1521-0111/87/6/890–906$25.00 http://dx.doi.org/10.1124/mol.114.096008MOLECULAR PHARMACOLOGY Mol Pharmacol 87:890–906, June 2015Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics

MINIREVIEW

G Protein Regulation of Neuronal Calcium Channels: Back tothe Future

Juliane Proft and Norbert WeissInstitute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic

Received September 18, 2014; accepted December 30, 2014

ABSTRACTNeuronal voltage-gated calcium channels have evolved as oneof the most important players for calcium entry into presynapticendings responsible for the release of neurotransmitters. In turn,and to fine-tune synaptic activity and neuronal communication,numerous neurotransmitters exert a potent negative feedbackover the calcium signal provided by G protein–coupled receptors.This regulation pathway of physiologic importance is also extensively

exploited for therapeutic purposes, for instance in the treatment ofneuropathic pain bymorphine and otherm-opioid receptor agonists.However, despite more than three decades of intensive research,important questions remain unsolved regarding the molecular andcellular mechanisms of direct G protein inhibition of voltage-gatedcalcium channels. In this study, we revisit this particular regulationand explore new considerations.

IntroductionWithin neurons, calcium ion (Ca21) represents an essential

important signaling molecule, responsible for regulation ofa large number of diverse cellular functions (Berridge, 1998).Voltage-gated Ca21 channels (VGCCs) have evolved as one ofthe most important players in the initiation of the Ca21 signalby converting electrical impulses into intracellularCa21elevation(Catterall, 2011). VGCCs are pore-forming multisubunit plasmamembrane complexes that are activated upon membrane de-polarization (i.e., action potentials) to permit entry of Ca21 alongits electrochemical gradient. To date, 10 genes encoding the pore-forming subunits of mammalian VGCCs have been identified(Fig. 1). Seven genes encode the high-voltage–activated chan-nel subfamily consisting of L-type (Cav1.1 to Cav1.4), P/Q-type(Cav2.1), N-type (Cav2.2), and R-type (Cav2.3), and three genesencode the low-voltage–activated channel subfamily composedexclusively of T-type channels (Cav3.1 to Cav3.3) (Ertel et al.,2000). The Cav pore-forming subunits of VGCCs share a similartransmembrane topology built of four homologous domains, eachof them containing six putative transmembrane helices (S1–S6),plus a re-entrant loop (P-loop) that forms the pore of the channel.

The four domains are connected via large cytoplasmic linkers(loops I-II, II-III, III-IV) and cytoplasmic amino- and carboxy-terminal domains, which form interaction sites for variousregulatory proteins. In addition to the Cav pore-forming sub-unit, high-voltage–activated channels contain ancillary sub-units (Arikkath and Campbell, 2003): b (b1 to b4, a 55-kDacytosolic protein of the membrane-associated guanylate kinasefamily), a2d (a2d1 to a2d4, a 170-kDa highly glycosylatedextracellular protein with a single transmembrane domain),and in some cases g (g1 to g8, a 33-kDa transmembrane pro-tein), which control channel trafficking, gating, and functionat the plasma membrane.To make use and regulate the amplitude, duration, and

subcellular localization of the Ca21 signal, VGCCs are undertight regulatory control. One of the most important regula-tory mechanisms involves G protein–coupled receptors (GPCRs),also known as seven-transmembrane domain receptors. GPCRsare a large protein family of integral membrane receptors(Vassilatis et al., 2003) that sense extracellular molecules suchas neurotransmitters and in turn activate intracellular sig-naling pathways by regulating the activity of heterotrimericG proteins. Heterotrimeric G proteins consist of a Ga subunitthat binds and hydrolyzes GTP into GDP, and Gb and Gg

subunits that remain constitutively associated and form theGbg dimer (Wettschureck and Offermanns, 2005). In the ab-sence of stimulus, GDP-bound Ga subunit and Gbg dimer

The work in N.W.’s laboratory was supported by the Czech ScienceFoundation [Grant 15-13556S] and the Institute of Organic Chemistry andBiochemistry (IOCB). J.P. is supported by a postdoctoral fellowship fromIOCB.

dx.doi.org/10.1124/mol.114.096008.

ABBREVIATIONS: AID, a interaction domain; AP, action potential; CSP, cysteine string protein; DRG, dorsal root ganglion; FHM-1, familialhemiplegic migraine type-1; FRET, fluorescence resonance energy transfer; GPBP, Gbg protein–binding pocket; GPCR, G protein–coupledreceptor; GST, glutathione S-transferase; HEK, human embryonic kidney; M2, muscarinic receptor 2; ORL-1, opioid receptor like-1; PIP2,phosphatidylinositol 4,5-bisphosphate; SNARE, soluble N-ethylmaleimide–sensitive fusion protein attachment protein receptor; VGCC,voltage-gated calcium channel.

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are associated with the receptor. Binding of an extracellularligand onto the GPCR induces a conformational change thatpromotes the exchange of GDP for GTP from the Ga sub-unit, resulting in the dissociation of the GTP-bound Ga andGbg dimer from the receptor. Intrinsic hydrolysis of GTP byGa subunit can be speeded up by GTPase-activating pro-teins such as regulators of G protein signaling that allowsreassociation of GDP-bound Ga subunit with the Gbg dimer,which terminates G protein signaling (Fig. 2). Although bothGTP-bound Ga subunit and the Gbg dimer mediate intra-cellular signaling by modulating the activities of neuronalVGGCs, this review is focused on the so-called direct voltage-dependent regulation mediated by the Gbg dimer. Interestedreaders may also refer to the recent review of Zamponi andCurrie (2013) for an interesting discussion on the regulationof VGCCs by Ga- and protein kinase–dependent signaling.

Description of the PhenomenonInterestingly enough, althoughCa21 entry at synaptic endings

can trigger transmitter release, numerous neurotransmitters re-leased from synaptic contacts and hormones secreted at proxi-mity of the synaptic cleft are in turn able tomodulate presynapticVGCCs via activation of GPCRs to terminate the Ca21 signaland neurotransmitter discharge. This regulation is not only of

physiologic importance, but it is also extensively exploited asa therapeutic avenue. For example, one of the most remark-able usages of G protein–mediated inhibition of VGCCs is themanagement of pain symptoms by specific opioid receptoragonists (e.g., natural opiates like morphine and its syntheticopioid derivatives).The first observation that synaptic activity is modulated

by neurotransmitters goes back to the late 1970s with thepioneer work of Dunlap and Fischbach (1978) on sensoryneurons, and later the phenomena was attributed to theinhibition of VGCCs (Dunlap and Fischbach, 1981). To date,up to 20 neurotransmitters and corresponding receptors havebeen described to modulate VGCCs (Table 1), including nor-adrenaline (Bean, 1989; Docherty and McFadzean, 1989;Lipscombe et al., 1989; McFadzean and Docherty, 1989),somatostatin (Bean, 1989; Ikeda and Schofield, 1989a,b),GABA (Deisz and Lux, 1985; Dolphin and Scott, 1987; Grassiand Lux, 1989), and acetylcholine (Bernheim et al., 1991;Shapiro et al., 1999).Based on the observation that application of pertussis toxin

on rat dorsal root ganglion neurons (DRG), or intracellularinjection of nonhydrolyzable GDPbS, prevents inhibition ofvoltage-activated Ca21 currents by noradrenaline or GABA,it was proposed that heterotrimeric G proteins certainlymediate GPCR-dependent modulation of VGCCs (Holz et al.,

Fig. 1. Structural organization and diversity of voltage-gated Ca2+ channels. VGCCs are integral membrane proteins composed of a Cav pore-formingsubunit surrounded by Cavb, a2d, and in some cases g auxiliary subunits. (A) Putative membrane topology of the Cav pore-forming subunit. It consists offour repeat homologous domains (I–IV) linked by large intracellular loops (loops I-II to III-IV), each composed of six transmembrane segments (S1 to S6).The S4 segment of each of the four domains, rich in positively charged residues, presumably constitutes the voltage sensor, whereas the P loops betweensegments S5 and S6 form the pore selectivity filter. Cter, C-terminus; Nter, N-terminus; PM, plasma membrane. (B) Schematic representation of theVGCC macromolecular complex embedded in the plasma membrane in association with typical auxiliary subunits. (C) Overview of the diversity ofVGCCs. High-voltage–activated (HVA) channels are composed of Cav1 (L-type) and Cav2 (P/Q-, N- and R-types) subfamilies, whereas low-voltage–activated (LVA) channels are only composed of Cav3 channels, so-called T-type channels.

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1986; Scott and Dolphin, 1986). Using an original approach,Forscher et al. (1986) further demonstrated that this regulationis spatially delimited and does not involve diffusible secondmessengers, suggesting proximity between the Ca21 channeland the GPCR. The functional importance of channel/GPCRcoupling inG protein–mediated inhibition of Ca21 currents willbe further discussed later in this review. A period of inten-sive work and controversy followed (1989–1996) to determinewhich of the G protein subunits mediate inhibition of theCa21 channel. Using specific antibodies and antisense oligonu-cleotides to block or knock down Ga subunits of heterotrimericG proteins, it was initially proposed that Gao is the mediatorof the inhibition (McFadzean et al., 1989; Baertschi et al.,1992; Campbell et al., 1993; Menon-Johansson et al., 1993).However, during the same period of time, various studiessuggested as well an implication of Gai (Ewald et al., 1989)or Gas and Gaq (Shapiro and Hille, 1993; Golard et al., 1994;Zhu and Ikeda, 1994). The divergent results led to thehypothesis that GPCR-mediated inhibition of VGCCs is notmediated by Ga subunits, but rather by a common G proteindeterminant, and Herlitze et al. (1996) and Ikeda (1996)eventually established that inhibition of Cav2 channels ismediated by the Gbg dimer concomitantly produced withGa-GTP following GPCR activation. Indeed, the overexpressionof Gb1g2 or Gb2g3 dimers in sympathetic neurons is sufficientto mimic noradrenaline-mediated inhibition of N-type currents,and prevents subsequent inhibition by a2-adrenergic agonists.The question is how can we incorporate those results in the

observation of other groups suggesting a functional implicationof Ga subunits? Interestingly enough, it appears that eventhough Gbg dimer is themediator of the inhibition, Ga subunitsmight have an important role in the ability and specificity ofGPCRs to functionally couple with VGCCs. Hence, inhibition ofneuronal VGCCs is usually mediated by GPCRs coupled to Gaior Gao (Holz et al., 1986; Scott and Dolphin, 1986) such as a2-adrenergic receptors, clarifying why depletion of the Gaosubunit in NG108-15 cells prevents noradrenaline-mediatedinhibition of Ca21 currents (McFadzean et al., 1989).

Landmarks of GPCR-Mediated Inhibition ofVGCCs

Inhibition of VGCCs by GPCRs involves the direct bind-ing of G protein bg dimer onto various structural molecu-lar determinants of the Cav2 subunit (see next section). Atthe whole-cell level, this regulation is characterized by vari-ous phenotypical modifications of the Ca21 current properties(Fig. 3). The most obvious is a decrease of the inward cur-rent amplitude (Boland and Bean, 1993; Wu and Saggau,1997) that usually varies from 15 to 80% depending on theCav channel/GPCR involved. Based on the observation thatGbg-mediated inhibition of VGCCs is less pronounced at de-polarized membrane potential, this regulation was namedvoltage-dependent. In some cases, this inhibition is also ac-companied by a depolarizing shift of the voltage-dependencecurve of current activation (Bean, 1989) and a slowing of activation

Fig. 2. GPCR activation cycle. In the inactive state of the receptor (A), the GDP-bound Ga subunit and the Gbg dimer are associated together on thereceptor. Conformational change of the receptor upon agonist binding (B) catalyzes the exchange of GDP for GTP on the Ga subunit, and GTP-boundGa subunit and Gbg dimer complex dissociate (C) and activate downstream signaling. Intrinsic hydrolysis of GTP to GDP, potentially stimulated byRGS proteins, allows reassociation of the Gbg dimer with GDP-bound Ga subunit (D), which terminates G protein signaling.

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(Marchetti et al., 1986) and inactivation kinetics (Zamponi,2001). In addition, short highly depolarizing voltage step,usually applied about 1100 mV before the current elicitingpulse (double-pulse protocol), is sufficient to reverse, at leastpartially, most of the landmarks of G protein inhibition andproduce a so-called prepulse facilitation (Scott and Dolphin,1990; Ikeda, 1991; Doupnik and Pun, 1994). Current in-hibition has been attributed to the direct binding of Gbg

dimer to the Cav2 subunit (referred to as ON landmark),whereas all the other landmarks, including the slowing ofcurrent kinetics and prepulse facilitation, can be described asvariable time-dependent dissociation of Gbg dimer from thechannel (referred to as OFF landmarks) and consequentcurrent recovery from inhibition (Elmslie and Jones, 1994;Stephens et al., 1998; Weiss et al., 2006). It is worth notingthat ON and OFF landmarks do not represent two indepen-dent regulations, but rather the transition from Gbg-boundchannels to Gbg-unbound channels, and vice versa. Further-more, although the dissociation of Gbg dimer was previouslydefined as voltage-dependent, it was then proposed thatchannel opening after membrane depolarization and associ-ated conformational changes of the Cav2 subunit was mostlikely the trigger for Gbg dissociation from the channel (Patilet al., 1996). More recently, this concept was further analyzed,and it was shown that the voltage dependence of the kinetic ofGbg dissociation correlates to the voltage dependence of the

channel activation (Weiss et al., 2006). Hence, it is likely thatthe trigger of Gbg dissociation is not the electrical membranepotential per se, but rather the conformational change thatoccurs within the Cav2 subunit during the opening of thechannel, making the regulation intrinsically channel opening-dependent rather than voltage-dependent.

What Are the Cav Channel MolecularDeterminants of G Protein Inhibition?

Various structural determinants of the Cav2 subunit havebeen characterized either as direct biochemical binding locifor Gbg dimer or of functional importance for GPCR-mediatedregulation of Ca21 currents. These determinants are locatedwithin the I-II loop of the Cav2 subunit, as well as in theamino- and carboxyl-terminal regions of the channel.Role of the I-II Loop of Cav2 Channels. The observa-

tion that Gbg dimer is able to functionally interact with theCavb subunit (Campbell et al., 1995) led some groups toquestion the role of the I-II loop of Cav2 channels in G proteinregulation. It is well established that Gbg dimer is able tointeract with the type II adenylate cyclase and the phospho-lipase Cb2 via a consensus motif QXXER (Chen et al., 1995).Interestingly, this consensus site is also present within theI-II loop of Cav2 channels, and is located in a proximal regionof the so-called a interaction domain (AID), a 18–amino-acid

TABLE 1Neurotransmitter- and receptor-mediated G protein modulation of Cav2 channels

Neurotransmitter Receptor Cav Channel Tissue/Species Reference

Ach M4 Cav2.2 SCG/rat Bernheim et al., 1991M2 Cav2.1 and Cav2.2 SCG/mouse Shapiro et al., 1999

Adenosine A1 Cav2.2 Ciliary ganglion/chicken Yawo and Chuhma, 1993Cav2.2 DRG/chicken Kasai and Aosaki, 1989Cav2.1 and Cav2.2 Cerebellum/rat Dittman and Regehr, 1996Cav2.2 and Cav2.3 Hippocampus (CA3→CA1)/rat Wu and Saggau, 1994

ATP/ADP P2Y Cav2.2 SCG/rat Brown et al., 2000;Filippov et al., 2000a

Dopamine D2 HVA DRG/chicken Marchetti et al., 1986Endocannabinoids CB1 Cav2.1 SCG/rat Garcia et al., 1998a

Cav2.1, Cav2.2, andCav2.3

Cerebellum Brown and Russell, 2004

GABA GABA B Cav2.1 and Cav2.2 DRG/rat Dolphin and Scott, 1987DRG/chicken Deisz and Lux, 1985; Grassi and Lux, 1989Cerebellum/rat Dittman and Regehr, 1996

Cav2.2 and Cav2.3 Hippocampus (CA3→CA1)/guinea pig

Wu and Saggau, 1995

Cav2.2 SCG/rat Filippov et al., 2000bGalanin GalR1 Cav2.2 Hypothalamus/rat Simen et al., 2001Glutamate mGluR1 Cav2.2 SCG/rat Kammermeier and Ikeda, 1999LHRH LHRH-R Cav2.2 SCG/bullfrog Elmslie et al., 1990; Boland and Bean, 1993;

Kuo and Bean, 1993Noradrenaline a2-Adrénergique Cav2.2 SCG/bullfrog Bean, 1989

Cav2.2 SCG/rat Garcia et al., 1998bNon-L NG108-15 Docherty and McFadzean, 1989; McFadzean and

Docherty, 1989NPY Y2 Non-L SCG/rat Plummer et al., 1991

Cav2.2 SCG/rat Toth et al., 1993Opioids -

Enkephalinsm Cav2.2 NG108-15 Kasai, 1992

Opioids -Dynorphins

k Non-L DRG/rat Bean, 1989

Serotonin 5HT-1A Non-L Spinal neuron/Lamprey Hill et al., 2003Somatostatin SS-R Cav2.2 DRG/rat Ikeda and Schofield, 1989b

SCG/rat Ikeda and Schofield, 1989aSubstance P NK1 Cav2.2 SCG/rat Shapiro and Hille, 1993VIP VIP-R Cav2.2 SCG/rat Zhu and Ikeda, 1994

Ach, acetylcholine; LHRH, luteinizing hormone-releasing hormone; NPY, neuropeptide Y; SCG, superior cervical ganglion; VIP, vasoactive intestinal peptide.

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sequence (QQIERELNGY–WI–AE) initially described as themain interaction locus of the Cavb subunit (Pragnell et al.,1994), suggesting a possible interaction between the I-II loopand the Gbg dimer. Consistent with this idea, injection inhuman embryonic kidney (HEK)293 cells of the syntheticpeptide of the Cav2.2 subunit FLKRRQQQIERELNGYL(peptide I-IIS2; Fig. 4) prevents Gbg-mediated inhibition ofCav2.2/a2/b1b channel, suggesting that Gbg dimer is able tointeract with the peptide (Zamponi et al., 1997). This in-teraction was then demonstrated by in vitro binding of Gb1g2dimer onto the glutathione S-transferase (GST)–AIDA fusionprotein with an affinity of 63 nM (De Waard et al., 1997). Forcomparison, Cavb1b binds to the GST-AIDA fusion proteinwith an apparent Kd of 5 nM that is a dozen times moreefficient than the binding of Gbg dimer (De Waard et al.,1995). In addition, the role of the QXXER domain in thebinding of Gbg dimer was further analyzed by site-directedmutagenesis. Hence, substitution of the arginine (R) by aglutamic acid (E) is sufficient to prevent the binding of Gb1g2dimer to the GST-AIDA fusion protein, but also preventsGTPgS-induced inhibition of Cav2.1/b4 channels expressed inXenopus oocyte (De Waard et al., 1997). This functional resultis, however, in contradiction with another observation that

the same amino acid substitution in the Cav2.1 channel ex-pressed in tsA-201 cells together with the Cavb1b subunitpromotes channel inhibition by GTPgS (Herlitze et al., 1997).At this point, it is worth noting that substitution of thearginine located in the QXXER motif to a glutamic acid notonly prevents the binding of the Gbg dimer, but also slowsdown the inactivation kinetics of the channel (Herlitze et al.,1997). Hence, the QXXER domain appears to be not only abinding locus of the Gbg dimer within the I-II loop of the Cav2subunit, but also an important molecular determinant of thefast inactivation of the channel. Considering that channelinactivation is by itself an important component for backmodulation of G protein inhibition, as we will see later, it isdelicate to conclude on the exact functional role of the QXXERdomain. It was also shown that substitution of the isoleucine(I) to a leucine (L) within the QQIER domain of the Cav2.1subunit decreases GTPgS-induced inhibition of Cav2.1/b1b

channel (Herlitze et al., 1997). Altogether, these resultsprovide a structural explanation for the weak sensibility ofCav1 channels, which instead contain a QQLEE motif, toGPCR inhibition (Bell et al., 2001). However, insertion of theconsensus QXXER domain into the Cav1.2 subunit is notsufficient to make the channel sensitive to G protein inhibition

Fig. 3. Putative kinetic model illustrating landmarks of Gbg-dependent regulation of Cav2 channels. This scheme relies on a single conducting state inwhich dissociation of the Gbg dimer has occurred, whereas the channel remains open. (A) Gbg-unbound closed channel. The transition from Gbg-boundclosed channel (B) to Gbg-unbound open conducting channel (D) requires a transition through an additional transient Gbg-bound open nonconductingstate of the channel (C) characterized by whole-cell current inhibition (G) (ON landmark). This transition state produces a time-dependent currentrecovery from Gbg-dependent inhibition, at the origin of the slowing of activation/inactivation kinetics of the whole-cell current (H) and an apparentdepolarized shift of the voltage dependence of activation (I) (OFF landmarks). Norm. ICa21, normalized calcium current. Dissociation of the Gbg dimerfrom the Cav channel subunit is intrinsically independent of the membrane potential and is rather triggered by conformational changes of theCav subunit upon channel opening. Inactivation can occur either in the absence (E) or presence (F) of Gbg but not necessarily with the same rate.

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(Herlitze et al., 1997), suggesting that, despite an importantrole in the biochemical coupling of Gbg dimer with the channel,the QXXER domain is not the sole molecular determinant forpotent channel inhibition by G proteins. Hence, additional mo-lecular loci of the I-II loop have been identified. For example,injection in HEK293 cells of the GVLGEFAKERERVENRRApeptide of the Cav2.2 subunit (peptide I-IIS1; Fig. 4), localizedupstream the AID domain and straddling the IS6 trans-membrane segment and the I-II loop is able to preventGbg-dependent inhibition of Cav2.2/a2/b1b channels, suggest-ing a second Gbg dimer-binding locus (Zamponi et al., 1997).Finally, the G protein interaction domain, localized 13 aminoacid residues upstream the AID domain, has also been pro-posed as a molecular locus for Gbg binding with an affinity of20 nM (De Waard et al., 1997), and the corresponding syn-thetic peptide is likewise able to prevent Gbg modulation ofCa21 currents (Zamponi et al., 1997). Interestingly enough,protein kinase C–dependent phosphorylation of the G proteininteraction domain on a threonine residue prevents bindingof Gbg, suggesting a functional crosstalk between direct (i.e.,mediated by Gbg dimer) and indirect (i.e., involving second

messengers) GPCR modulation of VGCCs (Zamponi et al.,1997).Altogether, it is unambiguous that the cytoplasmic I-II loop

of Cav2 channels contains various structural determinantsfor Gbg dimer binding. However, the functional importanceof those determinants in the modulation of the Ca21 currentsremains largely discussed, and other groups rather suggestedthat the I-II loop was not critical for Gbg-mediated inhibitionof Ca21 channels. Hence, it has been shown that the sub-stitution of the I-II loop of the Cav2.2 subunit by the I-II loop ofthe Cav2.1 channel (less sensitive to Gbg inhibition) or by theI-II loop of the Cav1.2 channel (insensitive to direct G proteinmodulation) does not alter somatostatine-mediated inhibitionof the Cav2.2/b1b channel (Zhang et al., 1996). Conversely, thereplacement of the I-II loop of the Cav1.2 subunit by the I-IIloop of the Cav2.2 subunit is not sufficient to restore a directG protein modulation of the Cav1.2/b2a channel by D2 do-paminergic receptors (Canti et al., 1999). Finally, the sub-stitution of the I-II loop of the Cav2.3 channel [rat rbE-IIisoform (Soong et al., 1993)]—that is less sensitive to directG protein modulation—by the I-II loop of the Cav2.2 subunit

Fig. 4. Schematic representation of the Cav molecular determinants involved in Gbg-mediated regulation of VGCCs. (A) Schematic representation ofthe location of the important Cav channel molecular determinants of Gbg-dependent regulation identified on the basis of functional evidence. SeveralGbg determinants are present within the I-II loop, including the region S2 containing the QXXER motif and the G protein interaction domain (GID) (inred). The AID of the Cavb subunit on the I-II loop is shown in blue. Furthermore, additional molecular determinants located within the amino- (Nter) andcarboxy- (Cter) terminal regions of the Cav2.1 and Cav2.2 subunits have been functionally documented. The EF hand, IQ (Ca2+-binding domain) andpre-IQ domains, and Ca2+-binding domain (CBD), which constitute the carboxy-terminal Ca2+ binding domains, are shown in blue. Gaq and Gao bindingsites are shown in purple. (B) Schematic representation of a presumable G protein bg-binding pocket within the Cav2 subunits involving the differentCav molecular determinants functionally and biochemically characterized, and most likely responsible for the variable sets of Gbg-mediated modulationof the channel (ON and OFF landmarks). The carboxy-terminal region of the Cav2 subunits also contributes in some cases to the biochemical coupling ofthe channel with the GPCR.

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induces a slowing of activation kinetics of Cav2.3/b2a channelupon activation of G proteins by injection of GTPgS, but no netinhibition of themaximal amplitude of the Ca21 current (Pageet al., 1997). Hence, it was proposed that the I-II loop could bethe channel molecular determinant mediating the slowing ofcurrent kinetics under direct G protein regulation, and thatcurrent inhibition per se could require other molecular de-terminants (Page et al., 1997). Considering that the apparentslowing of current activation kinetics is attributed to thedissociation of Gbg dimer from the channel (OFF landmarks)(Elmslie and Jones, 1994; Stephens et al., 1998; Weiss et al.,2006), the I-II loop might be the channel molecular deter-minant involved in the dissociation of Gbg dimer from theCav2 subunit.Role of the Amino-Terminal Domain of Cav2 Channels.

Interestingly enough, although Ca21 currents generated by theBII-2 brain rabbit isoform of the Cav2.3 subunit are subject todirect G protein modulation [either upon somatostatin applica-tion (Yassin et al., 1996) or by direct injection of GTPgS (Mezaand Adams, 1998)], currents generated by the rat rbE-II isofomthat presents a truncated amino-terminal domain remain in-sensitive (Page et al., 1997), suggesting an implication of theamino-terminal domain of the Cav2 channel in direct G proteinmodulation. Consistent with this idea, extension by polymerasechain reaction of the amino-terminal region of the rbE-II isoform,leading to a Cav2.3 subunit homolog to the rabbit one, issufficient to restore quinpirole (D2 dopaminergic agonist)-mediated inhibition of Ca21 currents (Page et al., 1998).Moreover, the substitution of the amino-terminal region ofthe Cav1.2 subunit by the amino terminal domain of the Cav2.2subunit makes the Cav1.2 channel sensitive to quinpirole-mediated inhibition (Canti et al., 1999). Further workidentified a highly conserved 11–amino-acid sequence(YKQSIAQRART) in the amino-terminal region of Cav2channels critical for direct G protein modulation (Cantiet al., 1999) (Fig. 4A), and alanine scan of the YKQ and RARmotifs abolished quinpirole-induced inhibition of Cav2.3/b2a/a2d

channels (Canti et al., 1999). In addition, it was proposed thatthe amino-terminal region of the Cav2.2 subunit might constitutea G protein–gated inhibitory module acting via interaction withthe cytoplasmic I-II linker of the channel (Agler et al., 2005).Morerecently, Cav2.2 amino-terminal–derived peptides have beenshown to prevent noradrenaline-induced G protein inhibition ofCa21 currents in superior cervical ganglion neurons, strength-ening the implication of the Cav2 amino-terminal region inG protein modulation in native environment (Bucci et al.,2011).Role of the Carboxy-Terminal Domain of Cav2 Chan-

nels. The carboxy-terminal region of the Cav2 subunits hasalso been proposed as an important determinant for directG protein regulation, and a binding domain of Gbg dimer wasidentified in the distal carboxy-terminal region of the Cav2.3subunit (Qin et al., 1997) that presents some homologies withthe corresponding region of the Cav2.1 and Cav2.2 subunits(Simen et al., 2001) (Fig. 4A). Hence, the substitution of thecarboxy-terminal region of the Cav2.3 subunit by the correspond-ing one of the Cav1.2 subunit is sufficient to abolish muscarinicreceptor 2 (M2)–mediated inhibition of the Cav2.3/b2a channel(Qin et al., 1997). Association of G protein b2 subunit with thecarboxy-terminal region of the Cav2.1 channel was also observedusing a fluorescence resonance energy transfer approach (FRET)(Hummer et al., 2003). In addition, based on the observation that

FRET signal between Cav2.1/Cavb1b FRET pairs is increasedin the presence of Gb2 subunit and requires the presence ofthe carboxy-terminal region of the channel, it was proposed thatbinding of G proteins to the carboxy-terminal region mayproduce a conformational change of the channel that mightcontribute to channel inhibition (Hummer et al., 2003). However,deletion of the carboxy-terminal region of the Cav2.2 subunitcontaining the binding determinant of Gbg dimer does not alterinhibition of Ca21 currents mediated by injection of GTPgS(i.e., agonist/receptor-independent) (Meza and Adams, 1998),but only alters somatostatin-mediated inhibition (i.e., agonist/receptor-dependent) of Cav2.2 channels (Hamid et al., 1999),suggesting that the carboxy-terminus might rather be involvedin the functional coupling of the channel with agonist-dependentactivation of G proteins. Interestingly, the binding determinantof Gbg dimer in the carboxy-terminal region of Cav2 channels islocalized close to the binding domain of Gao on Cav2.1 andCav2.2 subunits (Furukawa et al., 1998b) and Gaq on the Cav2.2subunit (Simen et al., 2001) (Fig. 4A), suggesting that thecarboxy-terminal region could be involved in the functional andbiochemical coupling of Cav2 channels with the GPCR via theGa/bg trimmer (Kitano et al., 2003; Beedle et al., 2004; Kisilevskyet al., 2008; Weiss, 2009). Paradoxically, the importance of thecarboxy-terminal region of Cav2 channels in the direct regulationby G proteins was poorly investigated compared with theintensive work that was done for the I-II loop and the amino-terminal region. One possible reason is that injection ofGTPgS, or overexpression of Gbg dimer to trigger directG protein modulation of Cav2 channels, was not suitable tohighlight the functional importance of the carboxy-terminalregion.Toward the Notion of Gbg Protein-Binding Pocket.

As previously seen, the Cav2 subunit contains various mo-lecular determinants for Gbg dimer binding, and it wasinitially proposed that several Gbg dimers could interactsimultaneously with the Cav2 subunit in a cooperative man-ner (Boland and Bean, 1993). However, further analysis re-vealed that the kinetics of Gbg dimer interaction with theCav2.2 channel (evidenced by the functional modulation of theCa21 current) can be described by amonoexponential functionwith a time constant directly correlated to the free Gbg dimerconcentration, suggesting that only a single Gbg dimerinteracts with the Cav2 subunit (Zamponi and Snutch,1998). Based on this observation, it was proposed that thevarious Gbg binding sites located within the Cav2 sub-unit could be spatially structured to form a unique interac-tion domain called the Gbg protein–binding pocket (GPBP)(DeWaard et al., 2005) (Fig. 4B), in which various binding lociof the GPBP could be responsible for a particular feature ofthe G protein regulation. Hence, the carboxy-terminal regionof the Cav2 subunit would play a critical role in the inhibitionof Ca21 currents by GPCRs (Qin et al., 1997; Furukawa et al.,1998a,b) through favoring the biochemical and functionalcoupling of the receptor with the channel via theGa/bg trimmer(Kitano et al., 2003; Beedle et al., 2004). In that context, thecarboxy-terminal domain of the Cav2 subunit would not bedirectly involved in the inhibition of the Ca21 currents, butwould rather allow the rapprochement of the GPCR with thechannel required for the direct G protein modulation (Forscheret al., 1986). The I-II loop of the Cav2 subunit represents animportant domain of interaction with the Gbg dimer via threemotifs clearly identified. However, the binding of Gbg dimer

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onto the I-II loop does not appear critical for the inhibition ofthe Ca21 current (ON landmark) (Zhang et al., 1996; Qin et al.,1997), but rather seems to be involved in the relaxation of theinhibition (OFF landmarks, i.e., unbinding of Gbg dimer fromthe Cav2 subunit) in response, for example, to a depolarizingprepulse (Herlitze et al., 1997; Simen and Miller, 2000). Fi-nally, the amino-terminal region of the Cav2 subunit appearsas the main determinant of direct G protein inhibition ofCa21 currents by Gbg dimer (Page et al., 1998; Canti et al.,1999). In that context, the GPBP emerges as a dynamicstructure, composed of various loci that on one hand bring theCa21 channel and the GPCR together, and on the other handdifferentially mediate the ON and OFF G protein landmarks.

How Does Gbg Dimer Inhibit Cav2 Channels?Mutagenesis studies have identified a number of amino

acid residues on the surface of the Gb subunit important forinhibition of the Ca21 channel (Ford et al., 1998; Mirshahiet al., 2002; Tedford et al., 2006). Interestingly, most of theseresidues are located on the Gb surface that interacts with Ga

and are essentially masked when Ga is present. However, themolecular mechanism by which binding of Gbg dimer to thechannel inhibits the Ca21 current remains largely unknown.It has to be mentioned that, besides the extensive work thatwas done to understand the molecular mechanisms of Gprotein regulation of VGCCs, it still remains unclear howthe binding of Gbg dimer to the Cav2 subunit inhibits theCa21 current. Based on the observation that the voltage-dependence curve of the Ca21 current under G proteinmodulation is significantly shifted to more depolarizedpotentials, it was initially proposed that the Ca21 channelundergoes a switch from a willing mode (i.e., easily activated)to a reluctant mode (i.e., hardly activated) upon GPCR ac-tivation (Bean, 1989). The channel returns to the willingstate after dissociation of the Gbg dimer from the Cav2subunit (Bean, 1989; Elmslie et al., 1990). Consistent withthis idea, single-channel recordings revealed latency in thefirst opening of the Cav2.2 channel upon M2 muscarinicreceptor activation, after which the channel presents a similarbehavior as the nonregulated control channel (Patil et al.,1996). Hence, it was proposed that the first opening latencycould correspond to the time for Gbg dimer to dissociate fromthe Cav2 subunit in response to the membrane depolarizationprior to channel opening (Patil et al., 1996). It is worth notingthat low-probability openings of Cav2.2 channels under Gprotein modulation have been observed, corresponding mostlikely to transient dissociations of the Gbg dimer from thechannel upon moderate membrane depolarizations (30 mV)(Lee and Elmslie, 2000). This simple model of regulationappears to be sufficient to support both the inhibition of theCa21 current and the depolarizing shift of the current/voltageactivation curve. However, the observation that the ampli-tude of gating currents of Cav2.2/a2/b1b channels is reducedupon injection of GTPgS led the authors to propose thatG proteins mediate inhibition of Ca21 current by alteringintrinsic gating properties of the channel (Jones et al., 1997).Gating currents are produced by the movement of the chargedvoltage-sensor domains, including positively charged S4 seg-ments, that move into the lipid bilayer in response to elec-trical membrane depolarizations and lead to the openingof the channel. It is conceivable that Gbg dimer could bind

either to the intracellular end of the S4 segment of the Cav2subunit, or to another structural determinant of the voltagesensor exposed on the intracellular surface of the channel,preventing the proper movement of the voltage sensor andthus inhibiting the channel. Consistent with this idea, it wasshown that the point mutation G177E localized in the thirdtransmembrane segment of the first domain (IS3) of theCav2.2a subunit (rat rbB-I isoform) and induces a tonicinhibition of the channel that can be reversed by a depolariz-ing prepulse as the classic G protein inhibition (Zhong et al.,2001). Interestingly, although this inhibition shares some ofthe features of the G protein modulation, activation ofG proteins does not produce additional inhibition. Similarly,introduction of the G177E mutation into the Cav2.2b subunit(rat rbB-II isoform) produces a tonic inhibition of the channeland prevents G protein–mediated modulation of the Ca21

current (Zhong et al., 2001). In contrast, the E177G mutationintroduced into the Cav2.2a subunit reverses tonic inhibitionof the channel and restores a normal G protein regulation.Hence, it was proposed that the negative charge introducedby the mutation G177E could interact with a positivelycharged residue of the S4 segment of the first domain of theCav2 subunit, pushing the channel to a reluctant mode viaa mechanism of voltage sensor trapping, suggesting that Gbg

dimer could possibly regulate the channel in a similar way(Zhong et al., 2001; Flynn and Zamponi, 2010). Intriguingly,Gem2, which belongs to the RGK (Rad, Gem, and Kir) fam-ily of small G proteins, is also able to inhibit Cav2.2 chan-nels (Chen et al., 2005). However, in contrast to the directG protein inhibition by Gbg dimer, depolarizing prepulsesdo not reverse Gem2-mediated inhibition (Chen et al., 2005).Remarkably, structure analyses of Rad, a homolog of Gem,indicate the presence of a molecular motif similar to the sevenrepeated motifs of the Gb subunit, suggesting a commonmechanism by which Gbg dimer and RGK proteins modu-late VGCCs. Another possible model of channel regulation byG proteins that does not require any alteration of gatingparameters was also proposed in which willing and reluctantmodes are intrinsic to the channel, whereas G proteins andother effectors simply shift the fraction of channels in thesetwo states without modifying their intrinsic gating properties(Herlitze et al., 2001). This willing to reluctant model couldalso potentially explain the decrease in gating currentsinitially observed during G protein regulation (Jones et al.,1997). Finally, based on the observation that sodium currentsrecorded through Cav2.2 channels are less affected thancalcium currents by G protein inhibition, it was proposed thatbinding of Gbg dimer onto the Cav2 subunit alters ionpermeation of the channel (Kuo and Bean, 1993). Hence,consistent with the observation that a 35–amino-acid peptide(residues 271–305) of the Gb2 subunit is sufficient to inhib-it Ca21 currents (Li et al., 2005), it is also conceivable thatGbg dimer simply inhibits the channel via a pore-modifiermechanism. However, such a mechanism could only contrib-ute partially to the overall current inhibition given the effectof Gbg dimer on single-channel gating properties (essentiallylatency of the first opening). Either way, the exact molecu-lar mechanisms by which Gbg dimer inhibits VGCCs re-main essentially unknown and certainly deserve furtherinvestigation. Recent structural information obtained fromvoltage-gated sodium channels (Payandeh et al., 2011; Zhanget al., 2012) will certainly provide key information to further

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analyze the binding of G proteins to the channel subunit andfind out the molecular determinant of the inhibition.

What Is the Functional Role of the Cavb Subunitin Direct G Protein Inhibition?

The involvement of the Cavb subunit in the direct regula-tion of VGCCs by GPCRs was extensively examined. Hence,the Cavb subunit and Gbg dimer share a structural bindingsite (QXXER located in the AID domain of the I-II loop) on theCav2 subunit, suggesting a possible crosstalk. The initialobservations that expression of the Cavb3 subunit in Xenopusoocytes decreases G protein inhibition of Cav2.1 and Cav2.2channels (Roche et al., 1995), and conversely that inhibition ofCav2.1 channels by m-opioid and M2 muscarinic receptors isincreased in the absence of Cavb subunit (Bourinet et al.,1996; Roche and Treistman, 1998a), led the authors to proposethat the Cavb subunit is antagonistic to the direct G proteinmodulation of Cav2 channels. In addition, it was shown thatbinding of Gbg dimer to the carboxy-terminal region of theCav2 subunit is abolished by the guanylate kinase domain oftheCavb2a subunit, which also preventsM2muscarinic receptor–dependent inhibition of the Ca21 current, providing a possibleexplanation for the functional antagonism existing betweenthe Cavb subunit and direct G protein inhibition of VGCCs(Qin et al., 1997). However, considering that the carboxy-terminal domain of the Cav2 subunit plays an important rolein the functional coupling with the GPCR, it is likely that theCavb-dependent antagonism observed relies on an alteredcoupling of the channel with the GPCR rather than on thedirect alteration of Gbg dimer binding to the channel. Incontrast to the antagonistic hypothesis, it was also proposedthat the Cavb subunit could rather be essential for potentG protein modulation of the Cav2 channels (Meir et al., 2000).Consistent with this idea, it was proposed using a FRETapproach that Gbg dimer induces Cav2.1 channel reluctanceby displacement of the Cavb subunit from the channel (Sandozet al., 2004). However, besides the fact that direct G proteinmodulation of Cav2 channels has been observed numeroustimes in the absence of the Cavb subunit (Roche et al., 1995;Bourinet et al., 1996; Canti et al., 2000), this observation isbalanced by a study using a similar FRET approach showingthat binding of Cavb1b and Gb to the Cav2.1 subunit is notexclusive, but rather synergetic (Hummer et al., 2003). Inaddition, it was shown that recovery of the Cav2.2 channelfrom G protein inhibition is not only influenced by the pres-ence of a Cavb subunit (Roche and Treistman, 1998b), but alsodepends on the Cavb isoform (b3 . b4 . b1b . b2a) (Cantiet al., 2000). Considering that channel activity is an importantfactor of the OFF G protein landmark, these results suggestthat the Cavb subunit could modulate G protein inhibitionindirectly by biophysical changes induced on the channel.Consistent with this idea, the respective kinetics of channelinactivation induced by various Cavb subunits nicely cor-relates with the kinetic of recovery from G protein inhi-bition (Weiss et al., 2007a). It was thus proposed that theCavb subunit, by controlling channel inactivation, indirectlyinfluences the speed of G protein dissociation from the channel.Hence, fast-inactivating Cavb subunits (example Cavb3) act asa cofactor to speed the recovery from the G protein inhibition.However, the situation becomes more complicated consideringthe fact that fast-inactivating Cavb subunits, by accelerating

the inactivation kinetics of the channel, also reduce the tem-poral window for G protein dissociation (Fig. 5). Hence, fast-inactivating Cavb subunits, while speeding up the rate of Gprotein dissociation, also reduce the maximal extent of currentrecovery from inhibition that leads to an apparent decrease ofthe prepulse facilitation. In contrast, a slow-inactivating Cavbsubunit (for instance, Cavb2a) that slows down channel in-activation has minor effect on the kinetic of G protein dis-sociation, but prolongs the temporal window of opportunity forG protein dissociation and leads to a far more complete currentrecovery from inhibition, evidenced by an apparent increasedprepulse facilitation (Weiss et al., 2007a). It is worth notingthat the amino-terminal domain of the Cav2 channels that hasbeen implicated in the direct G protein inhibition of VGCCsmediates Cavb-dependent fast inactivation of Cav2.2 channel(Stephens et al., 2000). In addition, the R387E mutation in theQXXER motif of the I-II loop that alters G protein regulation(De Waard et al., 1997; Herlitze et al., 1997) also affects chan-nel inactivation. Hence, it is possible that these molecularchannel determinants contribute to G protein regulation not

Fig. 5. Functional role of the Cavb subunit in Gbg-dependent modulationof VGCCs. In this scheme, the presence of a Cavb subunit does not affectthe binding of the Gbg dimer to the channel and thus does not alter themaximal extent of inhibition (ON regulation). In contrast, Cavb-mediatedinactivation of the channel delimitates a temporal window during whichthe Gbg dimer can dissociate from the open channel before it inactivates,letting Ca2+ flow into the cell. Hence, a slow-inactivating channel pro-duced, for example, by the Cavb2 subunit (A) presents a large temporalwindow for Gbg unbinding (Gbg-unbound open channel state) and currentrecovery, evidenced by increased OFF regulation landmarks (slowing ofactivation/inactivation kinetics) and apparent decreased ON regulation(apparent decreased current inhibition). In contrast, a fast-inactivatingchannel produced by Cavb3-subunit (B) rather undergoes inactivation,decreasing the occurrence of a Gbg-unbound open channel state, pro-ducing an apparent increased current inhibition (ON regulation) whilepreventing OFF regulation.

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only by providing an anchor for the Gbg dimer, but also viatheir intrinsic effect on channel gating.

How Do Synaptic Proteins Modulate DirectG Protein Inhibition?

The two types of Ca21 channels (Cav2.1 and Cav2.2) that aremost responsible for voltage-dependent release of neurotrans-mitter at nerve terminal endings biochemically associate withpresynaptic vesicles of transmitters (Bennett et al., 1992;Yoshida et al., 1992; Leveque et al., 1994) by interacting withsome of the proteins of the vesicular machinery releasecomplex (soluble N-ethylmaleimide–sensitive fusion proteinattachment protein receptors [SNAREs]), including syntaxin-1A and synaptosomal-associated protein 25 (SNAP-25). Mo-lecular characterization of channel/SNARE interaction hasidentified a synprint (synaptic protein interaction) locus inCav2.1 and Cav2.2 located within the intracellular loop be-tween domains II and III of the channels (Sheng et al., 1994;Rettig et al., 1996). It is believed that the functional relevanceof this interaction is to bring presynaptic vesicles close to theCa21 source for efficient and fast neurotransmitter release anddisruption of Cav2/SNARE complex alters Ca21-dependent re-lease of neurotransmitter (Mochida et al., 1996; Rettig et al.,1997; Harkins et al., 2004; Keith et al., 2007). Furthermore,direct binding of syntaxin-1A and SNAP-25 to the Cav2.1 andCav2.2 subunits also potently modulates channel gating byshifting the voltage dependence of channel inactivation towardmore negativemembrane potentials (Bezprozvanny et al., 1995;Wiser et al., 1996; Zhong et al., 1999; Degtiar et al., 2000; Weissand Zamponi, 2012; Zamponi and Currie, 2013), although thephysiologic relevance of this regulation is not fully understood.Interestingly, although the synprint site of Cav2.1 and Cav2.2subunits, and in general the II-III intracellular linker, are notessential molecular determinants of Gbg-mediated modula-tion of the channel activity, proteolytic cleavage of synatxin-1A with botulinium neurotoxin C1 in primary neurons wasshown to completely prevent GPCR-dependent inhibition ofpresynaptic Ca21 channels (Stanley andMirotznik, 1997). How-ever, because G protein modulation of transiently expressedCav2.2 channels in Xenopus oocytes (Bourinet et al., 1996) orHEK cells (Zamponi et al., 1997) does not require coexpressionwith syntaxin-1A, it is unclear why the cleavage of syntaxin-1A in native neuronal environment would lead to a total lossof G protein modulation. In contrast, Jarvis et al. (2000) re-ported that syntaxin-1A is not essential, but facilitates G proteinmodulation of the Cav2.2 channel. Indeed, whereas Cav2.2transiently expressed in tsA201 cells in combination withGb1g2 dimer is susceptible to a tonic inhibition that can beassessed (relieved) by a preceding strong membrane depolari-zation, coexpression of syntaxin-1A produced an even largertonic G protein inhibition, suggesting that syntaxin-1A facil-itates Gbg-dependent inhibition of the channel. However, itremains unclear whether syntaxin-1A facilitates G protein inhi-bition or promotes Gbg dimer dissociation from the channelupon prepulse depolarization. Indeed, as previously discussed inthe case of the Cavb subunit, it is possible that syntaxin-1A,by affecting channel gating, indirectly influences the kinetic ofG protein dissociation from the channel, producing a largerprepulse facilitation that could bemisinterpreted as the result ofan increased initial G protein inhibition. In support of this idea,coexpression of SNAP-25 that reverses syntaxin-1A–dependent

channel gating also reduces the magnitude of the prepulse–induced current recovery upon tonic G protein inhibition (Jarvisand Zamponi, 2001). However, this concept should also betoned down, as coexpression of a mutant syntaxin-1A that islocked permanently in an open conformation (the conformationadopted by syntaxin-1A when in interaction with SNAP-25 orsynaptobrevin-2) (Dulubova et al., 1999; Brunger, 2001) has noeffect on Cav2.2 gating, but still modulates Gbg-dependentinhibition of the channel (Jarvis et al., 2002). Conversely,syntaxin-1B affects Cav2.2 channel gating, but does not supportG protein modulation (Jarvis and Zamponi, 2001). Altogether,these results suggest that syntaxin-1A may present specificfeatures responsible for G protein modulation independently ofits effect on the channel gating, possibly by facilitating thecolocalization of G proteins with the Ca21 channel. Consistentwith this idea, a syntaxin 1/Gao/Cav2.2 complex has been de-scribed at presynaptic nerve terminals of chick ciliary ganglioncells (Li et al., 2004).Several other types of presynaptic proteins have been docu-

mented as important in the modulation of G protein inhibitionof VGCCs. For example, coexpression of Rim1 significantlydecreases direct [D-Ala2,N-MePhe4, Gly-ol]-enkephalin–inducedG protein inhibition of Cav2.2 channels expressed in HEK293cells (Weiss et al., 2011). Interestingly, careful kinetic analysesrevealed that Rim1 does not prevent inhibition of the channel(ON landmark, i.e., binding of Gbg dimer to the channel), butrather favors channel recovery from inhibition (OFF land-marks, i.e., unbinding of Gbg dimer from the channel), mostlikely by slowing down channel inactivation (Kiyonaka et al.,2007), increasing the time window for functional recovery fromG protein inhibition similarly to Cavb subunits (Weiss et al.,2007a, 2011). Modulation of G protein inhibition of VGCCshas also been documented with cysteine string protein (CSP).Although CSP has been shown to stimulate Ga subunit activityby promoting the exchange of GTP for GDP (Natochin et al.,2005) that in turn is expected to reduce free Gbg dimers, co-expression of CSP increases apparent Gbg-dependent inhibi-tion of Cav2.2 channel similarly to what was observed withsynatxin-1A (Magga et al., 2000). Although the molecularmechanism underlying CSP-dependent modulation of G pro-tein inhibition remains to be further explored, it is possible thatCSP brings G proteins together with the Ca21 channel becausebinding of CSP has been documented with Cav2.1 and Cav2.2subunits (Leveque et al., 1998; Magga et al., 2000), as well aswith G proteins Ga subunit and Gbg dimer (Magga et al., 2000).

How Does the Termination of the Signal Occur?To be efficient, cellular events have to be localized and

timely regulated. Hence, we have seen that direct G proteinmodulation of VGCCs requires the following: 1) the activationof the GPCR by specific extracellular ligands; 2) the releaseand binding of Gbg dimer to the channel, leading to a complexregulatory phenotype; and finally 3) that this regulation canbe temporally relieved, beside the continuous presence ofthe extracellular agonist, in response, for example, to a trainof action potentials. However, how does this regulation de-finitively end? Although the recapture and/or degradation ofthe agonist terminate the activation of the GPCR, how doesthe channel-bound Gbg dimer reassociate with the Ga sub-unit? Because channel recovery from G protein inhibition canoccur at membrane potentials above the reversal potential of

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Ca21, ion influx is not the driving force for Gbg dimer dis-sociation from the channel. From a biochemical and ther-modynamic point of view, it is conceivable that intrinsichydrolysis of GTP by the Ga subunit and formation of GDP-bound Ga subunit—which presents a high affinity for Gbg

dimer—could chelate free Gbg when dissociating from thechannel, according to a binding/unbinding thermodynamicsequilibrium. It is also possible that the GDP-bound Ga sub-unit is able to bind Gbg dimer while still on the Cav subunit.This concept implies the molecular determinants of Ga andGbg binding being accessible when the Gbg dimer is bound tothe channel. Binding of GDP-Ga to Gbg could produce aconformational change of the channel Cav subunit and/or Gbgdimer, unfavorable to the formation of Cav/Gbg complex, pos-sibly by altering an essential channel molecular determinantimportant for Gbg binding. Although this molecular deter-minant has not been identified yet, it is likely to contribute toGbg dissociation upon channel activation, and possibly in-volves channel structures sensitive to electrical membranepotential, like S4 segments, for example. However, this con-cept remains to be investigated to be able to better under-stand the molecular dynamic of GPCR-induced inhibition ofVGCCs.

Are Cav2 Channels the Only Calcium ChannelsSusceptible to Direct G Protein Inhibition?

It is thought that direct G protein modulation of VGCCs ismostly confined to neuronal Cav2 channels. Indeed, structure/function studies indicate that Cav1 channels that lack most ofthe believed important structural determinants involved inthe binding of the Gbg dimer to the channel, such as theQXXER motif of the I-II loop (Herlitze et al., 1997), are notsubject to direct G protein regulation (Roche et al., 1995;Bourinet et al., 1996; Zhang et al., 1996; Meza and Adams,1998). However, additional studies on native channels clearlydemonstrated an inhibition of L-type Ca21 currents mediatedby heterotrimeric G proteins. For example, injection of GTPgSin cerebellar granule cells induces inhibition of L-type Ca21

currents that is partly reversed upon channel activation bythe dihydropyridine agonist1(S)-202-791 (Haws et al., 1993).Moreover, inhibition of L-type Ca21 currents has been docu-mented upon agonist stimulation of GPCRs. Indeed, baclofenactivation of GABA-B receptors in retinal bipolar neuronsinduces inhibition of L-type Ca21 currents, an inhibition thatis potentiated by GTPgS injection (Maguire et al., 1989). Inaddition, activation of mGluR2/3 receptors by applicationof (2S,19S,29S)-2-(carboxycyclopropyl) glycine on cerebellargranule cells also inhibits L-type Ca21 currents (Chavis et al.,1994). Although these results do not provide evidence for adirect G protein inhibition, it is worth noting that GPCR-mediated inhibition of L-type Ca21 currents in nerve cells isfully abolished by pertussis toxin treatment, indicating theimplication of Gai/o proteins. However, evidence for a direct Gprotein modulation of Cav1 channels comes from the obser-vation that inhibition of L-type Ca21 currents in pancreatic bcells can be reversed by application of a depolarizing prepulse(Ammala et al., 1992). Although work performed on nerveand pancreatic cells mostly involved Cav1.2 and Cav1.3 chan-nels, G protein modulation of native Cav1.1 channel isoformhas also been described in skeletal muscle cells in whichL-type Ca21 current inhibition was observed upon b-adrenergic

stimulation by isoproterenol or GTPgS injection (Somasundaramand Tregear, 1993). In addition, it was shown that expressionof Gb1g2 dimer in vivo in adult skeletal muscle fibers spe-cifically inhibits L-type Ca21 currents and voltage-inducedCa21 release (Weiss et al., 2010). Although this inhibition isnot reversed by a classic depolarizing prepulse, the observationthat expression of some other Gbg dimer combinations (for in-stance, Gb2g2, Gb3g2, or Gb4g3) has no effect on theCa21 currentstrongly suggests that Gb1g2 dimer specifically mediatesmod-ulation of Cav1.1 channels. The situation is similar regardingthe low-voltage–activated Cav3 channels. Hence, application ofbaclofen on DRG neurons inhibits T-type Ca21 currents, thisinhibition being often counterbalanced by the concomitantpresence of a current potentiation (most likely mediated bydiffusible second messengers) (Scott and Dolphin, 1990).Similarly, it was shown that inhibition of T-type Ca21 currentby dopamine D1 receptor in rat adrenal glomerulosa cellsrequires the combined action of Gbg dimer and cAMP (Droletet al., 1997). More recently, the molecular mechanism ofT-type Ca21 current inhibition by G proteins was specified. Itspecifically affects Cav3.2 channel over the Cav3.1 isoform,and in contrast to Cav1.1 channels specifically requires theGb2g2 combination (Gb1g2 dimer having no significant effecton the Ca21 current) (Wolfe et al., 2003). The inhibition relieson the direct binding of Gb2g2 dimer to the II-III loop of theCav3.2 subunit, replacement of this loop by the correspond-ing Cav3.1 region preventing G protein modulation. Thisinhibition has been attributed to a diminution of the openingprobability of Cav3.2 channels, without other alteration ofchannel gating or expression (DePuy et al., 2006). It is worthnoting that similarly to what was observed for Cav1 channels,G protein–mediated inhibition of the Cav3.2 channel is notreversed by depolarizing prepulse, although mediated by thedirect binding of Gbg dimer to the channel.Taken together, these results suggest that likely most of

the VGCCs are affected by G protein inhibition. However,in contrast to Cav2 channels, it remains unclear whetherinhibition of Cav1 and Cav3 channels is mediated by a directbinding of Gbg dimer to the channel or required activation ofsecondary signaling pathways. Inhibition of Cav1 and Cav3channels that are usually not reversed by a depolarizingprepulse is also not characterized by a depolarizing shift of thecurrent/voltage activation curve (DePuy et al., 2006), typicalfeature of the direct Gbg-dependent inhibition. However,considering the presence of numerous molecular channeldeterminants that contribute to the phenotype of the directGbg regulation of Cav2 channels, it is possible the Cav1 andCav3 channels lack important molecular determinantsrequired to reveal OFF landmarks of the regulation. Eitherway, future studies will certainly uncover the molecularmechanisms by which G proteins modulate Cav1 and Cav3channels.

Voltage-Independent Inhibition of Cav2 ChannelsAlthough this review is essentially focused on the so-called

fast voltage-dependent regulation mediated by direct bindingof G proteins onto the channel, various studies on nativeneurons and heterologous expression systems have identifiedother types of inhibition that usually take tens of seconds todevelop and involve diffusible second messengers or surfaceremodeling of GPCR/channel complexes.

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Inhibition of VGCCs by Phosphoinositides. DirectGbg-dependent inhibition of VGCCs is usually fast and re-quires the activation of Gi/o-coupled receptors. In contrast,initial recordings performed on sympathetic neurons haveidentified a relatively slow and voltage-independent formof inhibition that is mediated by the activation of Gq-coupledreceptors (Gamper et al., 2004). Although various studies haveruled out the implication of typical Gq-dependent signalingpathways downstream of phospholipase Cb, inositol trisphosphate,diacyglycerol, and protein kinase C, it was proposed thatdepletion of membrane phosphatidylinositol 4,5-bisphosphate(PIP2) is the mediator of Gq-dependent inhibition of VGCCs(Delmas et al., 2005; Michailidis et al., 2007; Roberts-Crowleyet al., 2009; Rodriguez-Menchaca et al., 2012). Consistent withthis idea, loss of Cav2.2 channel activity typically observed inexcisedmembrane patches can be either reduced by applicationof PIP2 or in contrast enhanced by depletion of PIP2 (Gamperet al., 2004). In addition, the time course of the slow inhibitionof Ca21 currents produced by activation of muscarinic M1receptors in sympathetic neurons nicely correlates with thekinetics of PIP2 hydrolysis, whereas infusing of PIP2 into thecell via the patch pipette is sufficient to prevent M1 receptor-dependent inhibition of Cav2.2 channels (Gamper et al., 2004).More recently, using an exogenous voltage-sensitive 5-phosphatase that allows rapid hydrolysis of PIP2 intophosphatidylinositol 4-phosphate, it was shown that mem-brane depletion of PIP2 suppresses Cav1.2, Cav1.3, Cav2.1, andCav2.2 currents, supporting the idea that depletion of PIP2is sufficient to mimic the slow inhibition of calcium currentsobserved underGq-coupled receptor activation (Suh et al., 2010).However, it is worth noting that besides the fact that voltage-sensitive 5-phosphatase–induced PIP2 depletion is similarto the depletion produced by activation of muscarinic receptors,the amplitude of the Ca21 current inhibition is significantlyless, suggesting that another signaling pathway might alsocontribute to the slow Gq-dependent inhibition. Hence, it wasproposed that production of arachidonic acid by the action ofphospholipase A2 on PIP2 and othermembrane phospholipidselicits modulation of Cav2.2 channels (Liu et al., 2001; Liu andRittenhouse, 2003). Interestingly, it was also proposed thatdepletion of membrane PIP2 might reduce Gbg-dependentinhibition of Cav2.1 channels expressed in Xenopus oocytes,suggesting a crosstalk between voltage-dependent and voltage-independent inhibitions (Rousset et al., 2004). Althoughthe molecular mechanism by which PIP2 interferes with Gbgregulation is not fully understood, it was proposed that bindingof the carboxy-terminal tail of the Cav2.1 subunit to membranephosphoinositides might stabilize a Gbg-sensitive state ofthe channel (Rousset et al., 2004). However, considering thatdepletion of membrane PIP2 upon Gaq-dependent activationtakes several minutes, it is unlikely that this mechanismaccounts for the fast initial inhibition phase produced by Gbgdimer.Inhibition of Cav2 Channels by Channel/GPCRComplex

Internalization. Initially proposed by Forscher et al. (1986),G protein–dependent inhibition of VGCCs may require a tightGPCR-channel coupling, and evidence exists for direct biochem-ical interaction between the Ca21 channel and the GPCR. Hence,physical association of Cav2.1 channels with metabotropicglutamate receptors mGluR1 has been documented in cerebel-lar Purkinje neurons as well as in cellular expression systemsand involves the direct binding of the carboxy-terminal domain

of the Cav2.1 subunit with the carboxy-terminal region of thereceptor (Kitano et al., 2003). Similarly, a Cav2.2–opioid re-ceptor like-1 (ORL-1; also known as nociception receptor) sig-naling complex has been documented in small DRG neurons,and supports a tonic agonist-independent G protein inhibitionof the Ca21 channel evidenced by prepulse facilitation (Beedleet al., 2004). Similar observations have been reported form- andd-opioid receptors transiently expressed with Cav2.2 channel intsA201 cells, although the existence of these protein complexesremains to be explored in native conditions (Chee et al., 2008;Evans et al., 2010). Also, a physical interaction exists betweenCav2.2 channels and dopamine D1 and D2 receptors and re-quires other channel structural determinants, including theII-III intracellular linker (Kisilevsky et al., 2008; Kisilevskyand Zamponi, 2008; Weiss, 2009). Although the existence ofCav2-GPCR signaling complexes is unambiguous, the phys-iologic relevance of these interactions is not fully understood. Itwas proposed that association of the Ca21 channels withGPCRs might control channel density at the plasma mem-brane, providing an additional level of control of the Ca21

influx. Indeed, activation of ORL-1 receptors triggers anagonist-dependent cointernalization of Cav2.2–ORL-1 com-plexes into vesicular compartments both in tsA201 cells andDRG neurons (Altier et al., 2006; Evans et al., 2010). However,internalization of Cav2.2 channels is not accompanied by adiminution of the membrane Ca21 current, questioning thephysiologic relevance of this regulation (Murali et al., 2012). Inaddition, although m-opioid receptors also physically interactwith Cav2.2 channels, they do not cointernalize, indicating thatbiochemical coupling of the channel with the GPCR is notsufficient to mediate agonist-mediated internalization of theCa21 channel (Evans et al., 2010). It is also possible that theassembly of Cav2 channels and GPCRs provides a mechanismthat ensures spatiotemporal regulation of the Ca21 enteringsynaptic nerve terminals. In addition, tonic channel inhibitionmediated by channel-GPCR complexes could also representa mean to dynamically adjust the Ca21 influx to the electricalinput signal coming to the synaptic ending. Indeed, it wasshown that the extent of current facilitation (i.e., current re-covery from G protein inhibition) is dependent on both theduration (Brody et al., 1997) and the frequency of actionpotentials (AP) (Penington et al., 1991; Williams et al., 1997).Although low-frequency AP produces no or little recovery, in-creasing AP frequency significantly enhances recovery fromG protein inhibition and Ca21 influx and could contribute toshort-term synaptic facilitation or depression (Bertram et al.,2003).

Contribution of G Protein Modulation toChannelopathies

As previously discussed, gating properties of the Ca21 channelsignificantly affect direct G protein inhibition, essentially theOFF landmarks (i.e., the dissociation of Gbg dimer from theCav2 subunit). Hence, alteration of channel gating is likely toaffect G protein regulation and synaptic activity. A numberof congenital mutations in the gene CACNA1A encoding theCav2.1 channel cause familial hemiplegic migraine type-1(FHM-1), a rare and severe monogenic subtype of migrainewith aura, characterized by at least some degree of hemiparesisduring aura (Ophoff et al., 1996; Weiss et al., 2007b; Ducros,2013; Pietrobon, 2013). FHM-1 mutations generally affect

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structural determinants that are essential for channelgating, including the S4 transmembrane segments thoughtto carry the voltage sensor controlling channel activation,the S6 transmembrane segment involved in the control ofchannel inactivation, and the pore-forming loops. Biophys-ical analyses of channel gating revealed a hyperpolarizingshift of the voltage dependence of activation, as well as ad-ditional effects on channel inactivation kinetics, open pro-bability, and unitary conductance (Kraus et al., 1998, 2000;Hans et al., 1999; Mullner et al., 2004; Tottene et al., 2005;Tonelli et al., 2006). In addition, a knock-in mouse modelexpressing the human pathogenic FHM-1 mutation R192Qlocated in the first S4 segment of the Cav2.1 subunit revealeda decreased neuronal excitability threshold, increased Ca21

influx and cortical spreading depression (i.e., the mechanismunderlying migraine with aura) (van den Maagdenberg et al.,2004; Pietrobon and Moskowitz, 2013), and enhanced excit-atory transmission at cortical synapses (Tottene et al., 2009).Similar alterations have also been documented for the S218Lmutation (van den Maagdenberg et al., 2010). Whereas in-trinsic alteration of Cav2.1 channel gating most likely con-tributes to neuronal hyperexcitability, alteration of theG protein–dependent inhibitory pathway of presynapticCa21 channels may also contribute to synaptic hyperexcit-ability. Consistent with this idea, a decreased G protein in-hibition of R192Q Cav2.1 channels was reported (Melliti et al.,2003). Careful analysis revealed that the R192Q mutationdoes not affect the ON landmark, but rather favors the dis-sociation of Gbg dimer following channel activation (OFFlandmarks), thereby decreasing the inhibitory G protein path-way (Weiss et al., 2008). Similar results were observed withvarious other FHM-1 mutations (Weiss et al., 2008; Garza-Lopez et al., 2012, 2013), indicating that alteration of G proteinregulation of the Cav2.1 channel caused by FHM-1mutations isa common underlying mechanism that certainly contributes tosynaptic defects observed during the disease.Alteration of channel gating can also be caused by change

in regulatory subunits. Hence, an epileptic lethargic pheno-type in mouse resulting from the loss of expression of theCavb4 subunit is accompanied by a Cavb subunit reshuffling(Burgess et al., 1997). Considering that Cavb subunits sig-nificantly affect G protein inhibition of Cav2 channels in aCavb isoform-dependent manner (Weiss et al., 2007a), it islikely to contribute to the altered excitatory synaptic transmis-sion observed in those animals (Caddick et al., 1999; Hosfordet al., 1999).

Concluding Remarks and PerspectivesSince the first description of the phenomena by Dunlap and

Fischbach in the late seventies (Dunlap and Fischbach, 1978),great advances have been made in our understanding ofthe underlying molecular regulation of neuronal VGCCs byGPCRs and its importance in physiology. In this review, weprovided an appreciation of its tremendous complexity, arisingnot only from the numerous molecular channel and G proteindeterminants involved in the regulation, but also from thechannel subunit composition, GPCR subtype, interactions withsynaptic proteins and other intracellular signaling pathways,and most likely many more factors that have not yet beencharacterized. Although numerous channel/G protein–bindingdeterminants have been described, the molecular mechanism

by which Gbg dimer mediates inhibition of the Ca21 currentremains incompletely understood. It is likely thatmore discreteinteractions that have not yet been characterized support thisinhibition, and the recent structural information obtained fromstructurally similar channels will certainly help to find out themolecular basis of G protein inhibition. In addition, the use ofsmall molecules and peptides to selectively disrupt interactionof G protein bg dimer with some effectors has been demon-strated in vitro and in vivo on various models of heart failureand morphine tolerance (Bonacci et al., 2006; Mathews et al.,2008; Casey et al., 2010). A deeper biochemical and func-tional characterization of Gbg channel interaction will cer-tainly provide important information to identified moleculestargeting G protein inhibition of VGCCs with potential ther-apeutic benefits. From a more physiologic point of view,although the most evident outcome of G protein regulationof presynaptic Ca21 channel is a reduction of the Ca21 in-flux entering nerve terminals (ON landmark), the observa-tion that OFF landmarks might play an important rolein fine-tuning synaptic strength, possibly contributing toshort-term synaptic facilitation/depression, represents aninteresting concept in molecular neuroscience that certainlymerits to be further investigated. In addition, the notion thatG protein regulation is altered by pathologic mutations in theCa21 channel complex not only contributes to our understandingof the pathogenesis of neuronal Cav channelopathies, but alsoemerges as an important signaling pathway for potential newtherapeutic strategies.Finally, merely 20 GPCRs from over nearly 1000 estimated

from the sequencing of the human genome (including manyorphan receptors) (Fredriksson et al., 2003; Vassilatis et al.,2003; Zhang et al., 2006) have been described for modulatingVGCCs. Although investigations into this extraordinary fieldcontinue, it is likely that many new GPCRs can underliemodulation of not only neuronal VGCCs, but also channelsexpressed in other tissues, such as heart and skeletal muscle,and certainly represent a considerable source of potentialtherapeutic targets for the treatment of channelopathies ingeneral.

Authorship Contributions

Wrote or contributed to the writing of the manuscript: Proft, Weiss.

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Address correspondence to: Dr. Norbert Weiss, Institute of Organic Chemistryand Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Flemingovonám. 2., 166 10 Prague 6 – Dejvice, Czech Republic. E-mail: weiss@uochb.cas.cz

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