Biochimica et Biophysica Acta 1828 (2013) 1629–1643

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Review

Regulation of CaV2 calcium channels by G protein coupled receptors☆

Gerald W. Zamponi a, Kevin P.M. Currie b,c,⁎a Department of Physiology & Pharmacology, Hotchkiss Brain Institute, University of Calgary, Calgary, Canadab Department of Anesthesiology, Vanderbilt University School of Medicine, Nashville, TN, USAc Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN, USA

☆ This article is part of a Special Issue entitled: Calciu⁎ Corresponding author at: Departments of Anesthesio

Tel.: +1 615 322 8514; fax: +1 615 343 3916.E-mail address: kevin.currie@vanderbilt.edu (K.P.M.

0005-2736/$ – see front matter © 2012 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.bbamem.2012.10.004

a b s t r a c t

a r t i c l e i n f o

Article history:Received 16 August 2012Received in revised form 2 October 2012Accepted 4 October 2012Available online 12 October 2012

Keywords:Calcium channelG protein coupled receptorGβγTyrosine kinasePiP2Arachidonic acid

Voltage gated calcium channels (Ca2+ channels) are key mediators of depolarization induced calcium influx intoexcitable cells, and thereby play pivotal roles in awide array of physiological responses. This review focuses on theinhibition of CaV2 (N- andP/Q-type) Ca2+-channels by G protein coupled receptors (GPCRs), which exerts impor-tant autocrine/paracrine control over synaptic transmission and neuroendocrine secretion. Voltage-dependentinhibition is the most widespread mechanism, and involves direct binding of the G protein βγ dimer (Gβγ) totheα1 subunit of CaV2 channels. GPCRs can also recruit several other distinct mechanisms including phosphory-lation, lipid signaling pathways, and channel trafficking that result in voltage-independent inhibition. Currentknowledge of Gβγ-mediated inhibition is reviewed, including the molecular interactions involved, determinantsof voltage-dependence, and crosstalk with other cell signaling pathways. A summary of recent developmentsin understanding the voltage-independent mechanisms prominent in sympathetic and sensory neurons is alsoincluded. This article is part of a Special Issue entitled: Calcium channels.

© 2012 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16302. G protein coupled receptors and heterotrimeric G proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16313. Inhibition of CaV2 channels by G protein coupled receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16314. Voltage-dependent inhibition mediated by Gβγ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1631

4.1. Single channel investigations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16314.2. Alteration of gating currents by Gβγ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16324.3. Gβγ and channel inactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16324.4. Differential inhibition of CaV2 channels by Gβγ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1632

5. Structural determinants on Gβγ that govern modulation of CaV2 channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16336. Structural determinants on the channel α1 subunit that govern modulation by Gβγ . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16337. Contribution of the CaVβ subunit to voltage-dependent inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16348. Crosstalk between N-type channels, Gβγ, kinases and synaptic proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1635

8.1. Protein kinase C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16358.2. Synaptic proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16358.3. Calcium channel γ subunits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1636

9. Direct GPCR/N-type calcium channel interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163610. Voltage-independent inhibition of CaV2 channels by Gq-coupled GPCRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1637

10.1. CaVβ and intracellular Ca2+ modulate Gq-mediated inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163711. Kinase-mediated, voltage-independent inhibition of CaV2 channels in sensory neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . 163712. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1638

m channels.logy and Pharmacology, Vanderbilt University School of Medicine, 1161 21st Avenue South, Nashville, TN 37232-2520, USA.

Currie).

rights reserved.

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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1638References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1638

1. Introduction

Voltage gated calcium channels (Ca2+ channels) are keymediatorsof depolarization induced calcium influx into excitable cells, which inturn mediates a wide array of physiological responses including theactivation of calciumdependent enzymes, smoothmuscle contraction,pacemaker activity and neurotransmitter release [1–8]. Ca2+ channelsare also associated with a wide range of pathologies, including pain,epilepsy, migraine, cardiac arrhythmias and autism [9–14]. It is widelyknown that there are subtypes of Ca2+ channels with different phar-macological and biophysical properties, and distinct cellular and phys-iological functions [15–17]. In neurons, certain L-type Ca2+ channelisoforms are expressed at cell bodies and dendrites, and one of theirkey functions is the initiation of calcium dependent gene transcriptionevents [18–22]. Other L-type channel subtypes are expressed in co-chlear hair cells and photoreceptor nerve terminalswhere they regulateneurotransmitter release at ribbon synapses [23,24]. T-type calciumchannels are expressed in cell bodies as well as dendrites and one oftheir key functions is to regulate cellular excitability and neuronal firingproperties [25–27], in addition to participating in secretion [28–30].N-type and P/Q-type calcium channels are expressed at synaptic nerveterminals where their opening results in the release of neurotransmit-ters [1,19,31–34].

All Ca2+ channels are comprised of a pore forming Cavα1 subunitthat contains the major structural features required for permeation,activation, and inactivation. The mammalian genome encodes tendifferent Cavα1 subunits that fall into three major families — Cav1

Fig. 1. Schematic depiction of the topology and subunit composition of CaV2 voltage-gated Cunits. The intracellular β subunit interacts through its guanylate kinase-like domain (GK) witis largely extracellular and likely GPI-anchored to the plasma membrane. (B) Topology of theconsist of six transmembrane spanning α-helices (S1-S6) (blue or green cylinders) and a ‘P-channel (colored green), while S1–S4 (in particular S4 that has multiple charged residues) ccytoplasmic loops connecting domains I–IV are important for interaction with other proteinsCa2+ binding proteins (CaBP1, VILIP). These cytoplasmic domains are also targeted by seconAlternative splicing greatly increases the functional diversity of the channels. For example,channels by GPCRs in sensory neurons (see Section 11 for more details).

(L-type channels), Cav2 (N, P/Q- and R-types), and Cav3 (T-types)[17,35]. The CaV1 and CaV2 families are high voltage activated (HVA)channels, and are heteromers comprised of a pore forming Cavα1subunit as well as Cavα2-δ and Cavβ subunits [36–38] (Fig. 1). Inaddition, these channels associate with calmodulin which is now con-sidered part of the HVA channel macromolecular complex [39–44].The Cavα1 subunit determines the Ca2+ channel subtype and is alarge (~175–225 kDa) protein with four homologous transmembranedomains that are connected by cytoplasmic loops and bracketed bycytoplasmic N- and C-termini [37] (Fig. 1). These cytoplasmic regionsare key targets for second messenger regulation including proteinkinases and G proteins, as we discuss here in detail. The Cavβ subunitsare cytoplasmic proteins that associate with HVA α1 subunits at ahighly conserved region within the domain I–II linker (termed theAlpha Interaction Domain— AID) [45–47]. These subunits are encodedby four different genes (for reviews see [48,49]). The Cavα2-δ sub-units are transcribed from one of four different Cavα2-δ genes, proteo-lytically cleaved and then reconnected via a disulfide bond (for areview, see [50]). The α2 portion is located at the extracellular sideof the channel, whereas the δ portion either spans the membrane ormay be linked to the extracellular leaflet of the plasma membranethrough a glycosylphosphatidylinositol (GPI) anchor [51]. The func-tion of these ancillary subunits is to regulate channel properties andpromote Cavα1 subunit trafficking to and stabilization at the plasmamembrane [52–54] (for reviews see [48,49,55–57]. As we will outlinebelow, Cavβ subunits also alter second messenger regulation of thechannel complex [58–61]. Finally, it should be noted that most Ca2+

a2+ channels. (A) Cartoon showing the 3D topology along with channel auxiliary sub-h the I–II linker of theα1 subunit (at theα-interaction domain or AID). Theα2δ subunitpore forming α1 subunit. Four homologous repeats (domain I through domain IV) eachloop’ between S5 and S6. The S5–S6 helices and P-loop comprise the pore domain of theomprises the voltage sensor (colored blue). The intracellular N- and C-termini and theincluding the auxiliary β subunit, synaptic proteins, Gβγ, GPCRs, calmodulin and other

d messenger pathways including phosphorylation by PKC, CaMKII, and tyrosine kinases.alternative splicing of exon37 on the proximal C-terminus controls inhibition of CaV2.2

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channel subunits are subject to alternate mRNA splicing, thus greatly in-creasing the functional diversity of calcium channels [62–65]. Recentlydescribed RNA editing events that alter channel function add furthercomplexity [66]. This then makes it challenging to preciselyreconstitute all specific features of native calcium currents in transientexpression systems. In this review, we focus on the CaV2 family of chan-nels, and in particular their regulation by G protein coupled receptors.

2. G protein coupled receptors and heterotrimeric G proteins

G protein coupled receptors (GPCRs) are a large family of mem-brane proteins encoded by almost 800 human genes, and representan important class of therapeutic targets [67,68]. GPCRs are charac-terized by an extracellular N-terminus, seven transmembrane span-ning alpha helices, and an intracellular C-terminus which couples toheterotrimeric G proteins. Extracellular ligand binding to the receptorleads to activation of the G proteins and a myriad of downstreamintracellular signaling cascades. In human, sixteen genes encode G pro-tein α subunits (Gα), and these are classified into four major families:Gs, Gi, Gq, and G12, in addition to transducin (Gαt) which is found inthe retina. Five genes encode Gβ subunits, and twelve genes encodeGγ subunits (for reviews see [69–72]. Binding of agonist to the GPCRcatalyzes the exchange of GDP to GTP on Gα causing conformationalchanges/dissociation of the Gα and Gβγ heterodimer [71,73]. The liber-ated Gα and Gβγ are both capable of signaling tomultiple downstreameffectors, including voltage-gated Ca2+-channels as discussed in thisarticle. Signaling is terminated by the intrinsic GTPase activity of Gαand subsequent reassociation of the Gα-GDP subunit with the Gβγheterodimer. This GTPase activity can be accelerated by a family ofRGS proteins (regulator of G protein signaling) which thus influencethe extent and duration of downstream events [74]. Receptor desensiti-zation in the continued presence of agonist can also terminate signaling.Desensitization is complex, involving phosphorylation by PKA, PKC, orG protein coupled receptor kinases (GRKs) and uncoupling of the recep-tor from the downstream G proteins. Endocytic removal of the GPCRfrom the plasma membrane can also occur. GRKs recruited by Gβγphosphorylate the C-terminus of the GPCR leading to recruitment ofarrestins and the endocytic machinery [75,76]. As discussed below(Section 9), direct interaction of GPCRs and Ca2+ channels mightresult in co-internalization adding another dimension to channelmodulation.

3. Inhibition of CaV2 channels by G protein coupled receptors

Neurotransmitter mediated inhibition of Ca2+ channels was firstdemonstrated ~30 years ago by Dunlap and Fischbach who reportedthat norepinephrine reduced the duration of action potentials [77]and the amplitude of ICa [78] in chick sensory neurons. It is now ap-parent that a variety of different neurotransmitters/neuromodulatorsacting on their cognate GPCRs inhibit ICa, and that this is important forcontrolling neurosecretion (for reviews see [79–85]). It is also knownthat GPCRs can recruit several distinct signaling pathways that con-verge on Ca2+ channels. The most widespread and intensively stud-ied of these involves direct binding of Gβγ to the α1 subunit ofCaV2 channels. As detailed below (Section 4), Gβγ-mediated inhibi-tion shifts the voltage-dependence of channel activation, is less prom-inent at depolarized membrane potentials, and is transiently relievedby large depolarizing voltage steps. Consequently, this mechanismis often termed voltage-dependent inhibition. GPCRs can also elicitvoltage-independent inhibition of ICa which is mediated by severalother distinct and generally less well characterized pathways includ-ing phosphorylation, lipid signaling pathways, and channel trafficking(see Sections 9–11). While voltage-dependent inhibition is widespreadthroughout the nervous system, voltage-independent inhibition ismore variable in extent and mechanism but seems particularly promi-nent in sensory and sympathetic neurons. In this review we first

consider Gβγ-mediated inhibition, including recent developments andcrosstalk with other cell signaling pathways. Then we outline some ofthe voltage-independent mechanisms prominent in sympathetic andsensory neurons.

4. Voltage-dependent inhibition mediated by Gβγ

Voltage-dependent inhibition primarily targets CaV2.1 (P/Q-type)and CaV2.2 (N-type) channels, although CaV2.3 channels are alsoinhibited by similar mechanisms (see Section 4.4 below). The voltage-dependent nature of the inhibition was first demonstrated by Bean[86], who showed that the decrease in current amplitude was not dueto a loss of channels per se, but rather a shift in the gating propertiesthat could be overcome by strong depolarization. Several hallmarksare characteristic of this voltage-dependent mechanism: In wholecell recordings, the inhibition of peak ICa amplitude is diminishedat depolarized membrane potentials; activation kinetics are slowed;the voltage-dependence of activation is shifted to more depolarizedpotentials; and a conditioning prepulse to depolarized potentials relievesmost of the inhibition and normalizes channel kinetics (termed prepulserelief or prepulse facilitation). Fig. 2 shows an example of voltage-dependent inhibition of ICa. Prominent slowing of activation kineticsand prepulse relief of the inhibition is clearly seen. Voltage-dependentrelief of the inhibition can also occur at least to some extent duringmore physiologically relevant stimuli such as high frequency trains ofaction potential-like waveforms [87–92]. In turn, this might contributeto short term synaptic plasticity at some synapses [93].

Bean also introduced the “willing and reluctant” model to explainthese functional effects [86], a framework that persists to this day[94–97]. The channels exhibit two functional gating states, “willing”and “reluctant”. In the absence of Gβγ, the “willing” state predomi-nates, while binding of Gβγ favors the “reluctant” state which dis-plays the shifts in channel gating noted above. Voltage-dependentrelief of the inhibition is thought to reflect a shift of the channelsfrom “reluctant” to “willing” due to transient dissociation of Gβγ(Fig. 2B). This was supported by kinetic analyses of prepulse reliefas a function of agonist or Gβγ concentration. Increasing the concentra-tion of Gβγ did not alter the rate of relief during the prepulse, but didaccelerate the rate of reinhibition following the prepulse [98–101],as expected for voltage-dependent dissociation and rebinding of Gβγ.Further investigations revealed that the kinetics of reinhibition wereconsistent with binding and unbinding of a single Gβγ dimer with thechannel [101].

4.1. Single channel investigations

Single channel studies provided early evidence that the inhibitiondid not involve a diffusible second messenger. In the “cell-attached”(“on-cell”) recording configuration, bath application of agonist didnot inhibit the channels whereas agonist in the patch pipette did[99,102,103]. This led to the conclusion that the inhibition was“direct” or “membrane delimited”. Single channel recording also directlyrevealed “reluctant” gating of inhibited channels. Upon membrane de-polarization, the latency (delay) to first channel opening was increasedduring inhibition whereas there was little impact on other single chan-nel parameters [95,104]. As a result, the inhibited (“reluctant”) channelsappeared essentially silenced, unable to open until Gβγ dissociated andthe channels shifted to the “willing” state. Subsequently it has beenreported that CaV2.2 (N-type) but not CaV2.1 (P/Q-type) channels candisplay very brief channel openings from the “reluctant” state (i.e. with-out Gβγ unbinding), although the probability of such events was low[96,97].

Overall, the dominant effects of inhibition observed in all studies arethe shift in activation and prolonged latency to first channel opening.The slow activation kinetics seen in whole cell recording (Fig. 2) andlonger latency in single channel recordings reflect the conformational

Fig. 2. Functional effects of voltage-dependent inhibition on CaV2 channels. (A) “Whole cell” patch clamp recording of ICa from an adrenal chromaffin cell which express purinergicP2Y autoreceptors. Application of a P2Y receptor agonist (red trace) inhibited ICa compared to control conditions (black trace) with the hallmark features of voltage-dependentinhibition. Peak amplitude was reduced with prominent slowing of the activation kinetics, and both of these effects were reversed by a conditioning prepulse to +100 mV(green trace). (B) Voltage-dependent relief of inhibition reflects transient dissociation of Gβγ from the channel. Shown is an example of “whole cell” ICa recorded from recombinantCaV2.2 channels expressed with β1b, α2δ and Gβγ in HEK293 cells. Gβγ produced tonic inhibition of ICa that was reversed by a conditioning prepulse to +100 mV. The magnitudeof this reversal (prepulse facilitation) diminished as the interval between prepulse and test pulse (Δ) was increased (examples shown are with Δ=10 ms, 25 ms, and 200 ms). Asillustrated by the inset cartoon, prepulse facilitation is thought to reflect dissociation of Gβγ from an inhibitory binding site on the channel at the depolarized membrane potential.Upon return to the hyperpolarized membrane potential, Gβγ rebinds to (and re-inhibits) the channel. This re-inhibition of ICa is monoexponential, and the rate depends on the localconcentration of Gβγ.

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changes and subsequent dissociation of Gβγ from the channel uponmembrane depolarization. This diminished binding of Gβγ at depo-larized potentials also results in little inhibition of whole cell ICa whenneurotransmitter agonists are rapidly applied during a depolarizingvoltage-step [105].

4.2. Alteration of gating currents by Gβγ

Further evidence for altered activation comes from recording ofchannel “gating currents”. Gating currents are not due to ionic fluxthrough the channel pore, but rather reflect movement of the chargedvoltage-sensor domain of the channels in response to membranepotential changes. Expression of recombinant CaV2.2 in HEK293 cellsenables recording of these gating currents in isolation as the cells lackother endogenous voltage-gated channels. G proteins were found toreduce the amplitude, and shift the voltage-dependence of gating cur-rents to more depolarized potentials [106]. G proteins also produced asignificant separation in the voltage-dependent activation of gating cur-rent and ionic current [106]. These data suggest that Gβγ binding slowsmovement of the voltage-sensor and uncouples this movement fromopening of the activation gate. Modulation of gating currents by G pro-teins has also been reported in rat sympathetic neurons [107,108].

4.3. Gβγ and channel inactivation

In addition to these dominant effects on channel activation, evi-dence supports the idea that Gβγ can also modulate inactivation ofCaV2.2 channels [109,110]. Inactivation of Ca2+ channels is complexand mediated by several voltage-dependent and Ca2+-dependentmechanisms [111–113]. The precise molecular correlates remain some-what unclear, but fast voltage-dependent inactivation might involvea “hinged lid” type of mechanism in which the intracellular loopconnecting domains I and II of theα1-subunit serves as the “inactivationgate” [112,114] (but see [115]). The I–II loop is also an important bind-ing site for Gβγ on the channel [116–119] (Fig. 1) (see Section 6 formore discussion). Therefore, it is possible that binding of Gβγ disruptsthe movement of this putative inactivation gate, or its interaction with

other channel domains. Inactivation of CaV2.2 can also occur from inter-mediate closed state(s) of the channel favored during trains of briefrepetitive stimuli [104]. If Gβγ were to reduce the probability that thechannels populate this state (from which inactivation is preferred) itmight reduce the cumulative inactivation throughout a stimulus train.Further investigations are needed to determine quite how G proteinmodulation and channel inactivation interact.

Ca2+-dependent inactivation is mediated by calmodulin bound tothe C-terminus of the channel α1 subunit [42,120–123]. Strong intra-cellular Ca2+ buffering (EGTA or BAPTA in the patch pipette solution)blocks Ca2+-dependent inactivation of CaV2 channels indicating that itis mediated by a “global” elevation rather than a “local”microdomain ofCa2+. The reduction of Ca2+-dependent inactivation by Gβγ [109]might therefore result from fewer channels opening and a diminished“global” Ca2+ signal, although more complex interactions are also pos-sible, and direct in vitro binding of Ca2+-calmodulin to Gβγ has beenreported [124].

4.4. Differential inhibition of CaV2 channels by Gβγ

Originally demonstrated for N-type channels (CaV2.2) in sensoryand sympathetic neurons (for example, [78,125–127]), it subsequentlybecame clear that CaV2.1 (P/Q-type) channels are also modulated byGβγ in a similarmanner [128]. Initially it was thought that CaV2.3 chan-nels were insensitive to G proteins [129–131], although other studiesdid find some degree of inhibition [132–135]. Chimeric approachessuggested that the lack of (or poor) responsiveness of CaV2.3 residedin several regions within the N-terminus, domain I, and I–II linker ofthe channels [136–138]. Subsequently it was discovered that alterna-tive splicing of the N-terminus conferred G protein sensitivity to thechannels [139]. Truncation of 50 amino acids abolished inhibition,whereas a splice variant with full length N-terminus did display inhibi-tion, albeit to a lesser extent than CaV2.2 channels [139]. While inhibi-tion of CaV2.3 channels can occur (depending on splice variation), it isgenerally to a lesser extent and remains less well understood than forCaV2.1 and CaV2.2 channels. In part this might be due to difficulty inisolating these channels in neuronal cell types. The focus on CaV2.1

Fig. 3. Structural determinants on Gβγ that govern modulation of CaV2 channels.(A, B) Ribbon diagram renderings of the heterotrimeric G protein structure in panel A,and the Gβγ dimer in panel B (Gαi — green; Gβ1 — red; and Gγ2 — blue). Gβ adopts aseven blade β-propeller structure with an α-helical N-terminal domain that binds tothe α-helical N-terminus of Gγ. Gα interacts with multiple residues on the top face ofGβ and the side aspect of propeller blade 1. Many effectors bind to a protein interaction“hot spot” on the surface of Gβ that ismasked by Gα in the heterotrimer. (C, D)Molecularsurface rendering of the Gβγ dimer (Gβ — red; Gγ — blue). Panel C shows the Gαinteracting face of Gβγ, and panel D is rotated ~180° to show the opposite face of Gβγ.Residues marked in yellow have been reported to disrupt inhibition of CaV2 channels.Residues marked in green are involved in crosstalk between Gβ1 and PKC phosphoryla-tion of CaV2.2. Molecular graphics images based on data reported by Wall et al. [144](PDB ID: 1GP2) were produced using the UCSF Chimera package [256,257] from theResource for Biocomputing, Visualization, and Informatics at the University of California,San Francisco.

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and CaV2.2 channels is also driven by their prominence in triggeringneurotransmitter release, andmost of the following discussion revolvesaround those two channels.

Although the basic mechanism of inhibition is similar for CaV2.1and CaV2.2 (direct binding of Gβγ to the channel), subtle differenceshave emerged. As noted above, single channel recording showed thatCaV2.2 but not CaV2.1 channels display very brief duration, low prob-ability “reluctant openings” [96,97]. Differences are also apparentwith macroscopic (whole-cell) recordings: activation of GPCRs or ex-pression of Gβγ reduces the peak amplitude of ICa to a significantlygreater extent for CaV2.2 than CaV2.1 [129,140,141]. Moreover, trainsof action potential-like stimuli reverse a greater proportion of CaV2.1inhibition than CaV2.2 inhibition [92]. These effects can be explainedby differences in the affinity of Gβγ binding to the channels. Theapparent affinity of Gβγ for the channel can be inferred from prepulserelief and re-inhibition experiments, and is quite similar for the twochannels at hyperpolarized or very depolarized potentials. However,at moderately depolarized potentials (b+30 mV), within the physio-logically relevant range of action potentials, there is a significantdivergence in the affinity of Gβγ binding to the two channel types[96]. Subtle differences in binding of Gβγ to the channels are alsosuggested when comparing the inhibition produced by differentGβ subunits (Gβ1–5), all paired with the same Gγ2 subunit. Such ex-periments revealed a different rank order of inhibition for CaV2.1and CaV2.2 channels [142]. Point mutations on the Gβ1 subunit alsohave distinct effects on the inhibition of CaV2.1 and CaV2.2 channels[143].

It would appear that subtle differences in the binding affinity ofGβγ to the CaV2.1 and CaV2.2 channels result in differential inhibi-tion: CaV2.2 ICa is inhibited to a greater extent and this inhibition ismore resistant to reversal by high frequency bursts of action poten-tials. The relative expression level of the two channel types varies be-tween neurons, and even between neighboring synapses arising fromthe same neuron. Therefore, differential inhibition of CaV2.1 andCaV2.2 could lead to cell and/or synapse specific neuromodulationby GPCRs. Functional differences might also arise from variable inter-actions or crosstalk with other signaling pathways such as PKC (seeSection 8).

5. Structural determinants on Gβγ that govern modulation ofCaV2 channels

Gβγ is thought to be an obligate heterodimer and there are severalhigh resolution crystal structures in isolation or bound to interactingproteins including Gα, GRK2, and phosducin [144–148]. Fig. 3 showsa rendering of the heterotrimer (Gαiβ1γ2) (Fig. 3A), and heterodimer(the Gβ1γ2) (Fig. 3B) based on the structure reported by Wall et al.[144] (PDB ID: 1GP2). Gβ adopts a seven blade β-propeller structurewith an α-helical N-terminal domain that binds to the α-helicalN-terminus of the Gγ subunit (Fig. 3B). In the heterotrimeric com-plex, Gα interacts with multiple residues on the top face of Gβ andthe side aspect of propeller blade 1 (Fig. 3A). Gβγ interacts with mul-tiple downstream effectors and mutagenesis approaches have beenused to map the interaction sites important for binding to these tar-gets. Many effectors bind to a protein interaction “hot spot” on thesurface of Gβ that interacts with Gα, with overlapping subsets ofresidues involved in binding to different effectors [149]. A numberof residues identified in mutagenesis studies to contribute to inhibi-tion of Ca2+ channels are highlighted in Fig. 4. Most of these are onthe Gα interacting surface (Fig. 3C) and aremaskedwhen Gα is present[85,109,150–152], although residues on the reverse face of Gβ1 havealso been implicated [152–154] (Fig. 3D). Also of note, Asn35 andAsn36 on Gβ1 mediate the ability of PKC to antagonize inhibition ofCaV2.2 [153]. Thr422 on the rat CaV2.2 I–II linker has been identified asthe phosphorylation site for PKC that mediates this effect [155], so it is

tempting to speculate that this region of the channel and Gβγ come inclose proximity with one another (see Section 6).

Another study reported that a peptide mimicking the N-terminal25 amino acids of Gβ2 reduced inhibition of CaV2.1 [156]. The GβN-terminal peptide disrupted FRET interaction between the Gβ2 andGγ3 subunits suggesting a conformational shift or reorientation ofthe heterodimer that could disrupt interaction with the channels. Afew studies have also shown that the subtype of Gγ within the Gβγheterodimer can influence the extent of inhibition, with Gγ2 generallyeliciting greater inhibition than Gγ1, Gγ3 or Gγ13 [157,158]. Themolecular basis for why the Gγ subtype influences inhibition of ICa isnot clear, but it is interesting to note that the II–III linker (of the chan-nel α1 subunit) contains a G-gamma-like (GGL) domain [159].

6. Structural determinants on the channel α1 subunit that governmodulation by Gβγ

Although there is currently no crystal structure for voltage-gatedcalcium channels that could be used to visualize their interactionswith G proteins, site directed mutagenesis, chimeric, and biochemicalapproaches have been used to elucidate channel structural determi-nants involved in modulation. The first investigations involved chi-meras between Cav2.1 and Cav2.2 channels [140]. These chimeraswere expressed in Xenopus oocytes and their sensitivities to G pro-teins assessed via two electrode voltage clamp. These experimentsidentified domain I as a key determinant of G protein inhibition,along with the C-terminus of the channel. Subsequent biochemicalstudies using in vitro translated Gβγ subunits revealed two spatiallydistinct regions on the I–II linker of CaV2.1 as possible Gβγ targets[117]. The existence of two separate Gβγ binding domains in the

Fig. 4. Model depicting the molecular interactions that underlie Gβγ-mediated inhibition of CaV2 channels. Panels A and B (upper three images) depict a channel with a CaVβsubunit, while panel C (lower images) depicts the situation in which the CaVβ subunit is absent. Currently, data suggest that the binding site for Gβγ is comprised of multiplesites on the N-terminus, I–II linker, and probably C-terminus of the channel. Binding of Gβγ causes a conformational shift that promotes interaction of the N-terminus “inhibitorymodule”with the initial one-third of the I–II-linker (panel Aii). This (and perhaps other interactions) shifts gating charge movement to more depolarized potentials and uncouplesvoltage-sensor movement from channel activation. Strong membrane depolarization (panel B) leads to conformational changes that result in unbinding of Gβγ and loss of inter-action between the N-terminus and the I–II linker. This depends upon binding of a CaVβ subunit to the AID on the I–II linker that induces a rigid α-helical connection to theupstream IS6 region of the pore and voltage-sensor. In the absence of CaVβ subunit binding, inhibition still occurs (panel Ci) but cannot be reversed by strong depolarization(panel Cii).

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domain I–II linker was also observed in functional assays. Zamponiet al. [160] showed that intracellular dialysis of tsA-201 cells with~20 amino acid peptides directed against different regions of theI–II linker of both Cav2.1 and Cav2.2 channels prevented the abilityof exogenously delivered Gβγ subunits to mediate voltage dependentinhibition of the channels. The first site contains a QXXER consensussequence (QQIER in all three CaV2 family members) found in otherGβγ binding partners. This site also overlapped partially with theputative Cavβ subunit binding domain on the channel (the AID). Sub-sequent co-crystal structures of the Cavβ subunit bound to its interac-tion site on the isolated domain I–II linker revealed that only a part ofthe 20 amino acid stretch forming the putative Gβγ interaction site islikely to be accessible in the presence of a bound Cavβ subunit [46].This may suggest two possibilities: Either the Cavβ subunit partiallydissociates from regions involved in Gβγ binding, or alternativelyGβγ interacts with those residues that remain exposed after Cavβdocking. Further support for the involvement of the I–II linker camefrom scanning mutagenesis of the amino acids in each of the twobinding regions in rat CaV2.2 channels [161]. Mutation of two residues(Arg376 and Val416 to alanine) out of the thirty tested significantly re-duced the magnitude of voltage-dependent inhibition while mutationof Arg376 to phenylalanine increased inhibition. Irrespective of the pre-cise nature of the Gβγ interaction on the domain I–II linker, this gen-eral region has been implicated as being important for functionalchannel inhibition by a number of other groups [116,131,136]. Thesestudies contrast with the work of Qin and colleagues [134] whosedata implicated the C-terminus rather than the domain I–II linker asthe critical element for G protein modulation. While likely playingan auxiliary role, the C-terminus region does not appear to be essentialfor N-type channel inhibition as large parts can be deleted with onlysmall consequences on the extent of receptor mediated voltage-dependent modulation [155,162,163].

Several other groups attributed an important role to theN-terminus ofthe channel based on site directed mutagenesis work [136,138,151,164].The Dolphin lab identified the N-terminal 55 amino acids of CaV2.2,and in particular an eleven amino acid stretch (45–55) that is predictedto form an α-helix [165], to be critical for Gβγ-mediated inhibition ofthe channels. The Yue group demonstrated a direct interaction of theN-terminus with Gβγ [151] and that the N-terminus (residues 56–95)also binds directly to the I–II linker. Thus, the N-terminus contributesboth to binding of Gβγ, and as an “inhibitory module” which binds theI–II linker to perhaps mediate the shift from willing to reluctant gatingstates. Finally, a recent study revealed that a point mutation (S218L) inthe domain I S4–S5 linker of Cav2.1 that is found in patients with familialhemiplegicmigraine (FHM) facilitates recovery of the channels fromGβγinhibition, perhaps by facilitating the dissociation of the G protein dimer[166]. Two other FHM mutations (R192Q, Y1245C) have also beenreported to diminish Gβγ-mediated inhibition [167,168].

Taken together, several sites on both the CaV2 α1 subunit and theGβγ heterodimer have been implicated in voltage-dependent inhibi-tion. On the Cav2 α1 subunit, the domain I–II linker and N-terminusare essential structural elements (Fig. 4). Ultimately crystal structuredata will be needed to precisely determine how G protein subunitsinteract with these channel loci in the presence and absence of theCavβ subunit.

7. Contribution of the CaVβ subunit to voltage-dependent inhibition

The subtype of CaVβ can influence the extent and kinetics of Gβγ-mediated inhibition and this depends on the subtype of Gβ involved[169,170]. However, the precise role of CaVβ subunits in voltage-dependent inhibition of ICa has been unclear (for reviews see [49,79]).Overlapping binding sites for the two proteins have been identified onthe I–II linker, and one fundamental question that arose was whether

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CaVβ andGβγ can bind to the channel at the same time, orwhether theycompete in a mutually exclusive manner. Seemingly contradictory dataincluding FRET analyses suggested either competition [171] or synergis-tic binding [172]. Some of this confusion might stem from endogenousCaVβ subunits found in some heterologous expression systems (includ-ing Xenopus oocytes), or confounding shifts in the voltage-dependenceof activation by some CaVβ subunits (see [79]). Evidence from theDolphin and Yang labs outlined below now suggest that both proteinscan interact with the channel simultaneously, and that binding of theCaVβ subunit is required to confer voltage-dependent reversal to Gβγ-mediated inhibition (Fig. 4) [173–176].

The Dolphin lab introduced a mutation (W391A) into the AID onthe I–II linker of CaV2.2 channels which reduces CaVβ subunit bindingaffinity by ~1000 fold. [174]. While the extent of Gβγ-mediated inhi-bition was similar for mutant (W391A) and wild-type channels,prepulse reversal of the inhibition was almost abolished in the mu-tant. Expression of wild type CaV2.2 along with α2δ but withoutCaVβ resulted in similar findings, and the voltage-independent inhibi-tion in the absence of the CaVβ was blocked by overexpression oftransducin which acts to scavenge free Gβγ subunits [175]. Thus, inthe absence of CaVβ binding to the I–II linker, Gβγ-mediated inhibi-tion of the channels was still present but could no longer be reversedin a voltage-dependent manner. The experiments outlined aboveused the β1b subunit, but when β2a was expressed with the W391Achannels voltage-dependent relief of the Gβγ mediated inhibitionwas restored. Unlike β1b, the β2a subunit is palmitoylated at twoN-terminal cysteine residues, and mutation of these residues led toloss of voltage-dependent relief (i.e. the data resembled β1b). Theauthors proposed that palmitoylation increased the local plasmamembrane concentration of β2a such that low affinity interactionwith α1 could still take place and permit voltage-dependent relief ofthe inhibition.

The Yang lab came to similar conclusions for CaV2.1 channels[176]. In this case the authors mutated CaVβ to reduce the affinityfor the AID. The channels were expressed in Xenopus oocytes andmacroscopic currents were recorded from giant inside-out patchesthat contained many channels. Washing the cytoplasmic face of thepatches resulted in dissociation of CaVβ (due to the reduced bindingaffinity of the mutant), and this was confirmed by the expected shiftsin channel kinetics compared to wild type. In these channels lackingCaVβ, purified Gβγ still inhibited the currents but prepulse reversalwas abolished.

The CaVβ subunit consists of SH3 and GK domains separated by avariable HOOK region [48,49]. Binding to the AID on the I–II linkerof α1 is mediated by the GK domain, although interaction betweenthe SH3 and HOOK domains elsewhere on the α1 subunit might alsomodulate functional properties. In terms of Gβγ effects, voltage-dependent reversal was restored even by binding of the isolated GKdomain of CaVβ to the AID [175,176]. In the absence of such bindingthe AID adopts a random coil, but the presence of CaVβ induces anα-helical conformation that extends back to the interface with IS6[46,47,177,178]. The Yang lab introduced seven glycines betweenthe AID and IS6 to disrupt this α-helical structure and found thatthis prevented the ability of CaVβ to confer voltage-dependence tothe inhibition [176]. Conversely, introducing seven alanines (notexpected to disrupt the α-helix) maintained the ability of CaVβ toconfer voltage-dependence to Gβγ-mediated inhibition. It is possiblethat binding of CaVβ to the AID induces a rigid α-helical link withdomain IS6, and this transmits movement of the voltage-sensor andactivation gate (including IS6) to the I–II linker to alter the Gβγ bind-ing pocket at depolarized potentials. It is also worth noting thatGβγ-mediated inhibition was still present in both channel types lack-ing CaVβ, and in the CaV2.1 channels containing the seven glycine in-sert [175,176]. Apparently the rigid α-helical link to the upstreamactivation gate and voltage-sensor is not required per se to transducebinding of Gβγ into functional inhibition. CaVβ might also influence

Gβγ-mediated inhibition in other ways. For example, deletion of theHOOK domain promoted tonic inhibition of CaV2.2 channels, perhapsdue to increased affinity for the basal level of free Gβγ in the cells[175].

8. Crosstalk between N-type channels, Gβγ, kinases and synapticproteins

8.1. Protein kinase C

Most cell signaling events do not occur in isolation, but instead inan integrated fashion. G protein regulation of voltage-gated calciumchannels is no exception. This is exemplified by the modulation ofvoltage dependent Gβγ inhibition of N-type channels by proteinkinase C (PKC). In peripheral neurons, activation of PKC was shownto reduce the extent of subsequent G protein modulation by a numberof different receptor pathways, including GABA-B, adenosine andmuscarinic receptors [179–181]. Such an interference with G proteininhibition could be due to PKC dependent phosphorylation of the Gprotein interaction site on the channel, the G protein coupled recep-tor, or the G protein itself. The first hint supporting the first mecha-nism came from experiments showing that in vitro phosphorylateddomain I–II linker peptides could no longer effectively interact withGβγ peptides [160]. Subsequent work showed that a threonine resi-due within the putative I–II linker Gβγ interaction site was responsi-ble for this effect. When phosphorylated, or substituted for glutamicacid, this residue destabilizes the interaction of the channel withGβγ, and its substitution for alanine precludes the antagonistic effectsof PKC [155]. Interestingly, only Gβ1 mediated signaling (not otherGβ isoforms) was subject to this type of PKC crosstalk [182], andthis was attributed to a single locus unique to Gβ1 [153] (Fig. 3C).This observation suggests that activation of Gq-coupled receptorscan modulate signaling of certain types of Gβ1 linked receptors toN-type calcium channels. It should also be noted that PKC activationnot only results in antagonistic effects on Gβγ-mediated inhibitionbut, depending on N-type channel splice variant, can also promotedirect enhancement of current activity [155]. This is mediated byphosphorylation of both the above noted threonine residue and anadjacent serine and adds further complexity to the PKC-G protein sig-naling crosstalk.

8.2. Synaptic proteins

The two types of calcium channels that are most susceptible to theeffects of Gβγ also control neurotransmitter release at CNS synapses[1]. Both Cav2.1 and Cav2.2 channels physically associate with pro-teins that are involved in synaptic vesicle release, such as syntaxin1A and SNAP25. These SNARE proteins bind directly to a synaptic pro-tein interaction (synprint) site on the II–III linker (Fig. 1) whichserves to bring the channels into close proximity of the synaptic ves-icle release sites [183–189]. RIMs (rab3 interacting molecules) havealso emerged as important organizers of the presynaptic active zone[190], and bind CaV2 channels both directly and through RIM bindingproteins to control their density and localization at release sites[191–195]. Binding of syntaxin 1A to both Cav2.1 and Cav2.2 also re-sults in a hyperpolarizing shift in the voltage dependence of channelinactivation [185,196–198] (for a review see [199]). In addition tothis effect on channel gating, syntaxin 1A modulates G protein regu-lation of the channels. Coexpression of syntaxin 1A with N-type chan-nels in tsA-201 cells induces tonic inhibition mediated by Gβγ [200].Syntaxin 1A physically associates with Gβγ at a site distinct from thatinvolved in binding to the N-type channel [198,201], suggesting thepossibility that syntaxin 1A serves to colocalize the channels andGβγ to ultimately promote a form of tonic inhibition. In contrast,syntaxin 1B does not mediate such an effect, even though it is capableof binding to both the channel and Gβγ [202]. This may suggest that

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the spatial orientation of the syntaxin/G protein complex relative tothe N-type channel complex is critical for functional modulation.Gβγ interaction with SNARE proteins might also serve to directly reg-ulate neurotransmitter release in both lamprey and mammalian neu-rons and neuroendocrine cells [203–206].

Several other types of synaptic proteins have been shown to alter Gprotein regulation of N-and P/Q-type channels. Cysteine string protein(CSP) interacts with G proteins and the synprint site and mediates aneffect similar to that seen with syntaxin 1A [207]. In addition, CSPappears to stimulate Gα subunit activity by promoting the exchangeof GTP for GDP in a receptor independent manner [208]. In contrastto these enhancing effects of syntaxin 1A and CSP, coexpression ofRim1 with Cav2.2 in HEK293 cells promotes “deinhibition” (recoveryfrom Gβγ-mediated inhibition during depolarization) in addition tosubstantially slowing channel inactivation [209].

8.3. Calcium channel γ subunits

Another protein of note is stargazin, a member of the calcium chan-nel γ subunit family. Skeletal muscle CaV1.1 channels have been shownto associate with a γ1 subunit in addition toβ andα2δ subunits. Severalneuronal γ subunits have been identified although it remains uncertainthat these constitute bona fide channel subunits. Indeed the γ2 isoform(also called stargazin) and related proteins (γ3–7) associate with andmodulate glutamatergic AMPA receptors [210]. However, it has beenshown that stargazin can bind Gβγ in vitro, and acts to scavenge Gβγand reduce inhibition of CaV2.2 channels in Xenopus oocytes [211]. Alto-gether, these findings highlight the notion that Gβγmodulation of cal-cium channels does not occur in isolation, but is tightly controlled by awide range of cellular processes and signaling pathways.

9. Direct GPCR/N-type calcium channel interactions

Efficient signaling necessitates close proximity between GPCRs andeffectors such as ion channels. This can be accomplished through theformation of largemacromolecular signaling complexes between recep-tors, channels, G proteins, and kinase anchoring proteins [212–215].In addition, a physical association between receptors and channelsprovides for a possible mechanism by which receptors can control

Fig. 5. Trafficking-mediated modulation of CaV2.2 channels due to direct interaction with Gtheir C-termini. D1 and D2 receptors also interact with additional regions of the α1 subuntrafficking of the channels to the plasma membrane, and the D1 receptor appears to targeagonist application has been reported to promote co-internalization of the receptor/channeof voltage-independent inhibition [220] (but see [221]).

channel function in an agonist independent manner. This was firstshown by Kitano and colleagues [216] who identified a physical asso-ciation of Cav2.1 channels with metabotropic glutamate receptors thatresults in altered P/Q-type channel function. For N-type channels, theformation of a signaling complex between Cav2.2 and the NOP (a.k.a.nociceptin) receptorwas demonstrated in dorsal root ganglion neurons,and shown to promote tonic voltage-dependent modulation in theabsence of receptor ligand, presumably reflecting constitutive receptoractivity [217]. Similar observations have been reported for δ- andμ-opioid receptors coexpressed with N-type channels in tsA-201 cells[218,219].

Association of channels with receptors also provides for an addi-tional level of control through regulation of channel density in theplasma membrane. NOP receptors coexpressed with N-type channelsnot only promote the cell surface expression of the channels (Fig. 5A),but also trigger an agonist mediated co-internalization of the chan-nel/receptor complex into lysosomes [219,220], thus giving rise to anew form of voltage-independent inhibition (Fig. 5B). The extent towhich this occurs in neurons is up to some debate. While imagingstudies show a clear NOP receptor mediated internalization of chan-nels in cultured DRG neurons, and reduced calcium entry in responseto prolonged activation of receptors [220], there do not appear tobe clear effects on whole cell current densities in nociceptin treatedneurons [221]. It is possible that receptor mediated internalizationis offset by kinase pathways that augment the activities of channelsremaining in the plasma membrane. D1 and D2 dopamine receptorsalso associate with N-type calcium channels [222,223], but whileNOP receptors and Cav2.2 channels interact via their C-termini, D1and D2 receptors also interact with other regions of the Cav2.2 chan-nel α1 subunit such as the domain II–III linker. As with the NOP re-ceptor, D1 or D2 receptor coexpression facilitates trafficking of thechannels to the plasma membrane, and allows for receptor-channelco-internalization. In prefrontal cortex neurons, the D1 receptor ap-pears to target N-type channels to dendritic sites [222]. It is likely thatother types of GPCR may form complexes with N-type and perhapsP/Q-type calcium channels, however this will need to be confirmed ex-perimentally. Altogether, the formation of macromolecular signalingcomplexes between receptors and channels provides for previouslyunrecognized means for controlling channel activity/density.

PCRs. (A) Nociceptin receptors (NOP) interact directly with the Cav2.2 α1 subunit viait such as the domain II–III linker. Coexpression of these GPCRs with CaV2.2 facilitatest N-type channels to dendritic sites in the prefrontal cortex [222]. (B) Prolonged NOPl complex into lysosomes in cultured sensory neurons, thus giving rise to a new form

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10. Voltage-independent inhibition of CaV2 channels byGq-coupled GPCRs

Sympathetic neurons have been used extensively to investigatemodulation of CaV2.2 (N-type) channels, and at least two distinctpathways have been identified: the “fast” pathway, mediated by per-tussis toxin sensitive Gi/o-coupled GPCRs, is due to direct, voltage-dependent inhibition by Gβγ and a “slow”, voltage-independentpathway takes tens of seconds to develop, is mediated by Gq-coupled GPCRs, involves a diffusible second messenger, and is sensi-tive to intracellular [BAPTA] (for reviews see [224,225]). TypicalGq-coupled signaling pathways downstream of phospholipase Cβ in-cluding IP3/Ca2+, diacylglycerol, and PKC were shown not to mediatethe inhibition, and the pathway remained elusive for quite some time.In the past decade evidence has mounted implicating depletion ofplasma membrane PIP2 and/or generation of arachidonic acid as pos-sible mediators of this inhibition [226–229]. Here we outline the pro-posed mechanisms along with some recent developments.

PIP2 is required for a variety of ion channels to function (for reviewssee [229–232]). The first evidence that this included Ca2+channels wasthe demonstration that time-dependent “rundown” (loss) of CaV2.1channel activity in excised membrane patches was slowed by applica-tion of PIP2 and accelerated by depleting or sequestering PIP2[233]. Sim-ilar effects were subsequently reported for N-type (CaV2.2) channels,along with evidence that the “slow” inhibition by Gq-coupled GPCRsin sympathetic neurons was due to phospholipase C mediated PIP2hydrolysis [234]. For example, inhibition by muscarinic receptors wasblunted by including PIP2 in the patch pipette, whereas recovery frominhibition was slowed by blocking PI-4 kinases which replenish the de-pleted PIP2. The overall picture that has emerged is that PIP2 is requiredfor channels to open in response to membrane potential changes. Thismay involve dynamic low affinity interaction of PIP2 with the channelsand perhaps additional higher affinity binding to a distinct channeldomain. It has been postulated that such interactions might “crosslink”hydrophobic and hydrophilic domains and favor protein conformationsconducive to active channel states. Similar to Gq-mediated modulationof M-type potassium channels [226,230], this model proposes that de-pletion of local PIP2 by phospholipase C mediated hydrolysis removesthis permissive interaction and is both necessary and sufficient to inhib-it channel activity.

An alternative, although related, lipid signaling pathway has beenproposed by the Rittenhouse lab [227] who reported that arachidonicacid elicits bidirectional modulation of N-type channels; ICa was en-hanced at relatively hyperpolarized test potentials and inhibited atmore depolarized potentials [235,236]. The enhancement seems toinvolve extracellular actions of arachidonic acid [237], whereas theinhibition is mediated at the cytoplasmic face of the membrane. Ara-chidonic acid can be produced either by the action of phospholipaseA2 on PIP2 and other membrane phospholipids, or by the action ofdiacylglycerol-lipase on diacylglycerol [227]. It is postulated that acti-vation of these lipases by muscarinic receptors cleaves PIP2 and gen-erates arachidonic acid which binds to the channel. This binding hasthe opposite effect to PIP2 such that it stabilizes closed/inactivatedstates of the channel and thus leads to inhibition. The involvementof arachidonic acid is a matter of some debate, in part due toconflicting reports on the ability of DAG-lipase inhibitors to blockchannel inhibition [226,227,234,236].

A recent study from theHille lab has provided evidence in support ofthe PIP2 depletion model [238]. To avoid downstream and parallel sig-naling pathways subsequent to Gq-coupled receptor activation, the au-thors used controlled activation of exogenous polyphosphoinositide5-phosphatases which convert PIP2 into PI(4)P [239,240]. In one ap-proach, cells were transfected with a voltage-sensitive phosphatase(VSP) that enabled rapid (~1 s) and reversible depletion of PIP2. Anoth-er approach involved chemical dimerization to translocate transfectedyeast INP54p 5-phosphatase to the membrane and irreversibly deplete

PIP2. In both cases ICa was inhibited by PIP2 depletion, and the rate/extent of recovery from this inhibition tracked the rate/extent of PIP2resynthesis [238]. These data support the idea that depletion of PIP2 initself is sufficient to inhibit ICa. However, it was noted that the magni-tude of ICa inhibition was less than that produced by muscarinic recep-tors, even though the predicted depletion of PIP2 is comparable.Therefore, it is possible that another signal, perhaps arachidonic acid,also contributes to the Gq-mediated inhibition, perhaps through syner-gistic actions with PIP2 depletion [226,227].

10.1. CaVβ and intracellular Ca2+ modulate Gq-mediated inhibition

Notably, it has been reported that the CaV β2a subunit opposes in-hibition of ICa by arachidonic acid or PIP2 depletion. The β2a subunit ispalmitoylated at two N-terminal residues and it is this lipidation thatdiminishes the inhibition. Indeed, N-type channels containing β2a areenhanced rather than inhibited by Gq-coupled GPCRs and arachidonicacid [60,227,241]. Rittenhouse and colleagues postulated that thepalmitoyl groups interact directly with the α1 subunit of the channeland thereby mask an inhibitory binding site for arachidonic acid. Inhi-bition of ICa by PIP2 depletion (using a voltage-sensitive phosphatase)is also diminished in channels containing a palmitoylated β2a subunit[61]. It is speculated that the palmitoyl groups of CaVβ2a, the lipid tailof PIP2, and arachidonic acid compete for binding to a site(s) on theα1 subunit of the channel. Binding of the palmitoyl groups or lipidtails of PIP2 favors active channel conformations whereas loss of thisinteraction and/or binding of arachidonic acid favors closed/inactivechannel conformations.

Highlighting the complexity of neuronal Ca2+ channel regulation,it is noteworthy that even in the same cell, not all Gq-coupled GPCRselicit inhibition of ICa [234]. This correlates with the ability of the dif-ferent receptors to elicit significant IP3 mediated release of intracellu-lar Ca2+ stores which likely relates to the proximity of the GPCR, IP3receptors, and other components of a macromolecular signaling com-plex [226,242]. In turn, such Ca2+elevations are postulated to pro-mote phosphatidylinositol 4-kinase activity thereby preventing localPIP2 depletion and inhibition of ICa [226,234,243,244]. Thus, the ex-tent of this voltage-independent inhibition depends on the subunitcomposition of the channels (CaVβ isoform), and colocalization ofGPCRs and various phosphoinositide and Ca2+signaling componentsin macromolecular signaling complexes. Adding further to this com-plexity, another type of “fast”, voltage-independent inhibition ofN-type ICa mediated by a distinct pathway(s) perhaps involving bothGα and Gβγ signaling has also been described in these same sympa-thetic neurons [245,246].

11. Kinase-mediated, voltage-independent inhibition of CaV2channels in sensory neurons

Voltage-independent inhibition that appears to involve channelphosphorylation has also been described. For example, PKC hasbeen implicated in the inhibition of N-type ICa in chick sensory neu-rons [247,248]. In frog and mammalian neurons PKC can also potenti-ate ICa, might target multiple phosphorylation sites with opposingactions, antagonize Gβγ mediated inhibition, or modulate channeltrafficking (see Sections 8, 9) [155,180,181,249,250]. It was alsoreported that rapid activation of a tyrosine kinase by GABAB receptorsresulted in voltage-independent inhibition of N-type ICa in chick sen-sory neurons [251]. More recently, the Lipscombe lab demonstratedthat manifestation of this tyrosine kinase mediated inhibition inmammalian neurons is controlled by alternative splicing of the CaV2.2C-terminus (Fig. 6) [252,253]. There are two mutually exclusive formsof exon 37 (e37a and e37b). Gβγ-mediated, voltage-dependent inhibi-tion is identical in recombinant channels containing either e37a ore37b. However, channels containing exon 37a are also inhibited byanother voltage-independent pathway. This second pathway involves

Fig. 6. Alternative splicing of CaV2.2 controls voltage-independent inhibition of N-type ICa in sensory neurons. Two mutually exclusive forms of exon 37 encode the proximalC-terminus of CaV2.2. Expression of exon 37a is restricted to dorsal root ganglia, preferentially in nociceptive neurons, while exon 37b is widely expressed throughout the nervoussystem [254]. Gβγ-mediated, voltage-dependent inhibition of ICa is identical in channels containing either isoform of exon 37. An additional Gα-mediated, voltage-independentpathway involving pp60c-src tyrosine kinase inhibits channels containing exon 37a but not exon 37b. Thus alternative splicing of CaV2.2 results in cell-type specific alteration inthe magnitude and mechanisms of GPCR-mediated inhibition.

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rapid activation of pp60c-src tyrosine kinase and requires a tyrosineresidue (Y1747) present in exon 37a that is replaced by phenylalaninein exon 37b. Of particular note, expression of exon 37a is restricted todorsal root ganglia, and preferentially expressed in capsaicin sensitive,nociceptive neurons [254]. The gene encoding chicken CaV2.2 only hasone exon 37 which is similar to e37a and includes a tyrosine residue[252]. This then explains the restriction of this pathway to nociceptiveneurons in mammals and its prevalence in chick neurons. This expres-sion pattern also suggests that the e37a splice variant might be tailoredto play a role in pain transmission [255]. Recently, using an exon re-placement strategy in mice, it was shown that basal nociceptive trans-mission was unaltered by loss of e37a, but the analgesic effects ofintrathecal morphine were diminished [253].

12. Concluding remarks

In this review we have highlighted the complex inhibition of CaV2channels by G protein coupled receptors. Voltage-dependent inhibi-tion, mediated by direct binding of Gβγ to the Ca2+ channel α1subunit, is the most common and best understood mechanism. Mem-brane potential, firing patterns, channel subunit composition/splicevariants, and Gβγ heterodimer composition all modulate the extentand/or kinetics of voltage-dependent inhibition. Although less wellunderstood and perhaps less widespread, there are also several mech-anisms leading to voltage-independent inhibition of CaV2 channels.These include direct interaction with GPCRs, inhibition through lipidsignaling pathways, and channel phosphorylation. CaV2 channels arealso subject to a variety of other regulatory mechanisms, notablyCa2+-dependent feedback (both inactivation and facilitation). Thus,GPCRs in combination with Ca2+ channels sense and integrate a com-plex array of inputs in order to fine tune the spatiotemporal aspectsof Ca2+ entry that play such pivotal roles in cellular physiology andsynaptic transmission.

Acknowledgements

Work in the Currie lab is supported by the National Institutesof Health, National Institute of Neurological Disorders And Stroke[Grant R01-NS052446], and by the American Heart Association. GWZ

is supported by the Canadian Institutes of Health Research, is anAI-HS Scientist and a Canada Research Chair. The molecular graphicsimages in Fig. 3 were produced using the UCSF Chimera packagefrom the Resource for Biocomputing, Visualization, and Informaticsat the University of California, San Francisco (supported by NIH P41RR001081) [256,257].

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