PIP2-dependent inhibition of M-type (Kv7.2/7.3) potassium channels: direct on-line assessment of...

INVITED REVIEW

PIP2-dependent inhibition of M-type (Kv7.2/7.3) potassiumchannels: direct on-line assessment of PIP2 depletionby Gq-coupled receptors in single living neurons

Simon Hughes & Stephen J. Marsh & Andrew Tinker &

David A. Brown

Received: 20 March 2007 /Accepted: 20 March 2007 /Published online: 20 April 2007# Springer-Verlag 2007

Abstract The open state of M(Kv7.2/7.3) potassiumchannels is maintained by membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). They can be closed onstimulating receptors that induce PI(4,5)P2 hydrolysis. Insympathetic neurons, closure induced by stimulating M1-muscarinic acetylcholine receptors (mAChRs) has beenattributed to depletion of PI(4,5)P2, whereas closure bybradykinin B2-receptors (B2-BKRs) appears to result fromformation of IP3 and release of Ca2+, implying that BKRstimulation does not deplete PI(4,5)P2. We have used afluorescently tagged PI(4,5)P2-binding construct, the C-domain of the protein tubby, mutated to increase sensitivityto PI(4,5)P2 changes (tubby-R332H-cYFP), to provide anon-line read-out of PI(4,5)P2 changes in single livingsympathetic neurons after receptor stimulation. We findthat the mAChR agonist, oxotremorine-M (oxo-M), pro-duces a near-complete translocation of tubby-R332H-cYFPinto the cytoplasm, whereas bradykinin (BK) producedabout one third as much translocation. However, transloca-tion by BK was increased to equal that produced by oxo-Mwhen synthesis of PI(4,5)P2 was inhibited by wortmannin.Further, wortmannin ‘rescued’ M-current inhibition by BKafter Ca2+-dependent inhibition was reduced by thapsigar-gin. These results provide the first direct support for theview that BK accelerates PI(4,5)P2 synthesis in theseneurons, and show that the mechanism of BKR-induced

inhibition can be switched from Ca2+ dependent to PI(4,5)P2 dependent when PI(4,5)P2 synthesis is inhibited.

Keywords Phosphatidylinositol-4,5-bisphosphate . Tubby .

Phospholipase-Cδ-PH .Muscarinic receptors . Bradykinin .

M-current . Sympathetic neuron

Introduction

M-channels are a species of low-threshold, non-inactivatingvoltage-gated potassium channels [1] that play a crucial rolein the regulation of neuronal excitability [2, 3]. They arecomposed primarily of a heteromeric assembly of Kv7.2and Kv7.3 (KCNQ2 and KCNQ3) potassium channelsubunits [4, 5]. Mutations in the human KCNQ2 andKCNQ3 genes are responsible for a form of juvenileepilepsy (benign familial neonatal convulsions) [6], some-times with additional effects such as myokymia [7]. Theyare inhibited by stimulating heptahelical receptors thatcouple to Gq (or G11), most notably M1-muscarinicacetylcholine receptors (M1-mAChRs), with a consequen-tial increase in neuronal excitability [2, 3, 8, 9].

Like numerous other ion channels [10, 11], Kv7/M-channels require the presence of membrane phosphatidyl-inositol-4,5-bisphosphate (PI(4,5)P2) to open [12, 13], andhence shut down when membrane PI(4,5)P2 is reduced byactivation of an inositol 5-phosphatase [14] or charge-screened with basic peptides [12, 15]. Thus, it has beenproposed that their inhibition during mAChR activationresults from the depletion of membrane PI(4,5)P2 after itshydrolysis by Gq-activated phospholipase C (PLC) [12,16–19], rather than from the effect of a product of PI(4,5)P2hydrolysis as previously thought (see [3, 9] for reviews).The principal evidence for this is that: (1) recovery frominhibition is slowed or prevented when PI(4,5)P2 synthesis

Pflugers Arch - Eur J Physiol (2007) 455:115–124DOI 10.1007/s00424-007-0259-6

S. Hughes : S. J. Marsh :D. A. Brown (*)Department of Pharmacology, University College London,London WC1E 6BT, UKe-mail: [email protected]

A. TinkerBHF Laboratories, Department of Medicine,University College London,London WC1E 6BT, UK

is inhibited [16]; (2) inhibition is reduced when membrane PI(4,5)P2 is increased by over-expressing phosphatidylinositol-4-kinase (PI4-K) [19] or by activating phosphatidylinositol-4-phosphate-5-kinase (PI4P-5K) [14]; and (3) inhibition isincreased when PI(4,5)P2 is charge-screened [15]. Theseeffects are contrary to those expected if inhibition were toresult from a product of PI(4,5)P2 hydrolysis.

The question then arises; how much PI(4,5)P2 depletiondoes mAChR (or other Gq-coupled receptor) activationproduce? Although a rapid and substantial (75–93%) fall inPI(4,5)P2 after mAChR activation has been detected bybiochemical analysis in cell lines expressing M3 or M1mAChRs [13, 18, 20], the extent of depletion is moredifficult to determine in neurons. In previous experiments,we attempted to estimate this in single dissociated sympa-thetic neurons by observing the membrane-to-cytosoltranslocation of the green fluorescent protein (GFP)-taggedpleckstrin homology (PH)-domain of PLCδ (PLCδ-PH-GFP) [19]; this binds to the polar head groups of PI(4,5)P2in the membrane inner leaflet and then translocates to thecytosol when PI(4,5)P2 is hydrolyzed [21, 22]. mAChRactivation induced a substantial membrane-to-cytosol trans-location of PLCδ-PH-GFP that showed a close correlationwith M-current inhibition in time course and agonistconcentration dependence [19]. However, because PLCδ-PH also binds to cytosolic inositol 1,4,5-trisphosphate (IP3)[23], the product of PI(4,5)P2 hydrolysis, PLCδ-PH-GFPtranslocation only provides a measure of overall PI(4,5)P2hydrolysis, and is not a direct measure of the reduction inmembrane PI(4,5)P2 concentration. We tried to correct forIP3 binding (and hence estimate changes in PI(4,5)P2concentration) using an intracellular IP3 displacementassay; our corrected data suggested a maximal depletionof ∼83% [19]. As an indirect method, however, thisestimate is subject to considerable uncertainty, and impor-tantly, does not make any allowance for changes in the rateof PI(4,5)P2 synthesis during receptor-induced hydrolysis.

This is particularly crucial in relation to the action of thepeptide bradykinin (BK) because M-current inhibition insympathetic neurons by BK has been attributed to therelease of Ca2+ from intracellular stores by IP3 [24–26],rather than to PI(4,5)P2 depletion. This is because, unlikemuscarinic agonists, BK can increase intracellular Ca2+ inthese neurons [24, 26, 27] through tight coupling of theBK2 receptor to the IP3 receptor [27]. Not only can Ca2+

close M-channels [26, 28], it is also thought to limit orprevent PI(4,5)P2 depletion by BK [29], by simultaneouslyincreasing PI(4,5)P2 synthesis [20] (see also [30]). Howev-er, in our previous experiments [19], BK produced as greata translocation of PLCδ-PH-GFP as the muscarinic agonistoxotremorine-M (oxo-M).

In the present experiments, therefore, we have tried tofollow receptor-induced membrane PI(4,5)P2 changes in

single sympathetic neurons more directly using the PI(4,5)P2-binding construct ‘tubby’ [31]. This is a transcriptionfactor that is mutated in the tubby strain of mice showingmaturity-onset obesity [31]. The C-terminus of the nativetubby protein binds to membrane phosphatidyl polyphos-phates (PI(4,5)P2, PI(3,4)P2, and PI(3,4,5)P3) but not toPI(3,5)P2, PI(4)P, or PI. Hence, tubby associates withmembrane polyphosphates, then, upon Gq/PLC-activatedPI(4,5)P2 hydrolysis, dissociates from the membrane andmoves to the nucleus [31]. In contrast to PLCδ-PH-GFP,tubby translocation is not stimulated by products of PI(4,5)P2hydrolysis. Thus, it does not respond to an ionomycin-induced rise in intracellular Ca2+ [31] (cf. [22]) or to proteinkinase C (PKC) stimulation [31]; and it is not displaced fromthe membrane by a high concentration (100 μM) of IP3 [32](cf. [19]). Hence, translocation of tubby provides a good indexof membrane phosphatidylinositol polyphosphate reduction,of which hydrolytic breakdown of PIP2 forms the majorcomponent [33]. For our work, we used a yellow fluorescentprotein (YFP)-tagged tubby C-domain (tubby-cYFP) thatlacks the N-terminal nuclear localization signal [32].

Materials and methods

The tubby domain was amplified using the polymerase chainreaction from full-length tubby cDNA (tubby in pcDNA3,kindly provided by Professor L. Shapiro) and fused in frameusing a KpnI and XhoI digest to the YFP (eYFP-C1 andeYFP-N1 vectors, Clontech) at the C-terminus (tubby-cYFP).For most experiments, we used a version of tubby-cYFP witha point mutation R332H (tubby-R332H-cYFP) [32] (andunpublished). Point mutations were introduced using theQuickChange kit (Stratagene). All constructs were se-quenced to confirm their identity. The GFP-tagged PLCδ-PH domain cDNA construct (PLCδ-PH-GFP) was used asdescribed previously [19]. A cyan fluorescent protein (CFP)-tagged PLCδ-PH cDNA (PLCδ-PH-CFP, used with tubby-cYFP for simultaneous expression and imaging) was kindlyprovided by Professor T. Meyer [21].

Sympathetic neurons were dissociated from superiorcervical ganglia isolated from 17-day-old Sprague–Dawleyrats and cultured in vitro as described previously [19]. Neuronswere transfected with the tagged tubby and/or PLCδ-PHcDNA constructs by intranuclear injection 1–2 days afterplating [19] and were used for recording 1–2 days later.Fluorescence signals were recorded using a Nikon Diaphotinverted microscope equipped with an excitation monochro-mator and Openlab imaging software; images were subjectedto digital deconvolution [19], or using an SP2 Leica laserconfocal microscope. In the latter, tubby-R332H-cYFP wasexcited at 488 nm, and prismatic separation was used tomeasure emission between 520 and 570 nm. PLCδ-PH-CFP

116 Pflugers Arch - Eur J Physiol (2007) 455:115–124

(used in place of GFP to separate emission signals) wasexcited at 458 nm and emission measured between 470 and570 nm. There was no detectable cross-talk between CFP andYFP signals at these emission wavelengths.

Membrane currents were recorded using amphotericin-filled perforated-patch electrodes [19]. Cells were super-fused at 10–15 ml min−1 with an oxygenated solutioncontaining (in mM): NaCl, 136; KCl, 3; CaCl2, 2.5; MgCl2,1.5; Hepes, 5; glucose, 11 ( pH adjusted to 7.4 with NaOH).All experiments were performed at 32°C. M-currentamplitude was calculated by delivering a 1-s hyperpolariz-ing step from −20 to −50 mV at 5-s intervals, fitting thedeactivation tail-current with a bi-exponential curve andextrapolating to zero time [5].

Results

In our first experiments, we used the wild-type tubby-cYFP.This localized strongly to the membrane but (as with M3-

transfected Chinese hamster ovary (CHO) cells [32]) did nottranslocate on adding the mAChR agonist, oxo-M(10 μM). In contrast, this concentration of oxo-M produceda near-maximal translocation of PLCδ-PH-GFP, indicatingsubstantial PI(4,5)P2 hydrolysis [19]. Unlike PLCδ-PH-GFP [19], intracellular IP3 does not promote dissociation oftubby from the membrane [32] (and unpublished); we,therefore, suspected that the wild-type construct bound tootightly to residual membrane polyphosphoinositides toleave the membrane in the absence of this IP3 pull.

Accordingly, we turned our attention to the mutanttubby-R332H-cYFP, which was projected to have a loweraffinity for PI(4,5)P2 and which showed clear translocationin M3-mAChR-transfected HEK293 cells on application ofcarbachol [32] (and unpublished). This showed a substan-tial membrane localization at rest (Fig. 1), although withsome cytosolic and nuclear fluorescence—more so thanwith the PLCδ-PH-CFP construct (see Fig. 1 below and[19]). A slow nuclear penetration (but not concentrative

Fig. 1 Laser confocal images ofa single dissociated sympatheticneuron that had been co-injectedintranuclearly 24 h previouslywith plasmids encoding tubby-R332H-cYFP (a) and PLCδ-PH-CFP (b). Images on the left areglobal projection images of theentire neuron; images on theright are laser confocal slicesthrough the neuron soma. Notethat both YFP and CFP fluores-cence are concentrated in theouter plasma membrane relativeto the cytoplasm but that YFPfluorescence is also present inthe nucleus (but not in thenucleolus)

Pflugers Arch - Eur J Physiol (2007) 455:115–124 117

accumulation) of PLCδ-PH-GFP has been noted previously,and attributed to slow diffusion through nuclear pores [18].Although tubby-R332H-cYFP lacks the nuclear transloca-tion signal, it does retain the DNA-binding domain [31], so,over the 24-h expression time, it might diffuse into thenucleus like the PLCδ-PH construct and become concen-trated therein by DNA binding.

Effect of a mAChR agonist, oxo-M

Figure 2 shows that addition of 10-μM oxo-M nowproduced a clear increase in cytosolic fluorescence in cellsexpressing the mutant tubby, which paralleled the increasein cytosolic PLCδ-PH-CFP fluorescence (cf. [19]). Theonset and peak times of the two signals were closelymatched. However, in this cell (and in some others), therecovery of the tubby signal was slower than that of thePLCδ-PH-CFP signal, with a biphasic time course. Thissuggests that the initial decline in cytoplasmic PLCδ-PH-CFP fluorescence might be hastened by its release fromcytosolic IP3 after the latter’s rapid metabolism rather thansolely reflecting resynthesis of membrane PI(4,5)P2 (see[30] and also [14]). The greater noise of the PLCδ-PH-CFPsignal was due to the fact that, in the absence of a UV laser,we were restricted to a sub-optimal excitation wavelength

to avoid cross-talk with the YFP signal; this limited detailedcomparison of the recovery time courses.

The peak elevation of tubby-R332H-cYFP fluorescencewas rather smaller than that of PLCδ-PH-CFP. In a sampleof 22 neurons transfected with tubby-R332H-cYFP, oxo-Mincreased cytosolic fluorescence by 31.3±4.30% overbackground (ΔF/F0×100%). This compares with an in-crease of 76.5±6.24% in a separate sample of 12 cellstransfected with PLCδ-PH-GFP. This difference probablyreflects the greater cytosolic background fluorescenceobserved with tubby-R332H-cYFP, rather than less disso-ciation from the membrane. Pretreatment with wortmanninin a concentration that inhibits PI4-K [34] (and thereforereduces initial PI(4,5)P2 levels [16]) did not consistently orsignificantly enhance the subsequent membrane-to-cytosoltranslocation of tubby-R332H-cYFP produced by oxo-M asmeasured by the increase in cytosolic fluorescence (seebelow and Fig. 4). This implies that 10-μM oxo-M wassufficient to reduce membrane PI(4,5)P2 levels below thosethat bound a significant amount of tubby-R332H-cYFP.

In the above (and subsequent) experiments, we wereunable to observe the loss of tubby-R332H-cYFP fluores-cence from the membrane directly because the narrowmembrane width did not allow an adequate measurementof membrane fluorescence. However, in one cell, the

Fig. 2 a and b The mAChRagonist, oxo-M (10 μM; bar),produces a simultaneous trans-location of tubby-R332H-cYFPand PLCδ-PH-CFP fluorescencefrom membrane to cytosol in aneuron expressing both con-structs (see Fig. 1). Imagesabove are laser confocal slicesthrough the neuron soma takenbefore, during, and after appli-cation of oxo-M. c The time plotbelow shows the % increase inYFP and CFP fluorescence (ΔF/F0×100%) measured in an ROI(white square) within the cyto-plasm. Filled line, tubby-R332H-cYFP; red squares,PLCδ-PH-CFP. The greater‘noise’ in the PLCδ-PH-CFPplot is caused by the lowerintensity CFP fluorescence, dic-tated by the sub-optimalwavelength


membrane was infolded so that we could record the changein fluorescence in a region of interest (ROI), which in-corporated that area of membrane and compared it with thatin an equivalent-sized ROI in the subjacent cytosol. Asshown in Fig. 3, membrane fluorescence showed a cleardecrease with a time course matching that of the cytosolicincrease. Thus, changes in cytosolic fluorescence appear toprovide a good index of the inverse changes in membranefluorescence.

Effect of BK

We next compared the effects of BK and oxo-M. As previouslyreported [19], BK (100 nM) produced a similar increase incytosolic PLCδ-PH-GFP to that induced by 10-μM oxo-M(Fig. 4a). In contrast, BK produced only about one third ofthe rise in cytosolic tubby-R332H-cYFP fluorescence thatwas produced by oxo-M (11.3±0.87, n=6, compared to31.3±4.3, n=22; Fig. 4a).

This suggests that, as previously hypothesized for thesecells [19, 29], BK does not produce as substantial adepletion of PIP2 as that induced by oxo-M. Because itproduces a similar amount of PIP2 hydrolysis as oxo-M, asjudged from PLCδ-PH-GFP translocation [19], the lesser

PIP2 depletion may result from accelerated PIP2 synthesis,as shown in neuroblastoma cells [30]. To assess thispossibility further, we tested the effect of inhibiting PI(4,5)P2 synthesis with wortmannin (see above) on the BK-induced translocation of tubby. In these experiments, wefirst evoked a response to a maximal concentration (10 μM)of oxo-M, then, after fluorescence recovery, applied 20-μM

Fig. 3 Membrane tubby-R332H-cYFP fluorescence decreases in timewith the rise in cytosolic fluorescence during oxo-M application.Simultaneous records of changes in fluorescence recorded in an ROIover an infolded membrane region (arrow in a; red line in b) and in asize-matched ROI over the adjacent cytosol (black line in b) duringapplication of 10-μM oxo-M (bar). a Images taken through afluorescence microscope using digital deconvolution; b time plots offluorescence changes as in Fig. 2. ROIs are indicated by the whiterectangles over the right-hand image in a

Fig. 4 BK produces less translocation of tubby-R332H than oxo-M,but equal translocation when PIP2 synthesis is inhibited withwortmannin. a Comparative effects of oxo-M and BK on translocationof PLCδ-PH-GFP (left side) and tubby-R332H-cYFP (right side).Time plots of changes in cytosolic fluorescence (ΔF/F0×100%; seeFig. 2) produced by 1-min applications of 10-μM oxo-M and 100-nMBK in two separate neurons. Note that BK produced equaltranslocation of PLCδ-PH but less translocation of tubby-R332H.b Wortmannin promotes full translocation of tubby-R332H-cYFP byboth oxo-M and BK. Records above show time plots of % increase incytosolic fluorescence produced in two tubby-R332H-cYFP express-ing neurons by 1-min applications of 10-μM oxo-M and 100-nM BK(left side) and two 1-min applications of oxo-M (right side). In eachcell, the second application was preceded by 20-min perfusion with20-μM wortmannin (Wmn). Bar charts below show mean peakcytosolic tubby-R332H-cYFP fluorescence (measured from the initialbackground before wortmannin) produced by oxo-M and BK undercontrol conditions (left side; n=9) and after 20-min treatment with20-μM wortmannin (Wmn; n=7; right side). Bars show SEM. Thepeak fluorescence after application of BK was significantly greaterafter wortmannin (P<0.001), whereas that to oxo-M was not (P=0.91). There was no significant difference between responses to BKand oxo-M in the presence of wortmannin (P=0.70). Note: in two outof nine cells, the full protocol illustrated in the upper records could notbe completed, but results pre-wortmannin have been included in themean data


wortmannin for 20 min. We then either applied 100-nM BKor re-applied the oxo-M (Fig. 4b; we could only apply onedose of BK because of desensitization). Wortmannin aloneproduced a slow increase in cytosolic tubby fluorescence,compatible with a partial reduction in membrane PI(4,5)P2levels. Oxo-M applied after wortmannin produced the sameaverage peak cytosolic fluorescence as that observed beforewortmannin. Some cells—as in Fig. 4b—showed anenhanced response to oxo-M, but this was not a consistentobservation. This suggests that oxo-M produced near-complete PI(4,5)P2 depletion in the absence of wortmannin.However, the application of BK after wortmannin produceda much greater cytosolic tubby signal than when applied inthe absence of wortmannin, which now matched (oroccasionally exceeded) that initially produced by oxo-M.Thus, BK is capable of reducing membrane PI(4,5)P2 levelsto the same extent as oxo-M, but only if PI(4,5)P2 synthesisis inhibited.

M-current inhibition by BK after wortmannin

Inhibition of M-current in sympathetic neurons by BK hasbeen attributed to the release of and channel closure by Ca2+

rather than to PIP2 depletion (see “Introduction”). Thus,buffering of intracellular Ca2+ or inhibition of Ca2+ releaseby (for example) Ca2+ store depletion with thapsigargin hasbeen found to reduce or prevent BK-induced inhibition [24,25]. The above experiments support this idea in so far thatBK produced much less translocation of tubby-R332H-cYFP than that produced by oxo-M. However, because amuch greater translocation (and so, by inference, PI(4,5)P2depletion) occurred in the presence of wortmannin, wewondered whether BK-induced M-current inhibition couldthen be forced to switch to a PI(4,5)P2-depletion mecha-nism rather than a Ca2+-dependent mechanism bywortmannin.

To test this, we compared BK-induced M-currentinhibition with that produced by oxo-M in neurons thathad been treated with thapsigargin to suppress or reduce theCa2+-dependent component of BK-induced inhibition. Wethen conducted a series of similar experiments in thapsi-gargin-pretreated neurons subsequently exposed to wort-mannin. Because of desensitization, we limited our tests toa single application of BK. As shown in Fig. 5a, undernormal conditions, 100-nM BK produced nearly as muchM-current inhibition as 10-μM oxo-M (85.4±4.26% and90.9±4.26%, respectively, n=5; see also [19]). As previ-ously reported [24, 25], pretreatment with 5-μM thapsigar-gin for 10 min strongly reduced, and sometimes abolished,the response to BK without affecting that to oxo-M (37.4±10.4% and 81.5±8.18% [n=6] inhibition by BK in thepresence and absence of thapsigargin, respectively;

Fig. 5b). Thapsigargin did not appear to increase BK-induced tubby-R332H-cYFP translocation. Thus, in threetests, the ratios of BK-/oxo-M-induced cytosolic fluores-cence increases after pretreatment with thapsigargin were0.29, 0.27, and 0.24; these were within the range (0.33±0.13 [SD], n=8) recorded in the absence of thapsigargin.We did not measure intracellular Ca2+ depletion bythapsigargin in these experiments, but note that, in previousexperiments on these neurons, 0.1-μM thapsigargin suf-ficed to release Ca2+ from intracellular stores, and to slowrecovery from a Ca2+ load [35]. Addition of 20-μMwortmannin alone produced some inhibition of M-current(as previously noted [16], and in keeping with the slow risein cytosolic tubby-R332H-cYFP fluorescence seen in Fig. 4above). Subsequent addition of BK now produced asubstantial inhibition of M-current, again matching thatproduced by oxo-M (82.2±2.94% and 93.75±6.25% forBK and oxo-M, respectively, n=5; Fig. 5c). In theseexperiments, wortmannin itself tended to slow, but notfully prevent, recovery from BK- or oxo-M-inducedinhibition. When wortmannin was left on for a longer timethan 5 min, it produced a progressive and substantialdecline in M-current and totally prevented recovery fromagonist-induced inhibition, as previously reported [16].

Discussion

The C-terminal tubby construct used in these experimentsoffers the great advantage over the PLCδ-PH construct usedpreviously, in that it does not bind to the products of PI(4,5)P2 hydrolysis; hence, its dissociation from the membraneprovides an index solely of changes in membrane phos-phatidylinositol polyphosphate levels (see “Introduction”).Among membrane phosphoinositides, PI(4)P and PI(4,5)P2show the largest changes after mAChR stimulation [20].Thus, because tubby does not bind to PI(4)P [31],membrane-to-cytosol translocation of tubby-R332H-cYFPafter mAChR stimulation may be reasonably attributed tothe fall in PI(4,5)P2.

Unfortunately, the binding constant for PI(4,5)P2 bindingto tubby-R332H-cYFP is unknown, so membrane PI(4,5)P2changes cannot be calculated directly from the fluorescencesignals. Nevertheless, it seems likely that oxo-M produceda near-complete depletion of membrane PI(4,5)P2, as thecytosolic fluorescence increase produced by oxo-M was notenhanced to any significant degree when PI(4,5)P2 synthe-sis was additionally inhibited with wortmannin. Thus, thepresent results accord qualitatively with our previousestimate of ∼83% depletion by oxo-M in these cells basedon measurements of PLCδ-PH-GFP translocation whencorrected for IP3 binding [19].


In contrast, the cytosolic tubby-R332H-cYFP signalproduced by BK was only about one third of that producedby oxo-M. This is crucially important to the interpretationof how these two agonists inhibit the M-current in theseneurons. Thus, whereas the inhibition produced by oxo-Mcan be quantitatively accounted for by the large reduction inPI(4,5)P2 [19] (see also [17]) because of the hyperbolicrelation between PI(4,5)P2 concentration and M-channelopen probability [12], the lesser depletion by BK may bepredicted [19] to inhibit the M-current by ∼10% (assumingfor the moment a linear relation between the cytosolictubby-R332H-cYFP fluorescence signal and membrane[PI(4,5)P2]). Notwithstanding, BK inhibits the M-currentto almost the same extent as oxo-M.

Previous experiments have suggested that this additionalcomponent of inhibition is due to the release of intracellularCa2+ and consequent Ca2+-/calmodulin-dependent closureof the M-channels [24–27]; and it has been furtherhypothesized that BK simultaneously accelerates the syn-

thesis of PI(4,5)P2 ( possibly through Ca2+-dependentactivation of PI4-K [9, 19, 29]), thereby limiting depletion.The modest depletion of PI(4,5)P2 by BK compared withthat produced by oxo-M that we have observed providesimportant supporting evidence for the hypothesis that BKenhances PI(4,5)P2 synthesis in the cells, as in neuroblas-toma cells [30]. We have not specifically addressed the roleof Ca2+ release by BK in maintaining PI(4,5)P2 levels. Wewould, however, note that because thapsigargin reducedM-current inhibition (see also [24, 26]) without increasingPI(4,5)P2 depletion, if the release of Ca

2+ is responsible foraugmenting PI(4,5)P2 synthesis, then this process must bemore sensitive to Ca2+ than the M-channels.

Addition of wortmannin to inhibit PI(4,5)P2 synthesisled to a much greater depletion of PI(4,5)P2 by BK. Then,when the Ca2+-dependent component of inhibition had beenreduced by depleting the Ca2+ stores with thapsigargin,wortmannin could restore full M-current inhibition by BK.In this way, we essentially switched the mechanism of

Fig. 5 Effects of 10-μM oxo-Mand 100-nM BK on M-currentamplitudes a under control con-ditions, b after pre-incubationwith 5-μM thapsigargin for10 min, and c after thapsigarginpre-treatment and in the pres-ence of 20-μM wortmannin(Wmn, added for 5 min at timezero). M-current amplitude(recorded with perforated-patchelectrodes) was measured every5 s as the extrapolated amplitudeof the deactivation tail-currentobserved on stepping from −20to −50 mV. Time plot of M-current amplitude (1). Repre-sentative M-current deactivationtails recorded in the absence(C = control, black trace) andpresence (BK) of bradykinin (2).Bar charts showing mean %inhibition of M-current mea-sured under these three condi-tions in the number of cellsindicated (3); bars show SEM;asterisk, significant differencebetween oxo-M and BK. Sig-nificance levels (P) for differ-ences between oxo-M and BKare: a, 0.38; b, 0.006; c, 0.28


inhibition from primarily Ca2+ dependent to primarilyPI(4,5)P2 dependent. Thus, as with Ca2+-channel inhibition[29], BK, like other Gq-GPCR agonists, can potentiallyinhibit M-currents by reducing PI(4,5)P2 concentrations butdoes not normally do so in these neurons because it doesnot produce sufficient PI(4,5)P2 depletion. However, boththis and the Ca2+-dependent component of inhibition mightwell vary in extent between different neurons.

A consensus scenario for the inhibition of M-current inthese neurons by mAChR and B2-BKR stimulation thatmight be drawn from this and the preceding work (see“Introduction”) is depicted in Fig. 6. Under normalconditions, channels are maintained in the open state byPI(4,5)P2 (Fig. 6a). mAChR stimulation (Fig. 6b) catalyzesPI(4,5)P2 hydrolysis and thereby depletes PI(4,5)P2 to alevel that cannot sustain channel activity [17–19]. B2-BKRstimulation (Fig. 6c) also catalyzes PI(4,5)P2 hydrolysis to asimilar extent, but in this case, the receptors are closelycoupled to IP3 receptors [27], such that local production ofIP3 induces a release of Ca

2+ [24, 27]. This has two effects:it directly induces M-channel closure [28] by activatingchannel-attached calmodulin [25] (perhaps by reducingchannel affinity for PI(4,5)P2 [9]); and it acceleratesPI(4,5)P2 synthesis (possibly through neuronal calciumsensor (NCS)-1-activated PI4-K stimulation [19, 29]),thereby reducing PI(4,5)P2 depletion to a level, which itselfwould not induce appreciable channel closure. Only whenCa2+ release is reduced and PI4-K is inhibited is thedepletion of PI(4,5)P2 after B2-BKR stimulation sufficientto allow channel closure.

In conclusion, the present experiments provide importantdirect evidence regarding the magnitude of PI(4,5)P2 deple-tion in sympathetic neurons by Gq-GPCR stimulation, whichsupports inferences drawn from previous studies. Further, theresponses of tubby-R332H-cYFP to mAChR-Gq stimulationin sympathetic neurons accord with those previouslyobserved in HEK293 cells [32] (and unpublished), indicatingthat this construct may be well suited for studies on changesin membrane PI(4,5)P2 in many other cell types.

c

b

a

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PI

PI(4)-K

PI4(5)-K X

PIP2K+

PIP

PI

PI(4)-K

PI4(5)-K

Oxo-M

M1-mAChR

PLC

DAG

IP3

X

PIP2K+

PIP

X

PI

PI4(5)-K

BK

B2-BKR

DAG

IP3

CaM

Ca2+

PI(4)-K

Ca2+

Fig. 6 Diagrams to summarize suggested schemes for closure of M-channels by Gq-coupled receptors. a Resting state; b and c, responsesto activation of M1-mAChRs and B2-BKRs, respectively. See textfor details. PI Phosphatidylinositol, PIP phosphatidylinositol-4-phosphate, PIP2 phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2),PI(4)-K phosphatidylinositol-4-kinase, PI4(5)-K phosphatidylinositol-4-phosphate-5-kinase, Oxo-M oxotremorine-M, M1-mAChR M1 mus-carinic acetylcholine receptor (see [36, 37]), Gαq α-subunit of the Gproteins Gq (see [38]), Gαq/11 α-subunit of the G proteins Gq and/orG11 (see [39]), PLC phospholipase-C, PLCβ4 phospholipase-C β4(see [40]), DAG diacylglycerol, IP3 inositol-1,4,5-trisphosphate, BKbradykinin, B2-BKR B2-bradykinin receptor, CaM calmodulin

R


Acknowledgment We thank Professor L. Shapiro (Department ofPhysiology and Biophysics, Mount Sinai School of Medicine, NY10029, USA) for the gift of full-length tubby cDNA and Professor T.Meyer (Department of Molecular Pharmacology, Stanford UniversityMedical School, CA 94305, USA) for the gift of PLCδ-PH-CFP cDNA.This work was supported by a UKMedical Research Council postgraduatestudentship to S.H. and research grants from the Wellcome Trust and theEuropean Union (to D.A.B.) and the British Heart Foundation (to A.T.).

Proof note Pian et al. (this issue) provide further evidence thatbradykinin can increase PI(4,5)P2 synthesis in oocytes and in sino-atrial node cells.

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