials (391).

Studies using mock action potentials suggest thatmost of the Ca2� influx through T-type channels occursafter the peak voltage and corresponds to the tail current(218, 276, 334). This Ca2� influx might play a role in spikerepolarization and afterhyperpolarization mediated byapamin-sensitive Ca2�-dependent K� channels (440). Dueto differences in their rates of activation and inactivation,the three T-type channels respond quite differently totrains of mock action potentials (218). Both Cav3.1 andCav3.2 channels inactivate rapidly during trains deliveredat �20 Hz. In contrast, Cav3.3 continues to gate during100-Hz train frequencies. These differences suggest thatthe three channels play distinct roles in determining neu-ronal firing patterns and signal integration. Cav3.1 andCav3.2 channels are likely involved in short burst firing asseen in thalamacortical neurons, whereas Cav3.3 channelsare likely involved in sustained burst firing as seen inthalamic reticular neurons (70).

E. Auxiliary Subunits?

Auxiliary subunits of HVA Ca2� channels were firstidentified in purified preparations of skeletal musclechannels (see Ref. 326 for review). This channel complexwas found to contain at least four subunits: �1 (Cav1.1),�2� (�2�1), � (�1), and � (�1). Purified cardiac L-type andbrain N-type channels have a similar �1�2�� structure, butwith distinct �1- and �-subtypes. Additional members ofeach subunit class have been cloned (Fig. 4), such thatthere are now seven HVA �1-, four �-, three �2�-, and eight�-like subunits (reviewed in Ref. 171). The �-subunits playa critical role in channel function, controlling both chan-nel expression at the plasma membrane, voltage depen-dence, and pharmacology. The �2�- and �-subunits alsoplay a role in expression of functional channels. Muta-tions in Ca2� channel genes, or calcium channelopathies,have been linked to a variety of disease phenotypes inboth mice and humans (reviewed in Ref. 248).

Native T-type channels have yet to be purified, sotheir subunit structure is unknown. Two strategies havebeen used to infer its structure: to assay whether anti-sense oligonucleotides directed against HVA �-subunitscan alter native T-type channel function, and to testwhether the activity of the cloned T-type �1-subunit isaffected by coexpression with cloned HVA subunits. An-tisense depletion of �-subunits causes profound reduc-tions in HVA channel density; however, this treatment hasno effect on T-type channels recorded from the sameneurons (226, 234). Similarly, coexpression of cloned�-subunits has little or no effect on cloned T-type channelactivity (100). In contrast, coexpression with either �2�1

or �2�2 was found to double the size of Cav3.1-mediatedcurrents (100, 132). Concomitant immunolocalizationstudies supported the contention that this rise in currents



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was due to increased expression of �1-�2�1 complexes atthe plasma membrane (100). Similar results were ob-tained by coexpression of a �2�2 with mouse Cav3.1 (169),although the effects of �2�1 failed to reach statisticalsignificance (224). Coexpression with �2�1 causes no de-tectable changes in the biophysical properties of Cav3.1channels, while �2�2 has been reported to have minoreffects on inactivation (169). Mutations in the murine �2�2

gene have been linked to an absence of epilepsy pheno-type in the mouse strain ducky (23). Recordings fromPurkinje neurons isolated from ducky cerebella showedhalf the P-type current density observed in controls.These results, in combination with coexpression studies,suggest that �2�2 plays a greater role in HVA than LVAchannel expression.

Identification of the mutation in the mouse strainstargazer, which also exhibits an absence epilepsy phe-notype, led to the cloning of �2 (233). In silico cloning hasextended the �-like family to eight members (78). The�2-protein is only 25% identical to skeletal muscle � andmay play different physiological roles. Immunoprecipita-tion studies have shown that �2 may be a subunit of bothHVA Cav2.2 Ca2� channels and glutamate receptors of theAMPA class (74, 190, 366). Coexpression of �2 inhibits theexpression of Cav2.1 and Cav2.2 channels in the presenceof �2�1 and has minor effects on their biophysical prop-erties (190, 346). In contrast, �2 had little effect on Cav3.3expression, although it could slow the decay of the tailcurrent (145). The related isoforms, �3 and �4, have beenreported to have no effect on Cav3.3 currents (145). Incontrast, �2, �4, and �5 have been reported to accelerateinactivation of Cav3.1 currents and shift steady-state in-activation by �4 mV (210). To reiterate, the electrophys-iological activity of T-type �1-subunits expressed alone isvery similar to that observed with native channels. This isnot the case for HVA channels, which require �-subunitsfor proper gating (223), and �2�1- and �1-subunits forproper pharmacological activity (381). In conclusion,these studies indicate that HVA auxiliary subunits caninteract with Cav3 �1-subunits, but additional biochemicalstudies are required to establish this interaction in vivo.

V. PHARMACOLOGY

Cardiovascular L-type calcium channels are an estab-lished drug target in the control of blood pressure andcardiac rhythm. Selective Cav1 blockers have been devel-oped from over three drug classes, including dihydropy-ridines, phenylalkylamines, and benzothiazepines. In con-trast, there are no highly selective drugs or peptide toxins(369) that selectively block Cav3 channels. A comprehen-sive review of drugs tested on native T-type channels wasrecently published (158); therefore, this review focuseson drugs whose mechanism may involve block of T-typechannels.

A. Antihypertensives

Marketing of mibefradil (Posicor, Roche) as the firstselective T-type channel blocker stimulated considerableinterest in the pharmacology and physiology of thesechannels (412). Mibefradil was approved for use in essen-tial hypertension and stable angina pectoris until it waswithdrawn from the market due to drug-drug interactionsleading to irregular heart rhythms (219). Submicromolarconcentrations of mibefradil, formerly Ro-40–5967, pref-erentially block T-type channels, while higher concentra-tions are required to block cardiovascular L-type channels(282). Its 10- to 30-fold selectivity for T- over L-type chan-nels has been confirmed in a number of preparations,including recombinant channels expressed in mammaliancells (267, 323). The average IC50 from 11 studies formibefradil block of native T-type channels was 1.5 �M(see Table 1 in Ref. 267). Mibefradil potency is affected bythe concentration, and choice, of divalent cation, withless block in Ba2� than in Ca2� solutions (267). In 2 mMCa2� solutions, mibefradil blocked recombinant Cav3.2channels with an IC50 of 0.14 �M, whereas slightly higherconcentrations were required for block of Cav3.1 andCav3.3 (267). Mibefradil block of both HVA and LVA chan-nels is state dependent (41, 278). Single-channel measure-ments indicate the presence of open channel block, and inaddition, a longer lasting block that results in a reductionin active sweeps (280). Whole cell measurements indicatethat inhibition is enhanced by holding the membrane atpotentials where T-type channels inactivate, suggestingthe drug has higher affinity for inactivated states. Theapparent affinity for these states was estimated to be 83nM using native T-type currents (278), and similar resultswere obtained with recombinant channels (267). This pro-vides another mechanism for selectivity, since T-typechannels inactivate near the resting membrane potentialof most cells, while HVA channels require depolarization.A similar mechanism has been invoked to explain theselectivity of dihydropyridines for L-type channels in de-polarized vascular beds over L-type channels in heart.These results suggest that mibefradil selectivity in vivowas probably greater than estimated in vitro since moststudies used Ba2� solutions, and a significant fraction ofT-type channels is inactivated in vivo. Therapeutic plasmaconcentrations of mibefradil were found to be in the 0.5–1�M range, with 99.5% bound to plasma proteins such as�1-acid glycoprotein (433). Therefore, a significant frac-tion of T-type channels in vascular smooth muscle myo-cytes would have been blocked during treatment. Subse-quent studies have established that micromolarconcentrations of mibefradil are capable of blocking awide range of ion channels, including those for Na� (106),K� (138), and Cl� (304). It remains to be determinedwhether block of these other channels contributed to itsantihypertensive activity. This ability to block other ion



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channels complicates the interpretation of mibefradil ef-fects, and additional studies are required to establish therole of T-type channels in smooth muscle proliferation (355),cardiac remodeling (113, 422), and renal protection (28).

Akaike et al. (4) have extensively studied the sensi-tivity of neuronal T-type currents to calcium channelblockers. These studies demonstrated that many drugsblocked neuronal T-type currents at micromolar concen-trations. The order of potency (IC50 in parentheses, in�M) was flunarizine (0.7) � nicardipine (3.5) � nifedipine(5) � nimodipine (7) � methoxyverapamil (50) and dilti-azem (70). A similar rank order was found in a study ofseptal neurons, except nicardipine was found to be morepotent (IC50 � 0. 77 �M; Ref. 6). Because early studiesusing 0.1–1 �M dihydropyridines had shown selectiveblock of L-type channels with little block of T-type chan-nels, these authors concluded that there was a subset ofdihydropyridine-sensitive T-type channels. A similar con-clusion was reached from a study of thalamic and mes-enteric SMC T-type currents (246, 398). Felodipine is alsoa potent T-type blocker, displaying an apparent affinity forinactivated channels of atrial myocytes of 13 nM, but only700 nM for those of pituitary GH3 cells (83). Such high-affinity block led these authors to conclude that block ofT-type channels might contribute to felodipine’s antihy-pertensive properties. Amlodipine (Norvasc, Pfizer),which is currently the most widely prescribed dihydro-pyridine, is �12-fold selective for cardiac L-type channels(IC50 � 0.5 �M), blocking T-type channels with an appar-ent IC50 of 5.7 �M (323). Recombinant Cav3.2 channelswere slightly less sensitive (IC50 � 31 �M). Because am-lodipine serum concentrations are �14 nM (432), at least3% of the T-type channels will be blocked during therapy,and this fraction might be higher in depolarized cells.

The dihydropyridine structure-activity relationshipsof T- and L-type channels are different. This is mostclearly demonstrated by the stereoisomers of BAY K 8644;T-type channels are weakly blocked by micromolar con-centrations of both (237), while for L-type channels the(�)-isomer is an agonist (198). Stimulation of LVA cur-rents by BAY K 8644 is difficult to interpret because thiscompound shifts gating of L-type channels to more nega-tive potentials. In contrast, the stereoselectivity and po-tency of niguldipine were similar for both L- and T-typechannels; the racemic mixture blocked T-type currents inguinea pig atrial myocytes with an IC50 of 0.18 �M (341).Although the binding site for dihydropyridines is differentbetween L- and T-type channels, the mechanism of blockappears similar. Dihydropyridines appear to stabilize in-activated states of both channels, causing a negative shiftin the steady-state availability curve (31, 83, 221, 336). Inconclusion, dihydropyridines are selective for L-typechannels, and some analogs are capable of blocking T-type channels in the 1–10 �M range.

B. Antiepileptics

Generalized, or petit mal, absence epilepsies arecharacterized by periods of synchronized neuronal activ-ity, generating a 3- to 4-Hz spike-wave pattern on theelectroencephalogram. Considerable evidence suggeststhat spike-wave discharges are produced by synchronizedoscillations of cortical, reticular thalamic, and corticotha-lamic neurons (92, 136, 378). Spike-wave discharges ofcorticothalamic neurons are in part mediated by T-typechannels that produce LTS (93, 277, 377, 387). Ethosux-imide is a first-line drug in the treatment of absenceepilepsy (193). In vitro studies found that ethosuximidecan block T-type channels with little effect of HVA chan-nels in thalamic neurons (86). Because block occurred attherapeutically relevant concentrations (0.3 mM), it wasconcluded that T-type channel block might explain itsmechanism of action. Support for this hypothesis camefrom the following studies with related analogs: 1) T-typecurrent block was also observed at therapeutically rele-vant concentrations of methyl-phenylsuccinimide (MPS),the active metabolite of the antiepileptic drug methsux-imide, and 2) no block was observed using either theinactive analog succinimide or the convulsant analogtetra-methylsuccinimide (87). With one exception (215),most studies have failed to confirm ethosuximide block ofT-type currents at therapeutically relevant concentrations(403), leading to the suggestion that block of other ionicchannels may be more relevant (232).

Ethosuximide and MPS are capable of blocking allthree recombinant T-type Ca2� channels (137). As ob-served with mibefradil, block is enhanced up to 10-fold byholding the membrane at potentials that partially inacti-vate T-type channels. In addition, ethosuximide hasgreater affinity for open than rested channels, suggestingthat it is both a gating modifier and an open channelblocker. The effect of ethosuximide on other recombinantCa2� channels has not been rigorously tested, althoughblock of Cav2.3 (IC50 � 20 mM) has been reported (295).In particular, its affinity for partially inactivated HVAchannels has not been studied, so its selectivity is un-known. Studies with MPS suggest that it is only abouttwofold selective for LVA over HVA channels in thalamicneurons (87). The affinity (Ki) of ethosuximide for inac-tivated states was estimated to be 2 mM. Because thera-peutic plasma concentrations are close to 0.5 mM (57),only a fraction of channels is expected to be inhibitedduring therapy. This partial block may be relevant due to“pharmacological amplification” (297). This is due to therole of thalamic T-type channels in gating Na� channels,such that nominal block of the T-type channel is sufficientto block all-or-none action potentials (178).

T-type channels are also blocked by antiepilepticsthat are thought to work by blocking Na� channels. Per-haps the best studied is phenytoin, which clearly blocks



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both channels at therapeutic concentrations (413). Phe-nytoin appears to be selective for LVA over HVA currents(413). The mechanisms of block are also similar, withhigher affinity for inactivated states as evidenced by itsability to shift the h� curve (413). From this shift it can becalculated that phenytoin blocks inactivated channelswith a Ki of 7 �M. Studies using recombinant T-typechannels indicated that Cav3.2 was the most sensitive,although block was variable even in a clonal cell line,suggesting that additional factors may be important forblock (404). Zonisamide has also been reported to par-tially block T-type channels (60% max, IC50 �70 �M) withlittle effect on HVA currents (386).

Additional support for the hypothesis that an in-crease in T-type currents might underlie epilepsy comesfrom animal models. One extensively studied model is thegenetic absence epilepsy rats from Strasbourg (GAERS;Ref. 419), whose T-type currents were larger than thoserecorded from a seizure-free rat strain (409). Consistentwith the enhanced currents, enhanced expression ofCav3.2 was detected in the reticular thalamic nuclei, al-though the changes in current density (50%) were largerthan the changes in channel mRNA (20%; Ref. 394). Inaddition, a small increase in Cav3.1 expression in theventrobasal nucleus of thalamus was detected at the mes-sage level, but not with currents (151). Another interest-ing result of this study was that T-type channel mRNA wasvery similar, and in fact rose slightly, between juvenile (15days old) and adult (�40 days old) rats. Enhanced expres-sion of Cav3.2 mRNA was noted at both time points.Because the absence epilepsy phenotype of these animalsis not manifested until after 30 days, these data suggestthat maturation of the thalamocortical circuit plays amore important role in determining the onset than theintrinsic properties of these neurons (419, 431). Increasedexpression of T-type currents has also been found inhippocampal CA1 pyramidal neurons in three differentmodels: a kindling model of focal epilepsy (112), a pilo-carpine model of temporal lobe epilepsy (380), and an invitro model (301). These currents, as well as intrinsicbursting of these neurons, could be blocked by 100 �MNi2� (380), suggesting the involvement of Cav3.2 channels(230).

C. Anesthetics

Although the precise mechanism of action of generalanesthetics is unknown, many studies have demonstratedthat they are capable of modulating the activity of ionchannels, including ligand-gated GABA and glycine chan-nels. A newly described mechanism for inhalation anes-thetics is that they hyperpolarize neurons by potentiatingthe activity of leak K� currents carried by the two-poredomain channels such as TASK-1 (371). Isoflurane, halo-

thane, and nitrous oxide block DRG T-type currents attherapeutically relevant concentrations of anesthetics(401, 403). Isoflurane block of either DRG or recombinantCav3.1 occurs at concentrations (271–303 �M) that arelower than those achieved during anesthesia (404). Blockis not totally selective, as block of HVA currents by halo-thane occurs at similar millimolar concentrations (160,390). In contrast, nitrous oxide selectively blocked DRGLVA currents with no effect on HVA currents (401). Inaddition, nitrous oxide was found to be a selectiveblocker of Cav3.2 currents, with little effect on Cav3.1currents.

Octanol (20 �M), an aliphatic alcohol, was reportedto block LVA currents totally from inferior olivary neu-rons (245). Subsequent studies required much higher con-centrations for block (IC50 �200 �M; reviewed in Ref.158). Recombinant T-type channels can be totally blockedby octanol, with 50% inhibition occurring at 160 �M forCav3.1 and 219 �M for Cav3.2 (404). Studies on hippocam-pal CA1 pyramidal neurons indicated that octanol is non-selective, blocking T-, N-, and L-type channels to a similarextent. In conclusion, T-type currents are blocked at ther-apeutically relevant concentrations of some anesthetics,and this block may contribute to their anesthetic andanalgesic properties.

D. Antipsychotics

Antipsychotics are used in the treatment of psychoticdisorders such as schizophrenia, the main phase of manic-depressive illness, and other acute idiopathic psychoticillnesses (20). Most antipsychotics (neuroleptics) act byblocking D2 class dopaminergic receptors, although drugsof diphenylbutylpiperidine class can also block T-typechannels.

Among diphenylbutylpiperidines, penfluridol was themost effective at blocking T-type currents of human med-ullary thyroid cancer (TT) cells (108). Penfluridol blockedwith an IC50 of 224 nM, and similar results were obtainedwith adrenal zona fasciculata cells (109). It was also�10-fold more selective for LVA currents over HVA cur-rents as 500 nM penfluridol inhibited 82% of the LVAcurrent but only 20% of the HVA. Block of other channelshas not been studied in detail, although it should be notedthat these drugs are also potent blockers of K� channels(139) and bind with subnanomolar affinity to L-type chan-nels (208). Despite strikingly different structures, thiorid-azine, clozapine, and haloperidol have comparable activ-ity in TT cells, but are still much less active thanfluspiriline or penfluridol (108). Similar results have beenobtained using recombinant rat Cav3 channels, althoughthe apparent affinity was higher than observed with nativechannels (349). Cav3.1 channels were blocked with thefollowing order of potency (IC50 in parentheses, in nM):



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pimozide (43) � penfluridol (110) � flunarizine (530) �haloperidol (1,163). Similar results were obtained withCav3.2 and Cav3.3, although flunarizine block of Cav3.2required sevenfold higher concentrations. These com-pounds all shifted the steady-state inactivation curve tomore negative potentials, indicating that they bind pref-erentially to the inactivated state. Use-dependent blockhas been noted previously in studies of native channels,suggesting open channels may also have a higher affinitythan rested channels (15, 108). Pimozide binding to D2

receptors occurs over the same concentration range(KD � 29 nM) as block of the recombinant Cav3 channels,suggesting that T-type channels may be blocked duringtherapy (349).

VI. CONCLUSIONS

A. Physiological Roles

The expression of T-type channels in diverse celltypes suggests that they play a role in various physiolog-ical functions. The voltage dependence of activation andinactivation provides clues to these roles and places con-straints on when these channels will be active. In neuronswhere the resting membrane potential is in the �90 to�70 mV range, T-type channels can play a secondarypacemaker role; an excitatory postsynaptic potential(EPSP) opens T-type channels and generates a LTS,which in turn activates Na�-dependent action potentialsand HVA Ca2� channels. Therefore, T-type channels playan important role in the genesis of burst firing. In neuronswhere the resting membrane potential is relatively depo-larized (greater than �70 mV), T-type channels are inac-tivated and an EPSP directly activates Na� channels. Dueto their fast recovery from inactivation, T-type channelscan produce a rebound burst in depolarized neurons fol-lowing an IPSP. Electrophysiological recordings indicatethat these channels are preferentially localized to den-drites, and hence play a role in signal amplification (97,256, 265, 328). Large T-type currents have been found inthalamic neurons, where they play an important role inoscillatory behavior. The thalamus acts as a gateway tothe cerebral cortex, and inappropriate oscillations ofthese circuits, or thalamocortical dysrhythmias, havebeen implicated in a wide range of neurological disorders(242). T-type currents also appear to play a role in olfac-tion, vision, and pain reception (201, 317, 402).

Calcium influx not only depolarizes the plasma mem-brane but also acts as a second messenger, leading to theactivation of a plethora of enzyme and channel activities.T-type channels can cause robust increases in intracellu-lar Ca2�, especially in proximal dendrites (294, 460). Cal-cium and voltage synergistically open Ca2�-activated K�

channels, which contribute to spike repolarization and

afterhyperpolarizations (40, 244, 416). In addition to tran-sient increases in intracellular Ca2�, T-type window cur-rents may play an important role in slower increases inbasal Ca2�. This property appears to be important forhormone secretion from adrenal cortex and pituitary,myoblast fusion (45), and possibly smooth muscle con-traction.

B. Future Directions

Cloning of the T-type channel �1-subunits has pro-vided tools with which to study the distribution of thesechannels. As expected, mRNA for these channels wasfound to be distributed throughout the central nervoussystem; however, it was very surprising to find expressionin organs such as liver and kidney. Future studies arerequired to discern their role in these tissues. Such stud-ies would be aided by the development of high-affinityantibodies. Antibodies would also be useful in studyingthe distribution of these channels within neurons and intheir purification. HVA channels are multisubunit com-plexes; therefore, it seems likely that T-type channels willalso have auxiliary subunits. These hypothetical subunitsmay regulate surface expression or might be involved inhormonal regulation of channel activity.

Because mutations in many ion channel genes havebeen linked to inherited diseases, it seems likely thatmutations in T-type channel genes would lead to a phe-notype. Mutations that lead to enhanced expression ofchannels, or that reduce inactivation, might tip the bal-ance to overexcitability. Enhanced expression of T-typechannels have been observed in animal models of epi-lepsy (409), neuronal injury (79), cardiac hypertrophy(308), and heart failure (362). The opposite approach isalso being tested by creating transgenic mice that lackexpression of Cav3 genes. Studies with the Cav3.1 knock-out mouse have established the central role this isoformplays in generating burst firing and thalamic spike-wavedischarges (206). What will be the phenotype of otherCav3 knock-out mice?

Somewhat surprisingly the transcriptional regulationof Ca2� channel subunits has received scant attention.Preliminary studies indicate that the Cav3.1 promoter ismethylated in some tumors, which presumably leads todecreased expression (408). It has been noted that theCav3.2 gene contains a region with high homology to theconsensus sequence of neural restrictive silencer ele-ments, providing another avenue for future research(342). Transcriptional regulation also appears to be im-portant for hormonal upregulation of T channels (22, 447).Studies on gene regulation may also provide insights intothe control of T-channel expression during the cell cycle(220).

The pharmaceutical industry is using combinatorial



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libraries and high-throughput screening to search for newdrugs, and hopefully one of these will be a highly selectiveT-type channel blocker. Such a blocker might be useful inthe control of blood pressure, as evidenced by the utilityof mibefradil. If the drug penetrates the central nervoussystem, it might be useful in a variety of disorders thatinvolve thalamocortical dysrhythmias, such as epilepsyand neuropathic pain. Clearly, a highly selective blockerwould also be extremely useful in research and wouldlead to a greater understanding of the physiological rolesof voltage-gated Ca2� channels.

I thank my current and former fellows Drs. Jung-Ha Lee,Juan Carlos Gomora, and Janet Murbartian for help with record-ings of recombinant channels and proofreading. I thank JenniferDeLisle for countless trips to the library to photocopy articles. Ithank Drs. John Huguenard, David McCormick, Thierry Bal,Hee-Sup Shin, Andy Randall, and Chris Benham for permissionto use figures. I also acknowledge the valuable critiques andsuggestions provided by my collaborator Dr. Paula Barrett.

This work was supported in part by National Institutes ofHealth Grants HL-58728 and NS-38691 and an Established Inves-tigator Award from the American Heart Association.

Address for reprint requests and other correspondence: E.Perez-Reyes, Associate Professor of Pharmacology, Univ. ofVirginia, 1300 Jefferson Park Ave., Charlottesville, VA 22908-0735 (E-mail: [email protected]).

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doi:10.1152/physrev.00018.2002 83:117-161, 2003.Physiol RevEdward Perez-ReyesT-type Calcium ChannelsMolecular Physiology of Low-Voltage-Activated

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website at http://www.the-aps.org/.MD 20814-3991. Copyright © 2003 by the American Physiological Society. ISSN: 0031-9333, ESSN: 1522-1210. Visit ourpublished quarterly in January, April, July, and October by the American Physiological Society, 9650 Rockville Pike, Bethesda

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