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G Protein Modulation of Voltage-Gated Calcium Channels

Department of Pharmacology, University College London, London, United Kingdom

Abstract

I. Introduction

A. Molecular Subtypes of Calcium Channel

II. Role of CaV{beta} Subunits in Calcium Channel Function

A. Binding of CaV{beta} to the {alpha}1 I-II Linker

B. Binding of CaV{beta} Subunits to the N and C Termini of CaV{alpha}1 Subunits

III. Modulation of Calcium Channels

IV. Inhibitory Coupling between G Proteins and Voltage-Gated Calcium Channels in Native Tissue

A. The G Protein Subunits Involved in the Direct Inhibitory Modulation of Native and Heterologously Expressed Calcium Channels

B. Voltage Dependence of G Protein Modulation of Calcium Channels

C. The Role of the CaV{alpha}1 I-II Linker in G Protein Modulation of CaV2 Calcium Channels

D. The Essential Role of the CaV{alpha}1 N Terminus in G Protein Modulation

E. Basis for the Selectivity of Calcium Current Inhibition by Transmembrane G Protein-Coupled Receptors

F. Is There a Role for the C Terminus in Calcium Current Inhibition by G Protein-Coupled Receptors?

V. Essential Role of Cav{beta} Subunits in G Protein Modulation of Calcium Channels

A. Initial Evidence for the Role of CaV{beta} Subunits in G Protein Modulation in Native Neurons

B. The Involvement of CaV{beta} Subunits in G Protein Inhibition of Heterologously Expressed Calcium Channels

C. Does G{beta}{gamma} Displace CaV{beta} Subunits?

D. Potential Overlap of Determinants for CaV{beta} Subunit and G{beta}{gamma} Subunit Function

VI. Molecular Mechanism of G Protein-Mediated Inhibition

VII. Recovery from G Protein-Mediated Inhibition

VIII. Conclusion

	Abstract

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	I. Introduction

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Voltage-gated calcium channels (VGCCs¹) play a major role both in the normal functioning and in the patho-physiology of neurons and other excitable cells. Although they are also found at low levels in nonexcitable cells, their presence has been said to define an excitable cell (Hille, 2001). They were first identified in crustacean muscle by Fatt and Katz (1953), and subsequently extensively studied by a number of groups, including Hagiwara and Takahashi (1967). VGCCs were first classified according to their biophysical properties into low- and high-voltage-activated (LVA and HVA) channels (Carbone and Lux, 1984). Further study, with the additional aid of pharmacological tools, led to the classification of certain HVA channels as "long-lasting" or L-type channels, which were sensitive to the 1,4-dihydropyridine (DHP) class of drugs, and present in skeletal muscle, heart, smooth muscle, and neurons (Hess et al., 1984; Nowycky et al., 1985a).

In neurons it was clear that a component of the HVA calcium current was not L-type, for example, in Purkinje cells in the cerebellum (Hillman et al., 1991) and at presynaptic terminals (see Stanley and Atrakchi, 1990 for example). These additional current components were subclassified with the aid of several invaluable toxins. Two additional subtypes of calcium channel were thus identified: N-type channels, sensitive to -conotoxin GVIA (Nowycky et al., 1985b; McCleskey et al., 1987) and P-type channels, sensitive to -agatoxin IVA (Mintz et al., 1992). Another -agatoxin IVA-sensitive current component was subsequently identified in cerebellar granule cells and termed Q-type current (Randall and Tsien, 1995), but these two components are now combined as P/Q. There is also a residual or R-type calcium current component that is resistant to DHPs and the N and P/Q channel toxins (Randall and Tsien, 1995).

A. Molecular Subtypes of Calcium Channel

The molecular basis for the physiological subtypes of VGCCs was clarified after the identification of the subunits of voltage-gated calcium channels. This era started with the purification of skeletal muscle calcium channel complex also called the DHP receptor, which consisted of 1, 2, , , and subunits (Flockerzi et al., 1986; Hosey et al., 1987; Takahashi et al., 1987; Chang and Hosey, 1988; Hymel et al., 1988).

After identification of individual subunits, the sequencing of peptides derived from these subunits formed the basis for the subsequent identification and cloning of the cDNA for the DHP receptor, initially from skeletal muscle (Tanabe et al., 1987) and subsequently from heart by homology with the skeletal muscle sequence (Mikami et al., 1989) (Fig. 1A). The 1 subunits have 24 putative transmembrane segments, arranged into four homologous domains, with intracellular linkers and N and C termini (Fig. 1A). Ten different 1 subunits have been cloned that have specialized functions and distributions (for review, see Ertel et al., 2000) (Fig. 1B). Those that are of most concern to this review are the Ca_V2 subfamily of HVA calcium channels that shows classical modulation by G proteins, comprising Ca_V2.1 or 1A, the molecular counterpart of P/Q-type calcium channels (Mori et al., 1991), Ca_V2.2 or 1B (Dubel et al., 1992), the molecular counterpart of N-type calcium channels, and Ca_V2.3 or 1E (Soong et al., 1993), thought to contribute to the molecular counterpart of the R-type calcium current component (Piedras-Rentería and Tsien, 1998) (Fig. 1B, boxed).

View larger version (28K): [in this window] [in a new window]   FIG. 1. Subunits making up voltage-gated calcium channels. A, diagrammatic representation of the topology of VGCCs. Cyan cylinders denote transmembrane segments of 1 subunit. Red cylinders denote the charged S4 segment, and yellow denotes the pore region. Green cylinder represents transmembrane segment of subunit. Two domains of subunits [src homology (SH)-3 and guanylate kinase (GK)] were defined previously (Hanlon et al., 1999). Glycosylation is denoted by fork shapes. Redrawn with modification from Dolphin (1998). Not intended to represent exact sizes of various subunits. B, classification and nomenclature of CaV1 subunits, including nomenclature, main tissue localization, and pharmacology.

In the case of the N- and P/Q-type as well as the L-type HVA calcium channels, the Ca_V1 subunit has been shown to copurify with an intracellular subunit (Ca_V) (Liu et al., 1996; Scott et al., 1996). Four subunits have been cloned (1–4), with 1a being the skeletal muscle isoform of 1 (Ruth et al., 1989), 2 being cloned initially from cardiac muscle (Perez-Reyes et al., 1992), 3 present in cardiac and smooth muscle and neuronal tissue (Castellano et al., 1993b), and 4 cloned from brain (Castellano et al., 1993a). A number of splice variants have been identified, with one particular splice variant of 2, 2a, being N-terminally palmitoylated in certain species, giving it distinctive properties (Chien et al., 1996).

HVA calcium channels also copurify with an extracellular Ca_V2 subunit, which is attached by S-S bonds to a transmembrane subunit (Tanabe et al., 1987; Chang and Hosey, 1988; Witcher et al., 1993; Liu et al., 1996). Four 2 subunits have been cloned (Ellis et al., 1988; Klugbauer et al., 1999; Barclay et al., 2001; Qin et al., 2002).

Skeletal muscle calcium channels also copurify with a 1 subunit (Takahashi et al., 1987). Whether any of the recently cloned novel -like subunits (2–8) (Fig. 1B) are tightly associated with other types of VGCCs remains controversial (Letts et al., 1998; Black and Lennon, 1999; Klugbauer et al., 2000; Kang et al., 2001; Moss et al., 2002; Tomita et al., 2003).

	II. Role of CaV Subunits in Calcium Channel Function

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The intracellular Ca_V subunits have marked effects on the properties of HVA 1 subunits (Ca_V1 and Ca_V2 families), including trafficking of calcium channel complexes to the plasma membrane and modification of kinetic and voltage-dependent properties (Singer et al., 1991; De Waard et al., 1994; Chien et al., 1995; Brice et al., 1997; Bichet et al., 2000). My group has shown that the converse also applies, in that antisense-induced knockdown of Ca_V subunits from native neurons results in a reduction of the amplitude of endogenous calcium currents and slowed kinetics of activation (Berrow et al., 1995; Campbell et al., 1995b).

Most research indicates that all Ca_V subunits (except truncated splice variants described recently (Hibino et al., 2003; Hullin et al., 2003) increase the functional expression of HVA 1 subunits (for a recent review, see Dolphin, 2003). In theory, this could be attributable to effects on a number of channel properties, an increase in the open probability of the channel, an increase in single-channel conductance, an increase in the number of functional channels inserted into the plasma membrane, or a combination of several of these processes. Initial studies did not agree whether there was an increase in number of channels at the plasma membrane, measured as charge moved in isolated gating currents, with either no increase (Neely et al., 1993) or an increase being reported (Josephson and Varadi, 1996). Much early work on the roles of Ca_V subunits in calcium channel expression was performed in Xenopus oocytes, but these cells are now known to contain an endogenous Xenopus subunit that complicates the interpretation of these results (Tareilus et al., 1997; Canti et al., 2001). This endogenous Ca_V subunit was found to be both necessary and able to traffic at least some heterologously expressed Ca_V channels to the plasma membrane, since if endogenous subunit expression was reduced or eliminated by injection of 3 antisense oligonucleotides, Ca_V expression was largely lost (Tareilus et al., 1997; Canti et al., 2001).

In COS-7 cells, small currents were observed when Ca_V2.1, Ca_V2.2, and Ca_V2.3 were expressed alone, but exogenous subunits all increased Ca_V2.1, Ca_V2.2, and Ca_V2.3 maximum conductance about 10-fold (Berrow et al., 1997; Stephens et al., 1997; Meir and Dolphin, 1998; Stephens et al., 2000). COS-7 cells do contain mRNA for endogenous subunits (Meir et al., 2000), but the protein for corresponding subunits was not detectable by immunocytochemistry (Meir et al., 2000), although a low level might be found if higher sensitivity detection methods were used. Thus, at the moment there are no expression systems that definitively contain no Ca_V subunits that can be used conclusively to answer the question as to whether HVA calcium channels can be trafficked to the plasma membrane without a subunit.

The increase in current density brought about by Ca_V subunits can be attributed to a number of effects on biophysical properties as well as the important influence on trafficking. All Ca_V subunits hyperpolarize the voltage dependence of activation of all HVA VGCCs (Fig. 2), whereas all, except the 2a splice variant that is N-terminally palmitoylated, hyperpolarize the voltage dependence of steady-state inactivation (Birnbaumer et al., 1998). Where it has been studied, the subunits all produce an increase in mean open time, which is at least in part due to a hyperpolarizing shift in the voltage dependence of the mean open time (Wakamori et al., 1999; Meir et al., 2000). Although Ca_V1 subunits contain inherent determinants of voltage-dependent inactivation (Zhang et al., 1994; Herlitze et al., 1997; Cens et al., 1999; Spaetgens and Zamponi, 1999), association with different Ca_V subunit isoforms dictates their overall kinetics of inactivation (Olcese et al., 1994; Meir and Dolphin, 2002). At the whole-cell level, the inactivation rate was affected in the following order (highest first) 3 > 1b > 4 > 2 subunits. Retardation of inactivation has been shown to be particularly dramatic for the palmitoylated Ca_V2a subunit expressed with Ca_V1.2 (Chien and Hosey, 1998), Ca_V2.2 (Bogdanov et al., 2000; Stephens et al., 2000), or Ca_V2.3 (Qin et al., 1998).

View larger version (26K): [in this window] [in a new window]   FIG. 2. Properties of CaV2.2 voltage-gated calcium channels expressed in the absence and presence of a CaV subunit. Left, recordings of CaV2.2 channels transiently transfected in COS-7 cells in the absence of a coexpressed subunit (upper traces) or the presence of CaV2a (lower traces). The currents are activated by 40-ms voltage steps in 5-mV increments from a holding potential of -100 mV, and only those on the rising phase of the I-V relationship are shown for clarity. Right, I-V curve showing the increase in CaV2.2 current amplitude in the presence of CaV2a (open squares) compared with its absence (open circles). Recordings were in 10 mM Ba2+ extracellular solution. These I-V data were fitted with the equation: current = Gmax · (V - Vrev)/{1 + exp[(V - V1/2)/k]}, where Gmax is maximum slope conductance, V1/2 is the voltage at which 50% of the current is activated, Vrev is the null potential, and k is the slope factor. Coexpression of 2a increased Gmax and induced a hyperpolarizing shift in V1/2 and a reduction in k. In these examples, Gmax = 0.08 and 0.83 nS/pF in the absence and presence of CaV2a. The arrows indicate the voltage for 50% activation for the two curves, showing the hyperpolarization induced by 2a. Data replotted from Stephens et al. (2000).

A. Binding of Ca_V to the 1 I-II Linker

Ca_V subunits have been found to bind with very high affinity to the cytoplasmic intracellular linker between domains I and II of all HVA calcium channels, via an 18-amino acid motif called the interaction domain (AID) on the I-II linker (Pragnell et al., 1994). The AID sequence of rabbit Ca_V2.2 is QQIERELNGYLEWIFKAE, and the consensus sequence present in both Ca_V1.x and Ca_V2.x subfamilies is QQxExxLxGYxxWIxxxE.

A 41-amino acid sequence (BID) on the Ca_V subunit was identified as the minimal motif required to influence 1 subunit expression and to bind to the 1 subunit (De Waard et al., 1994, 1996). The consensus sequence of BID is K—E—PYDVVPSMRP—LVGPSLKGYEVTDMMKQALFDF; the underlined serines are consensus protein kinase C (PKC) phosphorylation sites. The residues in bold have been identified as particularly important for binding to Ca_V1 subunits (Walker and De Waard, 1998). This small BID sequence alone can produce an increase in calcium current density, albeit not to the same extent as the full-length protein (De Waard et al., 1994). The affinity between Ca_V subunits and a I-II linker fusion protein has been measured to be between 5 and 60 nM (De Waard et al., 1994), but has been proposed to be state-dependent (De Waard et al., 1995; Canti et al., 2001). In one study (De Waard et al., 1995), no dissociation was seen for 1b from the Ca_V2.1 I-II linker fusion protein after 10 h, but this may reflect a technical difficulty of overlay assays, because the bound protein may become directly anchored to the membrane. In our own binding studies using surface plasmon resonance, the affinity of 3 for the GST fusion protein of the I-II linker of Ca_V2.2 was about 20 nM, and the k_off was 5.2 x 10^-3 s^-1 (Canti et al., 2001). We found similar data (Fig. 3A) for 1b binding to the I-II linker of both Ca_V2.2 and Ca_V1.3 (Bell et al., 2001).

View larger version (21K): [in this window] [in a new window]   FIG. 3. Binding of 1b and G dimers to I-II linker of CaV2.2 and CaV1.3. A, GST fusion proteins of the I-II linker of CaV2.2 and CaV1.3 were bound to a Biacore 2000 sensor chip (Biacore International SA, Stevenage, Hertfordshire, UK) via a GST antibody, and the reversible binding of purified recombinant 1b (10 nM) was then examined. y-axis, R.U. (arbitrary units of mass bound to the sensor chip). B, the binding of increasing concentrations of purified bovine brain G was determined to the same fusion proteins. Data replotted from Bell et al. (2001).

We have studied the in vivo concentration dependence of the effects of Ca_V subunits. Our evidence supports the hypothesis that there are two distinct binding processes for subunits on Ca_V2.2 (Canti et al., 2001). One is a high-affinity process related to the effect of Ca_V on the maximum conductance of Ca_V2.2, presumably involving its trafficking to the plasma membrane, whose affinity corresponds closely to the in vitro affinity for the I-II linker (17 nM), which is coincidentally the concentration of endogenous 3 estimated to exist in Xenopus oocytes (Canti et al., 2001). The second process is of lower affinity (K_D 120 nM), associated with the voltage-dependent effects of the subunit, for example steady-state inactivation. One explanation for the discrepancy in these two calculated affinities is that a single binding site undergoes a marked reduction in affinity for Ca_V subunits once the Ca_V1 subunits have been trafficked from the endoplasmic reticulum and are inserted in the polarized plasma membrane. Alternatively, one might postulate the coexistence of two separate Ca_V subunit binding sites on each Ca_V2.2 molecule, but the binding of two Ca_V subunits has not been demonstrated directly (Canti et al., 2001). Whichever hypothesis is correct, it is highly likely that the Ca_V subunit interacts with other domains on the Ca_V1 subunit as well as the I-II linker.

B. Binding of Ca_V Subunits to the N and C Termini of Ca_V1 Subunits

Two other subunit interaction sites have been identified on various 1 subunits on the C terminus (Qin et al., 1997; Walker et al., 1998) and the N terminus (Walker et al., 1999; Stephens et al., 2000). These appear to be of lower affinity and may be selective for certain Ca_V subunits. Whether they represent part of a single complex subunit binding pocket made up of the I-II linker and the N and C termini remains to be established. However, the binding site found for 2a on the C terminus of Ca_V2.3 appeared to involve binding to the BID domain of 2a, the same as that which binds to the Ca_V1 I-II linker, making it an alternative, rather than an additional site, for an individual Ca_V subunit (Qin et al., 1997). Walker et al. (1999) showed that the N terminus of Ca_V2.1 interacted with Ca_V4 and 2a but not 1b or 3. The region of 4 involved was within its C terminus (amino acids 446–482). The C terminus of Ca_V4 also bound to the C terminus of Ca_V2.1. The N and C termini of Ca_V2.1 were found to occupy overlapping binding sites that were mutually exclusive, but either could bind in combination with binding to the I-II linker (Walker et al., 1999). This group also showed that Ca_V4 produced a smaller hyperpolarizing shift of Ca_V2.1 currents than did Ca_V3, and that this differential was due to the Ca_V2.1 N terminus. Stephens et al. (2000) showed that Ca_V2.2 N-terminal residues in the same region as the essential site for G protein modulation (see Section IV.D.) were involved in retardation of inactivation kinetics by 2a. Palmitoylated 2a has been suggested to retard inactivation by tethering the I-II linker so that it cannot mediate inactivation (Restituito et al., 2000; Stotz et al., 2000), but our data show an additional role for the N terminus of Ca_V2.2.

	III. Modulation of Calcium Channels

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There are several means by which VGCCs may be both up- and down-regulated by second messenger pathways, for example by phosphorylation (Nunoki et al., 1989; Dolphin, 1999; Catterall, 2000). These include regulation by kinases, for example, up-regulation of cardiac L-type channels by cyclic AMP-dependent protein kinase (Reuter, 1987) and regulation by protein kinase C (Stea et al., 1995). In this review, however, I shall concentrate on the classical G protein pathway and describe first how this was examined in native neurons.

For the neuronal channels, particularly N- and P/Q-types, a major mechanism of inhibitory modulation occurs via the activation of heterotrimeric G proteins by seven transmembrane G protein-coupled receptors (GPCRs). GPCR activation was first found to reduce action potential duration in dorsal root ganglion neurons in the 1970s (Dunlap and Fischbach, 1978). Subsequently, this effect was found to result from inhibition of voltage-gated calcium channels (Dunlap and Fischbach, 1981). Such modulation has since been observed in many types of neuron, including superior cervical ganglion neurons (Ikeda and Schofield, 1989) and submucosal neurons (Surprenant et al., 1990).

The GPCRs typically involved in this type of modulation include 2-adrenoceptors, µ and opioid receptors, GABA-B receptors (Fig. 4, A and B), and adenosine A1 receptors (Dunlap and Fischbach, 1978; Dolphin et al., 1986; Scott and Dolphin, 1986). The key features that typify this inhibition are a slowing of the current activation kinetics, which is thought to be due to a time- and depolarization-dependent recovery from voltage-dependent inhibition (Bean, 1989). The voltage dependence is manifested by a shift to more depolarized potentials of the current activation-voltage relationship and the loss of inhibition at large depolarizations, because of a shift from "reluctant" to "willing" channels (Bean, 1989). Removal of inhibition can also be induced by a depolarizing prepulse applied immediately before the test pulse (Ikeda, 1991). Additional mechanisms that are not voltage-dependent have also been described in various cell types manifested by a scaled reduction in the current and an inability of a depolarizing prepulse to reverse this component of the inhibition (for example, see Diversé-Pierluissi and Dunlap, 1993).

View larger version (17K): [in this window] [in a new window]   FIG. 4. Effect of the GABA-B agonist (-)-baclofen on action potential duration and calcium channel currents recorded from dorsal root ganglion neurons: involvement of G proteins. A, action potentials, whose duration was lengthened by blockade of potassium channels by extracellular tetraethylammonium (10 mM), were recorded in current clamp, induced by a brief depolarizing pulse. (-)-Baclofen (50 µM, Bac) was applied by pressure ejection and shortened the action potential duration, compared with the control (Con), before agonist application. The effect was readily reversible after terminating agonist application (Rec). B, calcium channel currents were recorded from these cells in voltage clamp, and application of (-)-baclofen (50 µM) reduced the current amplitude. C, when GTPS (17 µM) was included in the patch pipette, the current recorded was small in amplitude and more slowly activating than control. Data redrawn from Dolphin and Scott (1987) and Dolphin and Scott (1990). The Ba2+ concentration was 2.5 mM.

N- and P/Q-type calcium channels support synaptic transmission and are concentrated at nerve terminals. P/Q-type channels are most important for transmitter release at central terminals (Takahashi and Momiyama, 1993), although N-type channels are also present and particularly contribute earlier in development. In contrast, N-type channels are more prevalent in peripheral nerve terminals and are largely responsible for synaptic transmission in autonomic and sensory terminals (Mochida et al., 1996; Koh and Hille, 1997). Modulation of these channels by activation of GPCRs has been shown to occur both in cell bodies (Holz et al., 1986; Scott and Dolphin, 1986; Dolphin and Scott, 1987; Ikeda, 1991) and at presynaptic terminals (Takahashi et al., 1998). This mechanism may be responsible for at least some of the presynaptic inhibition of synaptic transmission mediated by a wide variety of GPCRs in many areas of the nervous system (Dolphin and Prestwich, 1985; Man-Son-Hing et al., 1989; Toth et al., 1993). Activation of GPCRs such as the GABA-B receptor will reduce calcium entry into presynaptic terminals via VGCCs by the same mechanism that is observed in cell bodies (Fig. 4A), and the effect should also be frequency- and potential-dependent. Inhibition will be reduced during a high-frequency train as a result of the voltage dependence of the inhibitory modulation. Relief of inhibition of calcium currents, evoked by action potential-like voltage waveforms, has been reported during high-frequency trains (Williams et al., 1997; Brody and Yue, 2000) and may contribute to the modulation of presynaptic inhibition according to input frequency.

	IV. Inhibitory Coupling between G Proteins and Voltage-Gated Calcium Channels in Native Tissue

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The role of G proteins in the inhibition of calcium currents by GPCR activation was first demonstrated some years later (Holz et al., 1986; Scott and Dolphin, 1986). It was also identified that there was a direct membrane-delimited link between G protein activation and N- or P/Q-type calcium channel inhibition. A key experiment enabling this idea to be accepted was the finding that inhibition of calcium channels recorded in the cell-attached patch mode only occurs when the receptor agonist is present in the patch pipette, and not when it bathes the remainder of the cell membrane (Forscher et al., 1986). This indicates that the inhibitory process is very localized and that a soluble second messenger is not involved. In most studies, it is found that this direct linkage only applies to the Ca_V2 family of calcium channels, but additional non-voltage-dependent pathways, which may be direct or via down-stream soluble intracellular messengers also occur in certain cell types and may additionally apply to L-type channels (Hille, 1992; Dunlap and Ikeda, 1998) and also to T-type channels (Wolfe et al., 2003).

A. The G Protein Subunits Involved in the Direct Inhibitory Modulation of Native and Heterologously Expressed Calcium Channels

The modulation of neuronal VGCCs in most native neurons is usually mediated by receptors coupled to pertussis toxin-sensitive G proteins (G_i and G_o subtypes) (Holz et al., 1986; Scott and Dolphin, 1986). The response to an agonist can be mimicked by nonhydrolyzable analogs of GTP such as guanosine 5'-O-(3-thiotriphosphate) (GTPS) (Fig. 4C) (Dolphin and Scott, 1987) and by photoactivation of a caged GTP analog (Dolphin et al., 1988). The effect of GTP analogs is, as expected, more extensive than that of receptor agonists and is irreversible because G proteins are permanently activated. To provide evidence that the effect of agonists is mediated by G proteins, initial experiments showed that the effect of agonists is enhanced and made irreversible by a low concentration of GTPS (Scott and Dolphin, 1986) and prevented by a GDP analog such as guanosine 5'-O-(2-thiodiphosphate) (Holz et al., 1986; Dolphin and Scott, 1987).

To identify which G proteins are involved in receptormediated inhibition of calcium channels in native systems (such as dorsal root ganglion neurons and sympathetic neurons), a number of studies were performed with blocking antibodies and antisense oligonucleotides complementary to G protein subunits, which showed that G_o was primarily responsible for the response (McFadzean et al., 1989; Baertschi et al., 1992; Campbell et al., 1993; Menon-Johansson et al., 1993). However, others found that both G_i and G_o were involved (Ewald et al., 1989), and in several studies, G_s- or G_q-coupled receptors produced similar modulation (Shapiro and Hille, 1993; Golard et al., 1994; Zhu and Ikeda, 1994). This led to the hypothesis that the species involved was the moiety common to all these G proteins, G rather than any particular G, and this was subsequently found to be the case (Herlitze et al., 1996; Ikeda, 1996), although previously other groups had directly investigated the involvement of G in calcium channel modulation and not found any effect of its infusion (Hescheler et al., 1987). However, there was a clear precedent for an effector role for G in the G protein-activated potassium channels (GIRKs). Although for many years a controversy reigned concerning which G protein subunit was responsible for modulation of the native GIRKs (e.g., Yatani et al., 1987), they were eventually shown conclusively to be activated by Gs (Logothetis et al., 1987; Kurachi et al., 1989; Clapham and Neer, 1993). Furthermore, most G combinations tested except transducin (G₁₁) are similarly effective (Wickman et al., 1994; Yamada et al., 1997). From the work of two groups (Herlitze et al., 1996; Ikeda, 1996), it became clear that transfection of either primary neurons or cell lines with G subunits mimicked agonist effects and led to tonic inhibition of the calcium current, which could be transiently reversed by a depolarizing prepulse, applied just before the test pulse, a hallmark of voltage-dependent inhibition of these channels.

Taking examples for illustration from our own work (Meir et al., 2000), the effect of coexpression of G with N-type calcium channels can be seen both at the whole-cell and at the single-channel level (Figs. 5A, and 6A). When Ca_V2.2 was coexpressed in COS-7 cells with 2a and G₁₂, the whole-cell currents were very small and slowly activating but were markedly enhanced by a depolarizing prepulse (Fig. 5A, left panel). The time constant of activation of the peak current at 0 mV in the presence of G was about 27 ms, compared with less than 5 ms in the presence of the G binding domain of -adrenergic receptor kinase 1 (-ARK1) to bind any free endogenous G. At the single-channel level, the slow activation of Ca_V2.2/2a channels was seen as a marked prolongation of the latency to first opening (Fig. 6A, compare traces in right panel with G with those in the left panel with -ARK1). Indeed, there were many instances where no openings were observed to a test pulse. The latency to first opening was significantly reduced by a large depolarization (Fig. 6A, right panel). G overexpression also occluded modulation by agonist (Ikeda, 1996). The G-mediated inhibition presents a picture that is very similar to that of agonist-mediated inhibition in terms of slowed activation and reversal by a large depolarizing prepulse. This is illustrated in an example from work by my own group, showing the effect of quinpirole-mediated activation of a coexpressed D2-dopamine receptor on the same channel combination (Fig. 7A) (Meir et al., 2000).

View larger version (18K): [in this window] [in a new window]   FIG. 5. Requirement for a subunit for the modulation of whole-cell CaV2.2 currents by G in COS-7 cells. A, slowed activation of CaV2.2/2a currents by G12 coexpression. Left top panel, voltage protocol, holding potential -80 mV, P1 and P2 test pulses to between -40 and +60 mV, with intervening prepulse to +100 mV; lower panel, example current traces for P1 and P2 to 0 mV for CaV2.2/2a/G12. Right, plot of time constant for activation (act) against voltage for P1 currents in the presence of G (•), compared with coexpression of -ARK1 minigene (). B, lack of slowed activation of CaV2.2 currents by G in the absence of CaV subunits. Left, example current traces for P1 and P2 to 0 mV for CaV2.2/G12. Right, plot and symbols as above. Note that traces in A are recorded in 1 mM Ba2+ and those in B are recorded in 10 mM Ba2+. Scale bars represent 10 pA/pF and 50 ms for both. Data replotted from Meir et al. (2000).

View larger version (33K): [in this window] [in a new window]   FIG. 6. Requirement for a CaV subunit for the modulation of CaV2.2 single-channel currents by G in COS-7 cells. A, effect of G12 (right) compared with -ARK1 (left) coexpression on CaV2.2 single-channel currents in the presence of CaV2a subunits, recorded from cell-attached patches in COS-7 cells. Upper voltage trace, holding potential -100 mV, test potential both in P1 and P2, +30 mV, separated by a depolarizing prepulse to +120 mV. Top, representative single-channel records from a single channel-containing patch. Scale bar 1 pA for all single-channel records. Bottom panel, single-channel ensemble average currents, with error bars only every 5 ms for clarity (n = 13). The slowed activation of CaV2.2/2a currents by G12 and reduced amplitude compared with coexpression of -ARK1 are evident, as well as its partial reversal by a prepulse. B, lack of effect of G12 (right) compared with -ARK1 (left) on CaV2.2 single-channel currents in the absence of CaV subunits. Top, representative single-channel records, 100-ms long; bottom, ensemble average currents (n = 13), showing no effect of G on current activation. Scale bar represents 0.1 pA and 100 ms and refers to all ensemble currents. C, comparison of the scaled ensemble averages in the presence of G and in the presence and absence of 2a. Scale bar represents 20 ms. Data replotted from Meir et al. (2000).

View larger version (22K): [in this window] [in a new window]   FIG. 7. Requirement for a subunit for the modulation of whole-cell CaV2.2 currents by activation of the dopamine D2 receptor in COS-7 cells. Whole-cell CaV2.2 currents recorded from transfected COS-7 cells. The voltage protocol consists of two 50-ms test pulses (P1, P2) to +10 mV from a holding potential of -80 mV, with an intervening 100-ms prepulse to +120 mV. The D2 dopamine receptor was activated with 100 nM quinpirole (during control and wash, the bath was perfused with extracellular solution). The traces shown are mean normalized currents before [P1 () and P2 (•)] and during [P1 () and P2 ()] application of quinpirole. Currents were normalized to the current at 20 ms after the onset of the test pulse in P1 before quinpirole application and then averaged. The scale bars apply to A and B and represent one normalized current unit and 50 ms. Mean ± S.E.M. are shown in the same symbols. A, effect of the dopamine D2 receptor agonist quinpirole (gray traces) on CaV2.2 currents in the presence of CaV1b subunits. The slowed activation of CaV2.2/2a currents by quinpirole and reversal by a depolarizing prepulse are evident. B, lack of voltage-dependent effect of the D2 receptor agonist quinpirole (gray traces) on CaV2.2 current activation kinetics in the absence of CaV subunits. A small degree of inhibition is observed, which is not reversed by a prepulse (compare P2 traces to those in P1). Data replotted from Meir et al. (2000).

The involvement of G dimers as mediators of the G protein-signaling pathway does not call into question the finding by many groups that G_o is involved in receptor-mediated inhibition in many native systems (Ewald et al., 1989; McFadzean et al., 1989; Campbell et al., 1993; Menon-Johansson et al., 1993; Degtiar et al., 1996), because G_o is present in very high concentrations, particularly in neurons. Thus, in the absence of the G_o subunit, GPCR-mediated signaling will be markedly attenuated since it depends on the G protein heterotrimer.

Nevertheless, a number of studies have further concluded that there is a specific role for G subunits. In some cell types, a marked specificity of different G combinations for signaling pathways between different receptors and calcium channels has been demonstrated (Kleuss et al., 1991; Degtiar et al., 1996). These findings might also be reconciled with the evidence that most G subunits are able to transduce the signal to calcium channels (Ikeda, 1996; Garcia et al., 1998; Ruiz-Velasco and Ikeda, 2000) by interpreting that a specific G protein heterotrimer combination may selectively couple to a particular receptor in intact cells, and the selectivity is therefore largely at the receptor-G protein interaction step or due to segregation into different compartments, rather than due to G specificity. In one study, however, it was concluded that there was an effector role for G_i3 subunits (Furukawa et al., 1998b). In this study, either G or G moieties were coexpressed in Xenopus oocytes with Ca_V2.2 or Ca_V2.1 channels and the µ-opioid receptor. Receptor-mediated inhibition was found to be enhanced by coexpression of G, which was therefore said to mediate this inhibition; but a more likely explanation of this finding is that expression of exogenous G increases the amount of G available for coupling to the receptor (Jeong and Ikeda, 1999; Canti and Dolphin, 2003) rather than that a specific G mediates the response. In another study in chick sensory neurons, it was originally suggested that following activation of 2-adrenoceptors, G dimers were responsible for the voltage-independent inhibition via activation of PKC and activated G for voltage-dependent inhibition via an unknown second messenger (Diversé-Pierluissi and Dunlap, 1993; Diversé-Pierluissi et al., 1995). It is unknown whether different pathways might be activated in avian neurons. Interpretation of the role of phosphorylation in mediating a pathway must always bear the proviso that phosphorylation might also occlude receptor-mediated effects by receptor down-regulation.

I have further examined whether G subunits play any role in mediating calcium channel inhibition, by the use of receptor-G fusion proteins. I found that there was no difference between ₂ adrenoreceptor-G_o and -G_i tandems and the wild-type ₂ adrenoreceptor in their ability to support G protein-mediated inhibition of N-type calcium channels in an expression system, and also no difference in the voltage dependence of the inhibition (Fig. 8), despite the fact that no G amplification would occur in the case of the tandems (Bertaso et al., 2003). This is in contrast to the selective inhibition by G_o rather than G_i in sympathetic neurons (Delmas et al., 1999), which may depend on localization within discrete membrane compartments in native cells (Delmas et al., 2000). Thus, coexpression studies in heterologous systems provide information on what is possible, but studies in native cells, using different means of interfering with the signal transduction pathway or its intermediates, provide information on what actually happens in any given cell. Both types of study are essential to place the correct interpretation on data from native cells.

View larger version (20K): [in this window] [in a new window]   FIG. 8. G protein modulation via receptor-G protein tandems. A, inhibition of CaV2.2/1b currents by activation of heterologously expressed 2A-adrenoceptors, either alone (top) or as tandem constructs with Gi (middle) or Go (bottom). The receptor-G protein tandems have a mutation at the C terminus of the G to prevent ADP-ribosylation and inactivation by pertussis toxin (C351I) (left panel). The charge carrier was 10 mM Ba2+. Clonidine (10 µM) was the agonist and the inhibition is shown either without (middle panel) or with (right panel) pertussis toxin pretreatment. The holding potential was -100 mV, and currents were evoked by 30-ms steps to 0 mV. B, mean data for inhibition by clonidine for the three conditions described above, given as a histogram. Solid bars, data obtained in the absence of pertussis toxin; open bars, data obtained in the presence of pertussis toxin. Data replotted from Bertaso et al. (2003).

One G subunit, G₅, when overexpressed in sympathetic neurons was less effective than other G subunit combinations at producing G protein modulation of VGCCs (Ruiz-Velasco and Ikeda, 2000). This may be because G₅ preferentially interacts with certain regulators of G protein signaling (RGS) proteins with G protein -like domains, rather than with G subunits themselves (Snow et al., 1998), and also couples selectively to the G_q family of G subunits (Fletcher et al., 1998).

B. Voltage Dependence of G Protein Modulation of Calcium Channels

It is commonly accepted that the relief of G protein-mediated inhibition by depolarization is a result of rapid dissociation of G dimers from the channel at depolarized potentials. This process is thought to be strongly voltage- and time-dependent (see Figs. 5A and 6A for examples) and, as suggested above, is also believed to cause the slow relaxation observed in response to a test pulse (Jones and Elmslie, 1997). Furthermore, re-establishment of inhibition after a prepulse, during a period at the holding potential, is likely to result from rebinding of G dimers. Whether these processes actually result in physical dissociation and reassociation between the G protein subunits and channel remains formally to be established. However, the finding that the rate of reblock is dependent on the concentration of activated G protein is consistent with this view (Elmslie and Jones, 1994; Stephens et al., 1998a; Zamponi and Snutch, 1998). The interpretation of these results is that the process involves binding from the pool of free G dimers.

C. The Role of the Ca_V1 I-II Linker in G Protein Modulation of Ca_V2 Calcium Channels

The combination of two findings, 1) that G dimers are the mediators of inhibitory modulation, and 2) that there is a functional interaction between Ca_V subunits and G (Campbell et al., 1995), led a number of groups to examine the intracellular I-II linker in detail. G dimers have been found previously to bind to sites on type 2 adenylyl cyclase and phospholipase C 2 (Chen et al., 1995), which have a characteristic central motif consisting of QXXER. Whereas this motif is not necessarily indicative of a functional G binding site, it is also found to occur in the I-II loop of Ca_V2.1, Ca_V2.2, and Ca_V2.3, intriguingly within the binding site described for the VGCC subunit (QQIERELNGY–WI-KAE) (Pragnell et al., 1994). Furthermore, it is modified in the cognate region in L-type channels (QQLEEDL-GY–WITQ-E).

It is clear that G binds to the I-II linker of G protein-modulated calcium channels. This has been shown by several groups using overlay assays (De Waard et al., 1997; Zamponi et al., 1997) and also by the use of a surface plasmon resonance-based system to measure reversible binding, where the on- and off-rates can be measured, showing an affinity of G for the I-II linker of Ca_V2.2 of 62 nM (Fig. 3B) (Bell et al., 2001). Furthermore, the I-II linker of the L-type channel Ca_V1.3 does not bind G, although it will bind Ca_V subunits (Bell et al., 2001). The residues in the I-II linker critical for G binding have been mapped (De Waard et al., 1997). As predicted, the AID part of the linker is one important domain, and some residues of the QQIER sequence were shown to be essential for G binding (De Waard et al., 1997).

The role of the I-II linker G binding site in the process of G protein modulation remains controversial. Initial electrophysiological studies that supported a major role for the I-II linker used peptides derived from the I-II linker region in the patch pipette and found that they blocked G protein modulation (Herlitze et al., 1997; Zamponi et al., 1997). However, peptides alone do not prove that the Ca_V1 I-II loop is the site of modulation but rather indicate whether the peptides bind to G and can therefore effectively compete for this mediator. Chimeric and mutant channels have also been made between those channels that showed the greatest G protein modulation, such as Ca_V2.2, and those that exhibited no or less modulation, in an attempt to define the regions involved in this process. Three groups found that the I-II linker was key to G modulation (De Waard et al., 1997; Herlitze et al., 1997; Zamponi et al., 1997). Zamponi et al. (1997) made chimeras between Ca_V2.1 and Ca_V2.2 by putting the I-II linker of Ca_V2.2 into Ca_V2.1, which resulted in an increased modulation, with Ca_V1b as the coexpressed subunit. However, both the channels used in this study were G protein modulatable. De Waard et al. (1997) found that a mutation that prevented G binding (R E in QQIER) also prevented inhibitory modulation of Ca_V2.1/4 by GTPS injection into oocytes, although in this study only a small amount of inhibition was observed with GTPS even in the wild-type Ca_V2.1. However, Herlitze et al. (1997) mutated the QQIER sequence in Ca_V2.1 to that in Ca_V1.2, which is QQLEE, and found a reduction of modulation of the channel coexpressed with 1b by GTPS, but not an abolition. Interestingly, in this study, they observed that Ca_V2.1 with the sequence QQIEE showed increased, rather than decreased, modulation by GTPS, in contrast to the results of De Waard et al. (1997).

Two other groups found that the presence of an I-II linker from a G protein-modulatable channel was either not essential for G protein modulation or not the most critical region (Zhang et al., 1996; Page et al., 1997, 1998; Canti et al., 1999). The results of Zhang et al. (1996) showed that neither the I-II linker from Ca_V2.1 nor that from Ca_V1.2 reduced modulation in a Ca_V2.2 backbone when coexpressed with 1. In agreement, the results of Page et al. (1997) showed that insertion of the I-II linker of Ca_V2.2 into a Ca_V2.3 construct (RbEII) (Soong et al., 1993), which had a truncated N terminus, did not restore the G protein modulation shown by Ca_V2.2/1b. Furthermore, Canti et al. (1999) showed that the I-II linker of Ca_V2.2 inserted into a Ca_V1.2 backbone and coexpressed with 2a did not allow any G protein modulation (Fig. 9). Both groups found that domain I of G protein-modulated channels was a key region in the process of G protein modulation (Zhang et al., 1996; Stephens et al., 1998b). The discrepancies do not appear to be due to the different G dimers or Ca_V subunits used, because the different groups have coexpressed channels with a variety of different Ca_V subunits,