Review

A role for membrane potential inregulating GPCRs?Martyn P. Mahaut-Smith1, Juan Martinez-Pinna2 and Iman S. Gurung3

1 Department of Cell Physiology and Pharmacology, University of Leicester, Medical Sciences Building, University Road,

Leicester, LE1 9HN, UK2 Division de Fisiologıa, Universidad de Alicante, Ap. 99, E-03080 Alicante, Spain3 Department of Physiology, Development and Neuroscience, Downing Street, University of Cambridge, Cambridge,

CB2 3EG, UK

G-protein-coupled receptors (GPCRs) have ubiquitousroles in transducing extracellular signals into cellularresponses. Therefore, the concept that members of thissuperfamily of surface proteins are directly modulatedby changes in membrane voltage could have widespreadconsequences for cell signalling. Although several stu-dies have indicated that GPCRs can be voltage depend-ent, particularly P2Y1 receptors in the non-excitablemegakaryocyte, the evidence has been mostly indirect.Recent work on muscarinic receptors has stimulatedsubstantial interest in this field by reporting the firstvoltage-dependent charge movements for a GPCR. Anunderlying mechanism is proposed whereby a voltage-induced conformational change in the receptor alters itsability to couple to the G protein and thereby influencesits affinity for an agonist. We discuss the strength of theevidence behind this hypothesis and include sugges-tions for future work. We also describe other examplesin which direct voltage control of GPCRs can account foreffects of membrane potential on downstream signalsand highlight the possible physiological consequencesof this phenomenon.

IntroductionG-protein-coupled receptors have widespread, fundamen-tal roles in cell signalling. Physiological activation of thissuperfamily of proteins normally results from the bindingof an extracellular agonist to a specific recognition site onthe receptor. The concept that GPCRs are also directlyregulated by the transmembrane potential has beenaround for some time [1–3] but only recently supportedby substantial experimental evidence. To date, most of theevidence has been indirect because it is derived fromassays of downstream signalling events [4–6] (summarizedin Table 1). Therefore the observation of voltage-dependentcharge movements for M1 andM2 G-protein-coupled recep-tors, which correlate with the receptor-activation state,represents amajor advance in this field. Within this articlewe examine the basis of intramembrane charge displace-ments in different membrane proteins and criticallyexamine the evidence for such events for GPCRs. We alsocompare the many examples of voltage dependence ofGPCR signalling, discuss the physiological relevance ofthis phenomenon and suggest directions for future work.

Corresponding author: Mahaut-Smith, M.P. (mpms1@le.ac.uk).

0165-6147/$ – see front matter � 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2008.

Voltage-dependent charge movement: a newobservation for GPCRsIntramembrane chargemovements (see Figure Ia in Box 1)have been reported during activation of a variety of mem-brane proteins. These small, transient currents can resultfrom the rearrangement of polar membrane components,the movement of resident mobile charges part-way acrossthe membrane or the movement of charged species be-tween the nearby solution and the membrane [7,8]. The‘gating currents’ of voltage-operated ion channelsrepresent one such charge displacement event (see FigureIb,c in Box 1 for a further explanation, including themeasurement of voltage-dependent K+ channel gating cur-rents). Charge movements have also been observed duringvoltage-dependent activation of the outer-hair-cell motorprotein prestin [9], ion-coupled co-transporters (where theyare referred to as pre-steady-state currents) [10] and, morerecently, a voltage-sensor-containing phosphatase fromCiona intestinalis (Ci-VSP) [11]. In addition, rhodopsinand cone retinal–opsin complexes display charge displace-ments known as early receptor currents (ERCs) afterstimulation by high-intensity light [12–14]. Figure 1 com-pares the charge movements activated by a series of vol-tage steps for M2 receptors (Figure 1a), prestin (Figure 1b),g-aminobutyric acid (GABA) transporters (Figure 1c) andCi-VSP (Figure 1d), and also after activation of rhodopsinby a flash of light (Figure 1e). Charge displacement eventsare small compared with transmembrane ionic currentsand often have rapid kinetics; therefore, Ben-Chaim et al.[15] used the ‘cut-open’ oocyte preparation to uncovervoltage-dependent charge movements for M1 and M2

GPCRs. This technique enables excellent temporal controlof membrane voltage and substitution of permeant ions onboth sides of the membrane in a large cell capable ofexpressing substantial amounts of heterologous protein[16]. Currents that arise from linear membrane com-ponents (see Figure Ic in Box 1) were subtracted usingvoltage steps over a range of potentials that is predicted tonot regulate the protein of interest (Ben-Chaim et al. [15]selected depolarizations from +40 mV). Evidence that theremaining currents relate to voltage-dependent chargemovement associated with muscarinic receptors includestheir lack of dependence on permeant ions and absence inwater-injected oocytes. In addition, a nonfunctionalmutated M2 receptor [double mutation in the highly con-served Asp(Glu)-Arg-Tyr (D(E)RY) motif of group I GPCRs;

05.007 Available online 11 July 2008 421

Table 1. G-protein-coupled receptors, heterotrimeric G proteins or Ins(1,4,5)P3-dependent Ca2+ release reported to be directlycontrolled by membrane voltage

Tissue GPCR Voltage-dependent

effect

Polarity Proposed voltage

sensor

Evidence Refs

Xenopus oocyte

(heterologous

expression)

M2 Ligand binding and

GIRK channel

activation

Depolarization

reduces

Receptor Gating currents [6,15]

Receptor chimeras

Ligand binding

Xenopus oocyte

(heterologous

expression)

M1 Ligand binding and

Ca2+ release

Depolarization

enhances

Receptor Gating current [6,15]

Receptor chimeras

Ligand binding

Xenopus oocyte

(heterologous

expression)

D2a GIRK channel

activation

Depolarization

reduces

Receptor Comparison to work of Parnas and

colleagues

[64]

Megakaryocyte P2Y1, TPa,

5HT2A

Ins(1,4,5)P3-

dependent Ca2+

mobilization

Bipolar:

Depolarization

enhances

Receptor P2Y1-deficient cells and

pharmacological modulators

[4,54]

Xenopus oocyte (native

receptor)

LPA Ins(1,4,5)P3-

dependent Ca2+

mobilization

Depolarization

enhances

Not studied [65]

Cerebellar granule

neurons

M3 Ins(1,4,5)P3

production and Ca2+

mobilization

Bipolar:

Depolarization

enhances

Downstream of

receptor

Effect still observed at high agonist

levels

[43]

SH-SY5Y neuronal cell

line

Cell line (type not

specified)

a2A-

adrenoceptor

Agonist-evoked

receptor

configurational

change (FRET

assay)

Depolarization

reduces

Ligand binding Shift of concentration:response

curve without affecting maximum

response

[21]

Lack of voltage dependence for

uncharged adenosine at A2A

receptors

Coronary-artery smooth

muscle

Muscarinic Ins(1,4,5)P3-

dependent Ca2+

mobilization

Bipolar:

Depolarization

enhances

Downstream of

receptor

GTPgS induces the voltage

dependence

[2]

Pancreatic acinar cell Muscarinic Ins(1,4,5)P3-

dependent Ca2+

mobilization

(oscillations)

Bipolar:

Depolarization

reduces

Unclear, but not

observed at high

agonist

concentrations

[66]

Lacrimal acinar cell Muscarinic Initial Ca2+

mobilization

Bipolar:

Depolarization

reduces

Receptor; possibly

ligand binding

Not observed at high ligand

concentrations

[67]

Rat brain synaptosomes

and cardiac myocytes

Muscarinic Ligand binding,

GTP/GDP binding

Depolarization G-proteins,

requiring voltage-

dependent Na+-

channel activation

Sensitivity to pertussis-toxin,

GDP[b]S and inhibitors of Na+-

channel activation

[68,69]

Visceral smooth muscle Muscarinic Ins(1,4,5)P3

production

Depolarization

enhances

Unclear [49]

Xenopus oocyte

(heterologous

expression)

mGlu1A Ligand binding and

Ca2+ release

Depolarization

enhances

Receptor Receptor and Ga subunit chimeras [70]

Altered ligand binding

Xenopus oocyte

(heterologous

expression)

mGlu3 Ligand binding and

GIRK channel

activation

Depolarization

reduces

Receptor Receptor and Ga subunit chimeras [70]

Altered ligand binding

Skeletal muscle Agonist not

required

Ins(1,4,5)P3

production, slow Ca2+

release and gene

transcription

Depolarization

enhances

Dihydropyridine

receptor

Blocked by nifedipine and pertussis

toxin

[52]

Also expression studies using a1

Ca2+-channel subunits

INS-1 insulin-secreting

cell line

Agonist not

required

Ins(1,4,5)P3

production and Ca2+

release

Depolarization

enhances

Unclear High K+ generates Ins(1,4,5)P3 in

Ca2+-free medium

[71]

Arterial smooth muscle Agonist not

required

Ins(1,4,5)P3-

production and Ca2+

mobilization (via both

Ins(1,4,5)P3R and

CICR)

Depolarization

enhances

Dihydropyridine

receptor

Blocked by nifedipene and PLC

inhibition

[63]

Neurones Multiple Gs-

and

Go-coupled

GPCRs

Voltage-gated Ca2+

channel

Depolarization

relieves GPCR-

evoked inhibition

of channel

Gbg-subunit

binding

to channel

Various molecular experiments

and direct intracellular application

of Gbg

[55]

aAbbreviations: CICR, calcium-induced calcium release; D2, dopamine receptor 2.

Review Trends in Pharmacological Sciences Vol.29 No.8

see later] did not show gating currents. Although thismutant shows lower (one-third) expression compared withthe wild-type receptor, it was predicted that this lowerdensity is not responsible for the lack of gating currents.Nevertheless, an important additional control in futurework is the assessment of voltage-dependent charge move-

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ment for proteins that operate independently of membranepotential at levels of expression comparable to theM1 orM2

wild-type receptor in the study of Ben-Chaim et al. [15].Evidence that the reportedM1- andM2-receptor voltage-

dependent charge movements have a role in protein acti-vation includes a close correlation with receptor affinity

Box 1. The basis of intramembrane charge movements and the measurement of ‘gating’ currents for voltage-gated K+ channels

(a) Schematic representation of events that can generate a charge-

displacement current in response to a voltage change. (i) Rearrange-

ment of polar membrane components across the membrane dielectric

due to a protein configurational change (this is mainly the movement

of charged amino acids, although, theoretically, any polar membrane

component could contribute). (ii) Movement of mobile charges; these

might already be bound to membrane components and move part-

way across the membrane (left) or move between the nearby solution

and the membrane (right). (b) The basis of voltage-dependent charge

movement during activation of voltage-gated K+ channels is reason-

ably well understood. Each K+-channel a-subunit has six transmem-

brane domains (S1–S6). The fourth transmembrane segment contains

multiple arginine or lysine residues, mostly every third residue, and

represents the main voltage-sensing region of the ion channel (the

‘S4 voltage sensor’). (ii) Four a-channel subunits combine to form a

functional channel in which voltage-dependent movement of the

structure formed by the S1–S4 domains ‘gates’ the opening of the

pore and, thus, allows transmembrane K+ currents. The charge

movement arising from movement of the S1–S4 structure generates

the transient gating current. (c) Steps involved in measuring voltage-

dependent charge movement during voltage clamp of a cell highly

expressing a voltage-gated K+ channel. (i) Subtraction of linear

membrane currents. Initially, the capacitive and resistive currents

through the passive membrane components are estimated using low-

amplitude voltage pulses over a range that does not activate voltage-

gated membrane components. The linear currents are scaled and

subtracted from records across the entire voltage range to leave only

the voltage-dependent currents. (ii) Separation of gating currents

from transmembrane K+ currents. K+ channels are then activated

using an appropriate voltage step and the large transmembrane K+

currents abolished by replacement of permeant ions (channel

blockers can also be used provided they do not interfere with gating).

After an increase in gain, small residual transient currents can be

resolved at the start and end of the voltage step that reflect movement

of charges during activation (on) and deactivation (off). Note the small

size of the gating currents compared with capacitive and transmem-

brane currents, thus the need for careful subtraction of ‘linear’

components and complete block of voltage-gated transmembrane

currents. A similar approach can be used to measure voltage-

dependent charge movements through other proteins that do not

form ion channels (Figure 1 in the main text), but care should still be

taken of endogenous ionic currents.

Figure I. Voltage-dependent intramembrane charge displacements and their measurement.

Review Trends in Pharmacological Sciences Vol.29 No.8

state, as judged from activation of a downstream signal[Figure 2a(i)]. The relationship between charge moved andvoltage-step amplitude is fit by the Boltzmann equationthat describes a two-state voltage-dependent process[Figure 2a(i)]. This estimates that depolarization will exert

a half-maximal effect on the muscarinic receptors at�44 mV and a maximal effect at +40 mV. Therefore, therelationship predicts that muscarinic receptors are suitedto detection of physiological changes in membrane poten-tial, such as action potentials. The shallow slope of the

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Figure 1. Comparsion of intramembrane charge movements exhibited by membrane proteins. Charge displacement currents for different membrane proteins activated

either by voltage steps (a-d) or light (e). (a) M2 receptors, (b) prestin, (c) neuronal GABA cotransporters, (d) Ciona intestinalis voltage-sensor-containing phosphatase (Ci-

VSP) and (e) rhodopsin. The proteins were heterologously expressed in Xenopus oocytes (a,c and d) or in human cell lines (b,e). The charge-displacement currents were

evoked by (a) depolarizations from �120 mV to +40 mV in 20-mV increments, (b) depolarizations from �70 mV to +50 mV in 20-mV increments, (c) voltage steps from

�40 mV to voltages ranging from �120 mV to +40 mV, (d) depolarizations from �80 mV to +160 mV in 10-mV increments, and (e) in response to a flash of light (500 nm;

arrow) at a constant holding potential. The inset in (a) shows an expanded trace of the currents for a step from �120 mV to �90 mV. Reproduced, with permission, from Refs

[10,11,15,61,62].

Review Trends in Pharmacological Sciences Vol.29 No.8

Boltzmann fit indicates that M1 and M2 receptors showonly a weak voltage dependence compared with voltage-gated ion channels [8], which is reflected in the low valency(0.85) of the charge moved in the muscarinic receptor [15].This is to be expected given the lack of a highly chargedtransmembrane domain [6,15] comparable with the mainvoltage-sensing sequence of ion channels (the ‘S4’ voltage-sensing domain: see Box 1 and later).

Identifying the voltage sensor within the GPCR cascadeIn their oocyte expression system, Ben-Chaim et al. [6,15]use G-protein-activated inwardly rectifying K+ (GIRK)channels or Ca2+-activated Cl� currents as readouts ofM2 and M1 receptor activation, respectively. When thesedownstream signalling events are stimulated by GTPgS oroverexpression of Gbg they show no voltage dependence,indicating that the main voltage sensor lies at the level ofthe receptor [6,15]. How might GPCR activation be influ-enced by a change inmembrane potential? By analogy withthe ability of depolarization to displace inhibitory extra-cellular Mg2+ bound to NMDA receptors [17], positivelycharged acetylcholine would be driven out of its bindingsite if any direct effect of depolarization was to occur

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directly on ligand binding. This could contribute to theresponse for M2 receptors, where depolarization reducesGIRK-channel activation [Figure 2a(ii) shows that theconcentration–response curve for this channel is shiftedto the right by depolarization]. However, M1-receptor sig-nals display the opposite polarity response to voltage[6,15]. In accordance with this effect, Ben-Chaim et al.[6,15] report that high K+-induced depolarization reducesthe binding of a radiolabelled ligand to M2 and enhancesbinding to M1. They propose that the voltage-dependentconformational change within the receptor alters itscoupling to the G protein, which in turn influences agonistbinding as a consequence of the different agonist affinitiesof the G-protein-coupled and -uncoupled states. Depolar-ization enhances G-protein coupling for M1 receptors, butreduces it for M2 receptors, thereby accounting for theopposite effects on ligand binding.

For voltage-gated ion channels, electrophysiologicalmeasurements of ions flowing through the open pore pro-vide a direct measurement of activation within the ‘gated’protein. However, as in many studies of GPCRs, Ben-Chaim et al. [6,15] assess M1- and M2-receptor activationindirectly using a downstream functional response.

Figure 2. Examples of voltage dependence of GPCRs or their signalling pathways. (a) M2 receptors expressed in Xenopus oocytes. (i) Voltage dependence of charge

movement (filled triangles, unbroken line) and voltage dependence of the proportion of receptors in the low-affinity state (open circles, broken line). The lines are fit to the

Boltzmann equation. (ii) Shift of the concentration–response relationship for ACh-evoked GIRK currents by the holding potential. (b) Depolarization-evoked Ca2+ release in

arterial myocytes in the absence of exogenous agonist under whole-cell patch clamp in Ca2+-free medium [voltage-clamp mode, (i)] or in response to high extracellular K+

[70-mM K+, current-clamp mode, (ii)]. (iii) Relationship between peak Ca2+ release and membrane voltage. (c) Voltage control of P2Y1-receptor-dependent Ca2+ mobilization

in rat megakaryocytes. (i) After initial ADP-evoked Ca2+ oscillations had subsided, incrementing amplitude depolarizing steps were applied. The average peak Ca2+ increase

evoked by each voltage step was obtained from a series of incrementing voltage steps (circles) or from individual steps (triangles) [15,44,51]. Reproduced, with permission,

from Refs [15,44,63]. Vm, membrane potential.

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Furthermore, these responses were interpreted in terms ofa two-state model of receptor affinity because previousstudies have indicated that the receptor occupies eithera low- or a high-affinity state (reviewed in Refs [6,18]). Thefact that charge movement and affinity ‘state’ show over-lapping relationships with a change in membrane poten-

tial [Figure 2a(i)] is currently the main evidence indicatingthat the two events are causally linked. To further test thehypothesis of Ben-Chaim et al. [15], more direct measure-ments of receptor efficacy and affinity are necessary in cellsunder electrophysiological voltage control. Elevated exter-nal K+ can influence GPCRs independent of a change in

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membrane potential [19] and must be avoided as a tool todepolarize cells. Direct measurement of GPCR intrinsicefficacy has been achieved using fluorescence resonanceenergy transfer (FRET) between fluorophores introducedinto the third intracellular loop (L3) and C-terminal tail[20,21]. This monitors the conformational change associ-ated with receptor activation. Combining this approachwith FRET-based studies of ligand binding using an N-terminal tag and a fluorescent ligand [22] could providefurther important information on the relationship betweenvoltage, efficacy and affinity.

Exactly what constitutes the charge displacementevent(s) within M1 and M2 receptors is presently unclear.Although the polarities of the voltage dependence of M1

and M2 receptors can be reversed by transposing their L3s[15], this does not alter the gating currents. The L3 Nterminus of M2 has a string of five charged residues whichis mostly neutral in the equivalent position ofM1, however,changing this sequence in M2 to that of M1 abolishes thevoltage dependence without affecting the gating currents.Thus, although L3 confers the voltage-dependent config-urational change of both M1 and M2 on their G proteins, itis not the voltage sensor. Interestingly, neutralization ofthe two charged amino acids within the highly conservedAsp-Arg-Tyr (DRY) motif abolishes the gating current forM2, and binding studies indicate that the receptor remainsin a high-affinity state when depolarized. However,because this double mutation eliminates activation of Gproteins and GIRK currents and displays reduced expres-sion levels, it is difficult to definitively address the role ofthe DRY motif in voltage sensing. GPCRs lack a regionanalogous to the arginine- and lysine-rich S4 domain of ionchannels or Ci-VSP, where several (at least three) basicamino acid residues are typically separated by two neutralresidues [8,23,24]. Nevertheless, the presence of an ‘S4-likesequence’ should not be considered to be an absoluterequirement for voltage-dependent configurationalchanges leading to protein activation. Amino acids outsidethis region contribute substantially to voltage control ofchannel activation [24–26]. Furthermore, intracellularlyderived anions are central to the voltage-dependent chargemovement and functional response of prestin [9]. Given thesubstantial voltage field that exists across the plasmamembrane [7,8] and the major role of electrostatics inprotein function [27], it is not surprising that voltagechanges can have widespread effects on transmembraneproteins even if they lack an S4-like series of positivecharges. It is also worth noting that changes in transmem-brane potential can cause physical movements of themembrane owing to the relationship between mobilecharge and surface tension, as predicted by the Lipmannequation [28]. The effect of such movement, which can bedetected by an atomic force microscope [28], on proteinfunction is unknown and might be worth investigatingbecause GPCRs have been reported to be directly activatedand inactivated by fluid shear stress [29,30].

Rhodopsin early receptor currentsAlthough the ERC is recorded in response to a flash of lightrather than a voltage change, this charge-displacementevent is important to discuss because rhodopsin is one of

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the best characterized GPCRs. In addition, some investi-gators have likened the ERC [12,13] (Figure 1e) to thegating currents of ion channels [12,31] because they areboth associated with functionally important protein con-formational changes. However, the relationship betweenthe events underlying the ERC and those responsible forthe muscarinic receptor voltage-dependent charge move-ment is unknown. In this regard, it is also worth notingthat, whereas the ERC results from light-induced isomer-ization of a chromophore covalently bound to rhodospin,muscarinic-receptor voltage-dependent gating ‘charge’events were recorded in the absence of agonist [15].

Although the precise molecular events underlying theERC remain to be defined, the initial depolarizing R1phase has been proposed to relate to the primary photo-chemical processes during isomerization [32], whereas theslower hyperpolarizing R2 phase that occurs on a milli-second timescale probably involves multiple electrostaticevents that lead to the activated state, metarhodopsin II[12,14,33]. These include deprotonation of the Schiff baseformed by the covalent linking of 11-cis-retinal to a lysinein transmembrane domain 7, protonation of the Schiff-basecounterion, configurational movements of transmembraneportions of the receptor and proton uptake at the cyto-plasmic surface [12,33]. Theoretically, any of these eventscould also be voltage dependent, although not all will occurduring activation of non-retinal-binding GPCRs. Protonuptake by the aspartic acid or glutamic acid within thehighly conserved D(E)RY motif at the cytoplasmic end ofthe third transmembrane helix has been suggested to be akey event in the activation of several rhodopsin-like class 1GPCRs [34–37]. By analogy with the proposed voltage-dependent movement of ions in and out of prestin and co-transporters [10,38], this proton uptake could be affectedby membrane potential. More work is necessary to inves-tigate the voltage dependence of the ERC, which has beendescribed as weak in one brief report [39] and having littlevoltage dependence between �50 and +80 mV in another[40].

Is voltage control of GPCRs physiologically relevant?The proposed relevance of GPCR voltage dependence canbe summarized into three categories:

(i) V oltage control of transmitter release at synapses

independently of Ca2+ influx (recently reviewed inRef. [18]). Presynaptic M2, metabotropic glutamatetype 3 (mGlu3) and GABAB receptors exert a tonicinhibitory effect on transmitter release, probably viadirect actions on the exocytotic machinery [18].Depolarization, which has been suggested to inhibitall three of these receptors [18], will, therefore, lead toenhanced transmitter release. Evidence in support ofthis hypothesis has recently been obtained fromstudies of transmitter release in which pertussis-toxin-induced uncoupling of inhibitory autoreceptorsfrom their G proteins exerts effects independently ofchanges in Ca2+ currents [41]. Theoretically, thisdirect voltage control of synaptic function couldcontribute to synaptic plasticity [18]. M2-deficientmice show behavioural and memory deficiencies andmajor changes in synaptic plasticity at the cellular

Review Trends in Pharmacological Sciences Vol.29 No.8

level [42]; however, the relevance of M2-receptorvoltage dependence in these knockout phenotypesremains to be ascertained.

(ii) S

ynergy between electrical and metabotropic influ-ences. For the majority of Gq-coupled receptors [e.g.P2Y1, thromboxane (TP)a, M1, M3], depolarizationenhances downstream Ca2+ mobilization [4–6,43].Although the receptor sensitivity to voltage is oftenbipolar, work on P2Y1 receptors indicates thatdepolarization has a greater effect compared withhyperpolarizations [44]. Consequently, the net resultof a single action-potential waveform is to enhanceCa2+ release, and trains of action potentials lead to aplateau elevation of Ca2+ [44]. The timecourse of themore rapid component of the gating currents reportedby Ben-Chaim et al. [15] (in the order of a few hundredmicroseconds) also indicates that individual actionpotentials might influence GPCR activity. This effectcould play a part in coincidence detection leading tosynaptic plasticity in neurones or in the interactionbetween hormonal and electrical influences in neuro-endocrine cells. For P2Y1 receptors, synergisticinteraction might be particularly important at lowlevels of agonist [45], whereby depolarization can stillinduce substantial Ca2+ mobilization. We have shownthat potentiation of P2Y1 receptors by depolarizationor inhibition by hyperpolarization is graded withoutevidence of a threshold [44], therefore even small-amplitude voltage shifts, such as synaptic potentials,could alter signalling at purinergic synapses. Inplatelets, voltage control of GPCRs could contribute tothe ability of P2X1 receptors to amplify ADP-evokedCa2+ increase [46] and to the potentiation of func-tional responses by Ca2+-impermeable a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)receptors [47].

(iii) R

egulation of Ca2+ oscillations. The bipolar nature ofthe voltage control described for some Gq-coupledreceptors enables oscillatory waveforms to generateCa2+ oscillations [48]. Although this voltage-depend-ent effect is not necessary for Ca2+ oscillations, itmight be a contributory or regulatory factor.

Dihydropyridine-sensitive and -insensitive mechanismsof voltage control of Ins(1,4,5)P3-dependent Ca2+ releaseDirect voltage control of GPCRs, heterotrimeric G proteinsor inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3]-dependentCa2+ release has been described in a variety of tissues(Table 1). Some of the earliest examples indicating voltagedependence of Ins(1,4,5)P3-dependent Ca2+ mobilizationare in excitable tissues, particularly smooth and skeletalmuscle, where elevated external K+ or trains of actionpotentials induce inositol lipid breakdown in the absenceof external Ca2+ or in the presence of voltage-gated Ca2+

channel blockers [49,50]. More recent studies in both typesof muscle indicate that dihydropyridine (DHPR) receptorsfunction as the voltage sensor to stimulate phospholipaseCvia heterotrimeric G proteins [51,52]. Figure 2b shows theeffect observed using simultaneous patch-clamp and[Ca2+]i studies of arterial smooth muscle. The role of

GPCRs in these responses remains unclear becauseexogenous agonists were not required, although in smoothmuscle the effect was potentiated by external ATP [53].The P2-receptor antagonist suramin failed to abrogate thiseffect of ATP; however, the role of P2Y receptors needsfurther examination because we have recently observedthat this antagonist can enhance, rather than prevent,voltage control of P2Y1 receptors [54]. In both non-excitableand excitable tissues, voltage control of Ins(1,4,5)P3-de-pendent Ca2+ release has been reported, which requiresthe presence of a Gq-coupled-receptor agonist [2,4,43] andis clearly independent of dihydropyridine receptors [4,43].The most extensively studied example is that of P2Y1-receptor-evoked Ca2+ mobilization in the non-excitablemegakaryocyte (Figure 2c). In this cell system, severalobservations support the hypothesis that the GPCR itselfacts as the principal voltage sensor. These include thecomparable latency of response to voltage and agonist[44], the ability of subthreshold concentrations of agonistor inactive competitive antagonists to induce voltage-de-pendent Ca2+ release [54] and the lack of voltage-depend-ent Ca2+ responses when GTPgS is included in the patchpipette in cells lacking P2Y1 receptors [4]. Some studiesconclude that events downstream of the receptor are vol-tage sensitive based upon a voltage dependence of GTPgS-induced Ca2+ release [2], or the effect still being observed atsaturating concentrations of agonist [43], although theseeffects do not definitively rule out a role for voltage controlat the level of the receptor. Nevertheless, the actions ofheterotrimeric G proteins can be voltage dependent, asexemplified by the relief of GPCR-induced inhibition ofvoltage-gated Ca2+ channels by large depolarizations,which is commonly accepted to be mediated by release ofinhibitory bg subunits [55]. Overall, therefore, theexamples of voltage dependence of Ins(1,4,5)P3-inducedCa2+ release can be divided into two groups: DHPR-sensi-tive and DHPR-insensitive mechanisms. In the case of thelatter, multiple points in the cascade might be voltagedependent; however, most evidence indicates that theprincipal voltage sensor is at the level of the GPCR, asindicated by the charge-movement studies of Ben-Chaimet al. [15].

Lessons from the megakaryocyte: an unusual cell?Unlike the approach of the Parnas group, who speculatedthat M2 receptors might be voltage dependent based uponearlier observations of synaptic transmission (reviewed inRef. [18]), we observed voltage control of Gq-coupled recep-tors in the megakaryocyte purely by serendipity [5]. Aseries of detailed studies has shown that voltage canmodulate Ins(1,4,5)P3-dependent Ca2+ mobilizationevoked by several different Gq-coupled receptors, but notby a tyrosine-kinase-coupled receptor, in this platelet-pre-cursor cell [5,44,48]. A similar voltage dependence ofIns(1,4,5)P3-dependent Ca2+ release has been observedin other cell types; however, the effects seem less robustcompared with the voltage-dependent responses observedduring stimulation of P2Y1 receptors in themegakaryocyte[2,43,44]. It can be hypothesized that P2Y1 receptors aremore voltage dependent as a consequence of the importantroles of the negatively charged phosphate groups of the

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agonist and several basic amino acids of the transmem-brane domains in ligand–receptor interactions [56]. Thecellular environment might also play a part because M3

receptors can be voltage dependent in one cell type (SH-SY5Y neuronal cells) but not in others (chinese hamsterovary and human embryonic kidney cells) [43]. The under-lying reason for this intercellular variation is unclear, butone possibility is that a specific balance of receptor, Gprotein and effector protein is necessary. In addition, themegakaryocyte possesses the demarcation membrane sys-tem, a unique series of plasma-membrane invaginationsthat tortuously extends throughout the extranuclear cellvolume and serves as a reservoir for future platelet pro-duction [57]. This membrane architecture is likely toincrease the number of surface receptors present pervolume of cytoplasm compared with a similar size cellwithout invaginations. Therefore, in the megakaryocyte,the effect of membrane potential on Gq-coupled-receptor-evoked Ca2+ release might be enhanced because depolar-ization generates more Ins(1,4,5)P3 per volume of cyto-plasm. Such conjecture also leads to the speculation thatvoltage dependence of GPCR signalling could be amplifiedin other cell types with invaginations (e.g. skeletal orventricular myocytes) or in small-cell compartments suchas dendritic spines, owing to their high surface area–volume ratio. Dendritic spines are rich in Ins(1,4,5)P3

receptors [58] and have an important role in synaptictransmission and plasticity [59,60].

Future directionsDirect control of GPCRs by action potentials or otherphysiological changes in transmembrane voltage couldimpact how this important family of receptors controlscellular responses. However, many questions remain aboutthe robustness of this effect, its relevance and the under-lying mechanism(s). In particular, the relationship be-tween the GPCR voltage-dependent membrane chargedisplacements, ligand binding and receptor activationneeds further investigation. To definitively address theseissues, more advanced measurements of receptor affinityand intrinsic efficacy, for example using FRET-basedassays, are required in cells under electrophysiologicalcontrol.

AcknowledgementsWe thank Richard Evans, John Challiss and Jon Mitcheson for commentson the manuscript, and Noel Davies for providing the theoreticalmembrane currents in Box 1 Figure Ic. Work in our laboratories isfunded by the Medical Research Council (http://www.mrc.ac.uk), theBritish Heart Foundation (http://www.bhf.org.uk) and GeneralitatValenciana (http://www.gva.es).

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