HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Dolphin, A. C. Search for Related Content PubMed PubMed Citation Articles by Dolphin, A. C.

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

Top Next

	I. Introduction

Top Previous Next

 
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

Top Previous Next

 
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

Top Previous Next

 
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

Top Previous Next

 
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, and the G combination, where used, was G₁₂ or G₂₃.

View larger version (26K): [in this window] [in a new window]   FIG. 9. Modulation of CaV1.2-CaV2.2 chimeras by activation of a dopamine D2 receptor. Left, diagrammatic representation of the chimeras made between CaV2.2 (rabbit BIII clone, represented by gray bars, top) and CaV1.2 (rat rbC-II clone, white bars, bottom). The four transmembrane domains are shown in black/gray stripes for CaV2.2 and solid black for CaV1.2. Right, the constructs were expressed in Xenopus oocytes together with auxiliary subunits and dopamine D2 receptors. Percentage of inhibition of peak calcium current at 0 mV by the agonist quinpirole (100 nM). Results taken from Canti et al. (1999).

Thus, the I-II linker of G protein-modulated channels has a clear ability to bind G (Fig. 3B), although there appear to be no motifs in the I-II linker whose presence, despite being essential for high-affinity binding to the I-II linker, is absolutely essential for the process of modulation of Ca_V2.x channels by these G dimers.

D. The Essential Role of the Ca_V1 N Terminus in G Protein Modulation

In reconstituted systems, consisting minimally of a VGCC 1/ combination, with or without an 2 and either an endogenous or an expressed G protein-coupled receptor, classical G protein modulation could be demonstrated for Ca_V2.2 and, to a lesser extent, for Ca_V2.1, but modulation of Ca_V2.3 was only observed by some groups (Yassin et al., 1996; Meza and Adams, 1998) but not by my group using a particular rat Ca_V2.3 clone (Page et al., 1997). My group subsequently identified the reason for this discrepancy; one of the initial clones from rat brain, rbEII (Soong et al., 1993), had a truncated 5' coding region, commencing at the second methionine, and this showed no G protein modulation (Page et al., 1998). PCR extension of the N terminus formed a full-length rat Ca_V2.3 clone homologous to the rabbit and human clones, and this full-length Ca_V2.3 is strongly G protein-modulated (Page et al., 1998). To examine whether the requirement for an intact N terminus was a general conclusion, not limited to Ca_V2.3, my group further showed that partial truncation of the highly homologous N terminus of Ca_V2.2 abolished its ability to be G protein modulated (Page et al., 1998). Furthermore, a chimeric channel consisting of only the cytoplasmic N terminus of Ca_V2.2 in a rat Ca_V1.2 backbone showed all the elements of classical G protein modulation, whereas Ca_V1.2 did not (Canti et al., 1999) (Fig. 9). We then extended this study to show that an 11-amino acid motif YKQSIAQRART in Ca_V2.2 N terminus that is also highly conserved in Ca_V2.1 and Ca_V2.3 was essential for G protein modulation. Within this sequence, mutation of either YKQ or RAR to AAA abolished G protein modulation (Canti et al., 1999). Elements of this 11-amino acid motif were also involved in interaction with Ca_V subunits, because deletion of this motif in Ca_V2.2 or mutation of certain residues countered the Ca_V2a-mediated retardation of inactivation (Stephens et al., 2000).

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

Activation of the G_q family of G proteins does not produce typical voltage-dependent inhibition of N-type calcium channels despite the production of G dimers, but instead produces a non-voltage-dependent inhibition (Kammermeier et al., 2000; Bertaso et al., 2003). The reason for this is unclear, but a number of hypotheses have been put forward. One possibility is that, as stated above, G_q frequently interacts with G₅ in native tissues, and G₅ is unique among the G proteins in that it does not interact with most G subunits (Zhou et al., 2000). A second possibility is that the inhibition is via G_q itself or a downstream effector. Indeed, the voltage-dependent inhibitory modulation of calcium currents resulting from activation of the pertussis toxin-sensitive G protein pathway or expression of G dimers can be reversed by coactivation of G_q (Zamponi et al., 1997; Simen et al., 2001; Bertaso et al., 2003). G_q activates phospholipase C and downstream signal transduction events including PKC. There is a threonine, Thr422, in a PKC consensus phosphorylation site just C-terminal to the AID motif in the sequence KRAATKKSR within the I-II linker of rat Ca_V2.2. It has been proposed that phosphorylation by PKC of this threonine residue counteracts G binding to the I-II linker and thus counters inhibitory modulation (Zamponi et al., 1997; Hamid et al., 1999). However, this was subsequently found only to hold true for G₁ and not other G subunits (Cooper et al., 2000). Furthermore, the sequence is not completely conserved in rabbit Ca_V2.2, which has alanine in place of threonine at the equivalent residue 422 (KRAAAKKSR in rabbit Ca_V2.2), although the same phenomenon of cross talk between G protein modulation and PKC activation occurs (Bertaso et al., 2003). Thus, the site(s) of phosphorylation by PKC responsible for the reversal of G protein modulation is not yet certain (Bertaso et al., 2003). It has been shown recently that G_q binds to the C terminus of N-type calcium channels, to which PKC was also found to bind, and this colocalization may facilitate the phosphorylation of the N-type calcium channel (Simen et al., 2001). It is also possible that, if there is a reduction in membrane levels of phosphatidylinositol (4,5)-bisphosphate (PIP₂) resulting from phospholipase C activation, this may play a role in the reversal or attenuation of G protein modulation, since PIP₂ has been shown to regulate calcium channels (Wu et al., 2002). However, it remains controversial whether PIP₂ levels are reduced substantially after activation of G protein-coupled receptors linked to phospholipase C, because no global change was observed in the heart (Nasuhoglu et al., 2002), but the reduction may be localized and also linked to increased synthesis of PIP₂ (Zhao et al., 2001).

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

A number of studies have suggested that the C terminus of certain Ca_V2 calcium channels is essential for their G protein modulation and indeed binds either G or G subunits (Qin et al., 1997; Furukawa et al., 1998a,b; Kinoshita et al., 2001). However, my group has found that the chimeric constructs containing domain I of Ca_V2.2 and the entire final three domains and C terminus of Ca_V1.2 is strongly G protein-modulated (Stephens et al., 1998b). Furthermore, truncation of the C terminus of Ca_V2.2 including the region homologous with that found to bind G in Ca_V2.3 (Qin et al., 1997) did not affect G protein modulation by GTPS (Meza and Adams, 1998), and a similar truncation of Ca_V2.2 was found by another group to reduce, but not to abolish modulation by somatostatin receptor activation (Hamid et al., 1999). Thus, any direct role for the C terminus is probably a minor one.

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

Top Previous Next

 
The identification of a QQIER motif, known to bind G dimers in other proteins, in the region of the 1 subunit I-II linker where the Ca_V subunit binds, suggested that the Ca_V subunit might be involved in G protein modulation.

A. Initial Evidence for the Role of Ca_V Subunits in G Protein Modulation in Native Neurons

To investigate the involvement of Ca_V subunits in G protein modulation, I first developed an antisense strategy to deplete dorsal root ganglion neurons of their Ca_V subunits by microinjection of an antisense oligonucleotide complementary to a region common to all subunits (Berrow et al., 1995). Antisense knockdown of the Ca_V subunit by about 90% resulted in an enhancement of the ability of the GABA-B receptor agonist (-)-baclofen to inhibit the residual currents (Campbell et al., 1995b). We hypothesized from these results that there might be a functional interaction between activated G protein and VGCC subunit for interaction with the relevant channels (Campbell et al., 1995b).

B. The Involvement of Ca_V Subunits in G Protein Inhibition of Heterologously Expressed Calcium Channels

The role of Ca_V subunits in G protein inhibition of expressed calcium channels has now been extensively examined (Bourinet et al., 1996; Qin et al., 1997; Roche and Treistman, 1998; Canti et al., 2000, 2001; Meir et al., 2000). In initial studies in Xenopus oocytes, there was reported to be less or even a complete loss of G protein inhibition following coexpression of a subunit (Bourinet et al., 1996; Qin et al., 1997), although these studies only examined inhibition at a single potential and need to be interpreted with caution because of the presence of an endogenous oocyte Ca_V subunit. The result was interpreted in terms of a competition or displacement of Ca_V by G at an overlapping binding site (Bourinet et al., 1996). However, since subunits shift the calcium current activation to more hyperpolarized potentials, it is inappropriate to measure G protein-mediated inhibition at a single potential. By studying the voltage dependence of receptor-mediated inhibition in Xenopus oocytes, my group has shown that this is a bell-shaped curve, peaking at about the voltage for 50% current activation (Canti et al., 2000). In the absence of coexpressed subunits, the maximum amount of inhibition induced by activation of the coexpressed dopamine D2 receptor was about 70% at -10 mV. This curve is hyperpolarized in the presence of coexpressed subunits, and the peak inhibition observed with 1b, 3, and 4 was little changed at 70, 62, and 59%, although it occurred at -20 mV, whereas with 2a coexpression, maximal inhibition was modestly reduced, being 51% at -10 mV (Canti et al., 2000). Thus, it is likely that this cannot represent a simple competition between Ca_V subunits and G dimers, but the interaction is dynamic and depends on the membrane voltage.

C. Does G Displace Ca_V Subunits?

In this section, the evidence will be assessed for two different views concerning the mechanism of inhibition of calcium channels by G proteins: 1) that Ca_V subunits do not dissociate during this process, which involves an allosteric rearrangement of the 1- interaction associated with the voltage-dependent binding and unbinding of G:Ca_V1-Ca_V + G Ca_V1--G as proposed in Meir et al. (2000); or 2) that G protein modulation is favored in the absence of Ca subunits and opposed by the presence of subunits, indicating that Ca_V and G compete for a single binding site on the 1 subunit, as in the reaction: Ca_V1-Ca_V + G Ca_V1-G + Ca_V as proposed by Bourinet et al. (1996) and Qin et al. (1997). Such a reaction would either require transient formation of an intermediate ternary Ca_V1--G complex or require that Ca_V dissociates before G binds, if they bind to the same site.

If G binding either displaced Ca_V or allosterically resulted in the physical dissociation of Ca_V, then the effects of the two species should oppose one another. Indeed, in many respects G dimers do appear to have the opposite effect from Ca_V subunits on calcium channel properties. All Ca_V subunits shift calcium channel activation to more hyperpolarized potentials (for review, see Birnbaumer et al., 1998) and G has the opposite effect (Bean, 1989). However, all Ca_V subunits except palmitoylated 2a hyperpolarize the steady-state inactivation by about 30 mV (Canti et al., 2000). In contrast, where it has been studied, little or no effect of G protein activation or G dimers has been observed on steady-state inactivation (Bean, 1989; Meir and Dolphin, 2002). This suggests that G does not simply displace Ca_V subunits and prevent their interaction with the channel.

For the Ca_V2 subfamily of channels, prepulse facilitation of G protein-modulated channels is thought to involve G unbinding from the channel, induced by depolarization, as described above. This finding can be used to test the hypothesis that there is a competition between G and Ca_V subunits, since if G unbinds during a prepulse, then Ca_V might be expected to bind in its place. If so, the rate of facilitation would be directly dependent on Ca_V concentration in the cytosol. Indeed, the rate of facilitation during a prepulse was markedly increased by the heterologous expression of all Ca_V subunits (Roche and Treistman, 1998; Canti et al., 2000), which might be construed as supporting this view.

My group therefore developed a means of testing this hypothesis by expressing increasing amounts of Ca_V3 cDNA with a constant amount of Ca_V2.2 cDNA in Xenopus oocytes. We first showed that there was a linear relationship between 3 cDNA injected and 3 protein expressed (Canti et al., 2001). The 3 subunit was used in these experiments because it is almost identical to the endogenous Xenopus 3 present in oocytes (Tareilus et al., 1997). We then performed an intracellular doseresponse curve for Ca_V subunits to examine the concentration dependence of the effect of subunits to increase the facilitation rate (Canti et al., 2001). This experiment is illustrated in Fig. 10. At high Ca_V concentrations in oocytes (between about 20 and 100 ng of 3/oocyte), the facilitation rate during the depolarizing prepulse can be fit by a single fast exponential (Fig. 10, A and B), which we have interpreted as corresponding to the G off-rate from the channel that has Ca_V bound. The reason for this interpretation is that with these concentrations of 3 coexpressed, the steady-state inactivation of the Ca_V2.2 channels is fit by a single Boltzmann function, which is hyperpolarized compared with that for Ca_V2.2 expressed without a subunit (Fig. 10C).

View larger version (39K): [in this window] [in a new window]   FIG. 10. Concentration dependence of the facilitation rate of CaV2.2 currents in Xenopus oocytes. CaV2.2 was expressed either in the absence of exogenous subunit or in the presence of increasing concentrations of coinjected 3 cDNA. The amount of 3 cDNA injected is linearly related to the amount of 3 protein expressed (Canti et al., 2001). A, top panel, voltage protocol used: P1 and P2 are identical test pulses to 0 mV, with P2 being preceded by a depolarizing prepulse to +100 mV, of varying duration. Lower panel, examples of three families of currents generated by increasing the duration of the depolarizing prepulse. Left, CaV2.2 in the absence of exogenous CaV; middle, CaV2.2 in the presence of an intermediate concentration of exogenously expressed CaV3 (45 pg of 3 cDNA); right, CaV2.2 in the presence of a maximal concentration of CaV3 (720 pg of 3 cDNA). Five millimolars Ba2+ is the charge carrier. B, prepulse potentiation was measured by subtracting the current amplitude in P2 from that in P1 and normalizing this to the asymptotic value. The graph shows the slow exponential time course of facilitation for CaV2.2 in the absence of exogenous CaV3 (closed squares), the time course of facilitation for CaV2.2 in the presence of a representative intermediate concentration of exogenous CaV3 (45 pg of 3 cDNA, closed diamonds), which can only be fit by two exponentials to generate a slow and fast time constant of facilitation (see Canti et al., 2001, for details), and the fast exponential time course for CaV2.2 in the presence of a maximal concentration of CaV3 (720 pg of 3 cDNA, closed circles). C, steady-state inactivation was measured by delivering a 15-s conditioning pulse to the potential shown, followed by a test pulse to 0 mV. The proportion of current available is given by I/Imax, where Imax is the current in the absence of a conditioning step, and the curves are fit by either a single or a double Boltzmann function. The graph shows the steady-state inactivation for CaV2.2 in the absence of exogenous CaV3, which is fit by a single Boltzmann function with a depolarized midpoint of about -40 mV (closed squares). It also shows the steady-state inactivation for CaV2.2 in the presence of a maximal concentration of exogenous CaV3 which is fit by a single Boltzmann function with a hyperpolarized midpoint of about -70 mV (720 pg of 3 cDNA, closed circles). The steady-state inactivation curves time for CaV2.2 in the presence of two representative intermediate concentration of exogenous CaV3 (45 pg of 3 cDNA, closed diamonds, and 15 pg of 3 cDNA, open squares) can only be fit by varying proportions of a double Boltzmann function with midpoints at -40 and -70 mV (see Canti et al., 2001, for details). D, variation of the proportion of the slow and fast time constants, determined as in B, slow (closed squares) and fast (closed circles) with the amount of CaV3 subunit protein expressed per oocyte. The 3 protein expression level was determined by Western blotting (see Canti et al., 2001, for details). The plots are fit by sigmoid curves, the midpoint of which (arrow) is calculated to represent a concentration of 120 nM 3 (see Canti et al., 2001, for details). E, variation of the values of slow (closed squares) and fast (closed circles) with CaV subunit concentration. Results taken from experiments described in detail in Canti et al. (2001).

At intermediate Ca_V concentrations, the facilitation rate is not well fit by a single exponential (Canti et al., 2001), but can be fit by the sum of the same invariant fast exponential plus a slow exponential (Fig. 10, A and B). The value of the fast time constant, interpreted above as the G off-rate, is invariant over 100-fold change of 3 concentration and is therefore highly unlikely to involve any process requiring actual binding of 3 from the bulk solution. However, one aspect of the process does show a dependence on Ca_V concentration. The proportion of current showing the fast facilitation rate (Fig. 10D) shows exactly the same dependence on Ca_V protein concentration as the proportion of current with a hyperpolarized steady-state inactivation (see the biphasic steady-state inactivation curves at intermediate Ca_V concentrations in Fig. 10C). This agrees with our interpretation that the fast time constant of facilitation represents the behavior of a population of channels that has Ca_V bound. Reciprocally, the proportion of current showing a slow time constant of facilitation decreases as Ca_V concentration is increased, as does the proportion of current with a depolarized steady-state inactivation (Fig. 10C). We interpret this component as representing Ca_V channels without a bound Ca_V. Unlike the component with the fast time constant of facilitation, this slow component of facilitation has a time constant that does vary with Ca_V concentration (Fig. 10D). We have interpreted this finding as representing Ca_V subunit binding to the population of free channels during the depolarizing prepulse, after which G then unbinds rapidly with the invariant fast time constant (Canti et al., 2001). From this study we conclude that there is not a simple competition between G and Ca_V subunits, but rather that under normal circumstances, G dissociates at depolarized potentials from and rebinds at hyperpolarized potentials to channels that have Ca_V bound. Only under circumstances when Ca_V is limiting, which might rarely occur in native tissues, does Ca_V bind with higher affinity during depolarization and thence induces G unbinding.

Thus, Ca_V must have a higher affinity for the channels in their depolarized state. This phenomenon of depolarization-dependent displacement of the 1- equilibrium toward increased Ca_V binding would only be observed under conditions where Ca_V is limiting, and it remains unknown whether this is ever the case in native tissues. In agreement with this interpretation, we could still observe significant tonic facilitation under conditions where G was minimized for Ca_V2.2 channels expressed in oocytes without a Ca_V subunit, in sharp contrast to the lack of tonic facilitation in the additional presence of exogenous 1b (Fig. 11) (Canti et al., 2000).

View larger version (25K): [in this window] [in a new window]   FIG. 11. Facilitation of CaV2.2 currents in the absence and presence of CaV subunits expressed in Xenopus oocytes. The protocol shown at the top was used to generate control P1 and P2 currents. Tonic G protein modulation has been removed by a previous application of quinpirole followed by recovery, as described previously (Canti et al., 2000). Constructs are expressed in the absence (A) or presence (B) of coexpressed 1b subunit. Ba2+ (5 mM) is the charge carrier. The prepulse was 200 ms in A and 50 ms in B. Lengthening the prepulse to 200 ms had no additional effect on facilitation in B. Expression was in Xenopus oocytes, which contain an endogenous low level of subunit. Note the presence of control facilitation (P2 con/P1 con) when subunits are not coexpressed, interpreted as resulting from enhanced binding of endogenous subunits during the depolarizing prepulse. In B, where 1b is coexpressed, the lack of control facilitation can be interpreted as suggesting that most channels are already associated with coexpressed 1b subunits at the holding potential of -100 mV. C, control facilitation is quantified, for the two conditions, no coexpressed (n = 4, open circles) where facilitation reaches 2.6, and with coexpressed 3 (n = 5, closed circles) where facilitation is 1.4 at the same potential. Results taken from experiments described in Canti et al. (2000).

It was found that the reinhibition rate following a prepulse (for the Ca_V2.2/1b/2-1 combination) was increased as the concentration of G protein in the patch pipette was increased (Zamponi and Snutch, 1998), but in my group, we have observed that this rate is also increased slightly by overexpression of any Ca_V subunit together with Ca_V2.2, compared with the rate in the presence only of endogenous oocyte Ca_V (Canti et al., 2000). If there were direct competition between G and Ca_V for an overlapping binding site, then Ca_V should unbind during this process, before G rebinds. During the process of reinhibition following a prepulse, the depolarizing shift in activation and consequently the inhibition observed would result from Ca_V unbinding, rather than G binding, and therefore should not be dependent on G concentration. Elevation of Ca_V would be expected to slow the overall reinhibition rate, which is the opposite of what is observed. The conclusion must be that G preferentially rebinds to the Ca_V1- combination, and this species predominates when Ca_V is overexpressed.

Our model for the functional interplay between G dimers and Ca_V subunits does not support the idea that there is a simple competition between Ca_V and G for binding to the channel, or that Ca_V dissociates from the channel during G protein modulation, but rather that under normal conditions where the channels all have a Ca_V bound, G allosterically disrupts the effect of Ca_V on Ca_V1 channels. Conversely, depolarization, such as occurs during a prepulse, results in a state-dependent conformational change between Ca_V1 and Ca_V, which decreases the stability of G binding.

The observation that at some potentials G protein modulation is enhanced in oocytes in the absence of overexpressed Ca_V in Xenopus oocytes (Bourinet et al., 1996), or following antisense depletion of Ca_V subunits in sensory neurons (Campbell et al., 1995b), may be explained as follows. The slowed current activation in the presence of G is one of the main components of G protein-mediated inhibition, and is a reflection of the fact that G-bound channels either do not open upon depolarization until G dissociates, or show a very brief reluctant opening (Patil et al., 1996; Lee and Elmslie, 2000). Since a reduction of Ca_V produces slowing of overall facilitation rate of G protein-modulated channels during the prepulse, then the same will be true for the smaller degree of G unbinding that occurs during the test pulse. This is likely to be the reason that a reduction in Ca_V levels results in the observation of enhanced inhibition during a test pulse, or at least a shift in the voltage dependence of inhibition to more depolarized potentials (Canti et al., 2000). Conversely, an elevation of Ca_V reduces the inhibition observed. It should be noted that the direct effects of Ca_V subunits on inactivation during the test pulse, while not directly influencing the process of G protein modulation, will also influence the net amount of modulation exhibited (Meir et al., 2000; Meir and Dolphin, 2002).

In an expression system (COS-7 cells) in which (unlike Xenopus oocytes) no endogenous Ca_V subunit protein was detected by immunocytochemistry (Meir et al., 2000), coexpression of G with Ca_V2.2 in the absence of Ca_V resulted in calcium channel currents that were rapidly activating and not facilitated by a prepulse. This was observed both at the whole-cell level (Fig. 5B), and at the single-channel level (Fig. 6B). However, G did produce a small reduction in the current amplitude, compared with currents recorded in the absence of G. For all these sets of currents, their depolarized activation clearly showed that no Ca_V was associated. The additional presence of heterologously expressed Ca_V subunits was required for the relief of G-mediated inhibition by a depolarizing prepulse (Figs. 5A, 6A, and 7A) (Meir et al., 2000).

At the single-channel level, in the cell-attached patch mode, when only one channel is present, the effect of G and Ca_V can be compared in the absence of the confounding effect of Ca_V on the number of channels expressed. The main effect of coexpression Ca_V2a on Ca_V2.2 channel properties is an increase in the mean open time and a hyperpolarizing shift in the latency to first opening (Meir et al., 2000). For the model in which Ca_V is displaced by G to be correct, the currents in the presence of G should display the same properties as those in the absence of Ca_V, that is, the combination Ca_V2.21/G. This is not the case as the main effect of G in the presence of Ca_V is an increased latency to first opening. For the competition hypothesis to be true, if it is assumed that the N-type channels do not open before G unbinds, then before the first opening when the Ca_V2.21//G combination is coexpressed, only G should be bound; however, the channels do not show the same properties as the Ca_V2.21/G combination (Meir et al., 2000).

We concluded from that study that Ca_V subunits were essential for the process of facilitation or G dissociation. In the same system, receptor-mediated inhibition via activation of the D2 dopamine receptor was also examined. It was much reduced in the absence of coexpressed Ca_V subunits (Fig. 7B), and reversal of this inhibition by a 100-ms prepulse was lost, implying that in the absence of Ca_V subunits, G dimers are able to bind and produce a small non-voltage-dependent inhibition of the Ca_V2.2 current, but their unbinding is not influenced by voltage (Meir et al., 2000).

D. Potential Overlap of Determinants for Ca_V Subunit and G Subunit Function

There is overlap in the determinants for G protein modulation and Ca_V binding or function for all three of the sites discussed above: the I-II linker, the C terminus, and the N terminus of Ca_V2 calcium channels. The G binding site on the Ca_V1 subunit intracellular I-II loop (De Waard et al., 1997; Zamponi et al., 1997) partially coincides with binding sites for auxiliary Ca_V subunits (Pragnell et al., 1994). However, the main amino acids that are critical for Ca_V subunit interaction are not within but adjacent to the QxxER consensus sequence implicated in G binding (Herlitze et al., 1996; De Waard et al., 1997).

Within the N terminus of Ca_V2.2 between amino acids 45 and 55, four individual point mutations (S48A, I49A, R52A, and R54A) were isolated, which significantly compromised modulation of Ca_V2.2 by G proteins (Canti et al., 1999). My group has subsequently shown that both the Ca_V2.2-R52,54A and Ca_V2.2-R52A constructs also exhibited compromised 2a-mediated retardation of inactivation as did Ca_V2.2-Q47A, which was shown previously to undergo normal G modulation (Stephens et al., 2000). Taken together with our initial study that identified this site (Canti et al., 1999), the results indicate that the Ca_V2.2 amino terminus contributes determinants for both Ca_V subunit and G dimer function. However, the differentiating effect of Ca_V2.2-Q47A suggests that although the overall region involved may partially coincide, the determinants are not identical.

A partial overlap in Ca_V subunit and G binding sites has also been proposed for the Ca_V2.3 carboxyl-terminal site (Qin et al., 1997). However, whereas deletion of the majority of this Ca_V2.3 site affected G modulation, it allowed retention of full sensitivity to 2a (Qin et al., 1997), suggesting that this is not the prime mediator of the subunit response (see also Jones et al., 1998).

	VI. Molecular Mechanism of G Protein-Mediated Inhibition

Top Previous Next

 
It is still not understood how G binding to the Ca_V1 subunit results in inhibition of the calcium current. It is first necessary to establish the nature of the inhibitory effect at the level of a single calcium channel. It was initially suggested either that the G protein-bound channels opened with different gating properties (Kasai and Aosaki, 1989) or that there was a modal shift in gating (Delcour et al., 1993; Delcour and Tsien, 1993). However, the view that now prevails is that G protein-bound channels are reluctant to open, and that dissociation of bound G protein from the channel is required to convert them into willing channels (Bean, 1989; Elmslie et al., 1990). The simplest model would involve opening only the free and not the G protein-bound channel (for review, see Dolphin, 1991). At the single-channel level there is a prolonged latency to first opening in the presence of an agonist (Patil et al., 1996). However, once a channel had opened, no difference was observed on subsequent open probability or gating pattern compared with nonmodulated channels (Patil et al., 1996). This result is in agreement with the hypothesis that the delay to first opening is due to dissociation of the G dimers from the channel, allowing it to open, and that the G protein-bound channel does not open even with large depolarizations. This indicates either that the G binding is itself strongly voltage- or state-dependent or that G binds to a site on the channel that produces a voltage-dependent inhibition. More recently, it has been suggested that reluctant or G-bound channels can open, albeit with a low probability (Lee and Elmslie, 2000).

It is still unclear how many G dimers are required to bind to each Ca_V2 channel to produce inhibition. Models have suggested that more than one activated G protein may be bound per channel in a cooperative manner (Boland and Bean, 1993), but more recently it was suggested that a single G was bound per channel (Zamponi and Snutch, 1998).

The mechanism by which bound G prevents the channel from opening is also unknown. In one study, the effect of G protein activation on voltage sensor movement in Ca_V2.2 was examined. This revealed that GTPS produced a depolarizing shift in the voltage dependence of charge movement that could be reversed by a large depolarizing prepulse and also induced the appearance of a slow component of "on" gating charge (Jones et al., 1997). The greatest effect was the large separation on the voltage axis between gating charge movement and channel opening. Thus, G is acting both to slow voltage sensor movement and to inhibit the subsequent transduction of this movement into channel opening (Jones et al., 1997). In partial agreement with this, from a study exploiting a spontaneous mutation in Ca_V2.2 channels, G177E in domain IS3, which converts channels into a form that behaves as if it is tonically G protein-modulated in the absence of G dimers, it was suggested that the normal role of G dimers is voltage sensor trapping (Zhong et al., 2001).

There are a number of potential mechanisms whereby the N terminus might exert its essential role in G protein modulation. Three possibilities will be considered here. The first possibility is that, bearing in mind the effect of the N terminus of Ca_V2.x channels on the actions of Ca_V subunits, it might form part of a complex Ca_V subunit binding pocket, into which G dimers could intercalate. However, the interaction of the N terminus with subunits is unlikely to be of high affinity; as in a yeast two-hybrid assay, the N terminus did not interact with Ca_V subunits or with the I-II linker of Ca_V2.2 (Canti et al., 2001), although as described above (Section II.B.), the N terminus of Ca_V2.1 has been shown to bind to 4 subunits in overlay assays (Walker et al., 1999).

The second possible mechanism is that the N-terminal motif identified in Ca_V2.x channels might also form the effector of G protein modulation, as suggested in Canti et al. (1999). It might, for example, create a blocking particle, in a manner somewhat analogous to the ball and chain model of potassium channel block by the N terminus (MacKinnon et al., 1993). However, the N-terminal motif is not at the extreme N terminus, because it represents amino acids 44 to 55 in Ca_V2.2, and amino acids N-terminal to this motif (1–44 in Ca_V2.2) are not required for G protein modulation (Canti et al., 1999). The sequence of the N-terminal ball peptide in K_V1.1 (Shaker B) is MAAVAGLYGLGEDRQHRKKQ, and there are no similarities with the N-terminal motif of Ca_V2.2 (YKQSIAQRART), apart from the presence of a number of essential positively charged residues and a KQ motif. In the case of K_V channels, the ball peptide inserts into the inner vestibule of the pore of the open channel and produces inactivation by open-channel block. If this type of action is involved in G protein modulation of Ca_V2 channels, the inhibition is not an open-channel block mechanism; rather it is both retarding voltage sensor movement and preventing channel opening in response to the voltage sensor movement, and the effect is relieved by a large depolarization. One might envisage that the N-terminal motif is held in place, for example, to anchor the voltage sensor(s) by the binding of a G dimer, and its association is weakened by an altered interaction between the Ca_V1 and Ca_V subunits induced by depolarization.

A third possibility is that the relevant part of the N terminus may form a PIP₂ binding site, since RAR is similar to motifs containing positively charged residues in GIRK channels involved in binding the negatively charged head groups of PIP₂, resulting in membrane association. In GIRK channels, PIP₂ binding is thought to lead to association of residues on the N and C termini with the inner surface of the plasma membrane, producing a channel conformation that favors activation. The interaction of these regions of GIRK channels with PIP₂ is found to be stabilized by G, and the presence of PIP₂ in the membrane is a prerequisite for G modulation (Huang et al., 1998; Logothetis and Zhang, 1999; Zhang et al., 1999). If such a mechanism were to occur for calcium channel modulation, PIP₂ might be expected to coregulate the channel together with G dimers. Indeed, this has been studied (Wu et al., 2002), and PIP₂ was found to have a dual effect, both preventing calcium channel rundown in patches and producing an inhibitory modulation. Whether the inhibitory effect of PIP₂ directly interacts with G modulation remains unclear.

	VII. Recovery from G Protein-Mediated Inhibition

Top Previous Next

 
The speed of termination of GPCR-mediated inhibition of calcium currents depends on a number of factors. The rates of onset and offset of an agonist-mediated response are slower than the dissociation and reassociation rates of the activated G protein-channel complex, obtained by the prepulse protocol (Zhou et al., 1997). The onset of agonist-mediated inhibition using 10 µM noradrenaline was found to have a time constant of 0.7 s, whereas for reinhibition following a prepulse, the time constant was about 0.2 s. This difference is accounted for by the time taken for agonist binding to the receptor and for G protein activation. The rate of recovery from the agonist-mediated response (time constant of approximately 6 s for 10 µM noradrenaline) is very much slower than the facilitation rate, representing the dissociation of G measured during the depolarizing prepulse (Zhou et al., 1997). This discrepancy may be explained both by the off-rate of the agonist, which for some drugs may be very slow, the slower dissociation of G from the calcium channel at polarized rather than depolarized potentials (the basis for the voltage dependence of inhibition), and by the slow decay of the free G concentration, which determines the rebinding rate. This will depend on lateral diffusion in the membrane and reassociation of G and G-GDP, which will in turn be dependent on the rate of hydrolysis of activated G-GTP to G-GDP. The intrinsic hydrolysis rates of most heterotrimeric G proteins are much slower than the measured off-rate of this response. A family of RGS proteins has been identified that stimulates the GTPase activity of the G moiety of specific heterotrimeric G proteins (Watson et al., 1996; Dohlman and Thorner, 1997; García-Palomero et al., 2001). Overexpression of certain RGS proteins accelerated the off-rate of the response (Jeong and Ikeda, 2000). They also slowed the rate of recovery of inhibition after prepulse facilitation, indicating that they had reduced the level of free G by increasing the G-GDP available to rebind G dimers. Endogenous RGS proteins are likely to be involved in recovery from inhibition as expression of an RGS-insensitive G_o in sympathetic neurons resulted in a dramatic slowing of the rate of recovery of calcium currents after inhibition by noradrenaline (Jeong and Ikeda, 2000). Of interest in this regard, my group has shown that the GTPase activity of G_o in neuronal membranes is blocked by an antibody against Ca_V subunits (Campbell et al., 1995a). It remains to be determined whether there is an association between RGS proteins or G protein subunits and Ca_V subunits. It will also be fascinating to examine whether GPCRs are included in such macromolecular signaling complexes.

	VIII. Conclusion

Top Previous Next

 
A number of experiments indicate that the Ca_V2 calcium channel 1 I-II linker is involved in the modulation of the Ca_V2 family of calcium channels by G dimers. However, several pieces of evidence suggest that this is not the main site involved in mediating the effects of G, since the N terminus is essential in this regard (Page et al., 1998; Canti et al., 1999). Goals for the future include elucidation of the molecular mechanism of modulation by G dimers since there is still little understanding of the way in which G protein binding is converted into an effect on latency of channel opening (Patil et al., 1996). It will also be of interest to evaluate whether the G protein subunit plays a role in terminating the signal transduction process, which may be the case for GIRKs (Schreibmayer et al., 1996), and to examine the exact mechanism of the functional interaction between Ca_V subunits and G dimers in the inhibition of the Ca_V2 family of calcium channels.

	Acknowledgements

 
The work from my laboratory described in this review has largely been funded by grants from the Wellcome Trust. I thank and acknowledge the contribution of members of my group past and present, from Rod Scott, with whom I started this work, through Liz Fitzgerald, Nick Berrow, Alon Meir, Karen Page, Carles Canti, Damien Bell, and many others who have made substantial contributions.

	Footnotes

 
Address correspondence to: Prof. A. C. Dolphin, Department of Pharmacology, University College London, Gower St., London WC1E 6BT, UK. E-mail a.dolphin{at}ucl.ac.uk

DOI: 10.1124/pr.55.4.3.

¹ Abbreviations: VGCC, voltage-gated calcium channel; HVA, high-voltage-activated (channel); DHP, 1,4-dihydropyridine; AID, interaction domain; PKC, protein kinase C; GST, glutathione S-transferase; GPCR, G protein-coupled receptor; GTPS, guanosine 5'-O-(3-thiotriphosphate; GIRK, G protein-activated potassium channel; -ARK1, -adrenergic receptor kinase 1; RGS, regulators of G protein signaling; PIP₂, phosphatidylinositol (4,5)-bisphosphate.

	References

Top

 

Baertschi AJ, Audigier Y, Lledo P-M, Israel J-M, Bockaert J, and Vincent J-D (1992) Dialysis of lactotropes with antisense oligonucleotides assigns guanine nucleotide binding protein subtypes to their channel effectors. Mol Endocrinol 6: 2257-2265.[Abstract/Free Full Text]

Barclay J, Balaguero N, Mione M, Ackerman SL, Letts VA, Brodbeck J, Canti C, Meir A, Page KM, Kusumi K, et al. (2001) Ducky mouse phenotype of epilepsy and ataxia is associated with mutations in the Cacna2d2 gene and decreased calcium channel current in cerebellar Purkinje cells. J Neurosci 21: 6095-6104.[Abstract/Free Full Text]

Bean BP (1989) Neurotransmitter inhibition of neuronal calcium currents by changes in channel voltage-dependence. Nature (Lond) 340: 153-155.[CrossRef][Medline]

Bell DC, Butcher AJ, Berrow NS, Page KM, Brust PF, Nesterova A, Stauderman KA, Seabrook GR, Nurnberg B, and Dolphin AC (2001) Biophysical properties, pharmacology and modulation of human, neuronal L-type (1D, Ca_V1.3) voltage-dependent calcium currents. J Neurophysiol 85: 816-827.[Abstract/Free Full Text]

Berrow NS, Brice NL, Tedder I, Page K, and Dolphin AC (1997) Properties of cloned rat 1A calcium channels transiently expressed in the COS-7 cell line. Eur J Neurosci 9: 739-748.[CrossRef][Medline]

Berrow NS, Campbell V, Fitzgerald EG, Brickley K, and Dolphin AC (1995) Antisense depletion of B-subunits modulates the biophysical and pharmacological properties of neuronal calcium channels. J Physiol (Lond) 482: 481-491.[Abstract/Free Full Text]

Bertaso F, Ward RJ, Viard P, Milligan G, and Dolphin AC (2003) Mechanism of Action of Gq to inhibit Gbeta gamma modulation of Ca_V2.2 calcium channels: probed by the use of receptor-Galpha tandems. Mol Pharmacol 63: 832-843.[Abstract/Free Full Text]

Bichet D, Cornet V, Geib S, Carlier E, Volsen S, Hoshi T, Mori Y, and De Waard M (2000) The I-II loop of the Ca2+ channel alpha(1) subunit contains an endoplasmic reticulum retention signal antagonized by the beta subunit. Neuron 25: 177-190.[CrossRef][Medline]

Birnbaumer L, Qin N, Olcese R, Tareilus E, Platano D, Costantin J, and Stefani E (1998) Structures and functions of calcium channel subunits. J Bioenerg Biomembr 30: 357-375.[CrossRef][Medline]

Black JL and Lennon VA (1999) Identification and cloning of putative human neuronal voltage-gated calcium channel gamma-2 and gamma-3 subunits: neurologic implications. Mayo Clin Proc 74: 357-361.[Abstract]

Bogdanov Y, Brice NL, Canti C, Page KM, Li M, Volsen SG, and Dolphin AC (2000) Acidic motif responsible for plasma membrane association of the voltage-dependent calcium channel B1b subunit. Eur J Neurosci 12: 894-902.[CrossRef][Medline]

Boland LM and Bean BP (1993) Modulation of N-type calcium channels in bullfrog sympathetic neurons by luteinizing hormone-releasing hormone: kinetics and voltage dependence. J Neurosci 13: 516-533.[Abstract]

Bourinet E, Soong TW, Stea A, and Snutch TP (1996) Determinants of the G protein-dependent opioid modulation of neuronal calcium channels. Proc Natl Acad Sci USA 93: 1486-1491.[Abstract/Free Full Text]

Brice NL, Berrow NS, Campbell V, Page KM, Brickley K, Tedder I, and Dolphin AC (1997) Importance of the different subunits in the membrane expression of the 1A and 2 calcium channel subunits: studies using a depolarisation-sensitive 1A antibody. Eur J Neurosci 9: 749-759.[CrossRef][Medline]

Brody DL and Yue DT (2000) Relief of G-protein inhibition of calcium channels and short-term synaptic facilitation in cultured hippocampal neurons. J Neurosci 20: 889-898.[Abstract/Free Full Text]

Campbell V, Berrow N, Brickley K, Page K, Wade R, and Dolphin AC (1995a) Voltage-dependent calcium channel B-subunits in combination with alpha1 subunits have a GTPase activating effect to promote hydrolysis of GTP by G alpha_o in rat frontal cortex. FEBS Lett 370: 135-140.[CrossRef][Medline]

Campbell V, Berrow N, and Dolphin AC (1993) GABA_B receptor modulation of Ca²⁺ currents in rat sensory neurones by the G protein G_o: antisense oligonucleotide studies. J Physiol (Lond) 470: 1-11.[Abstract/Free Full Text]

Campbell V, Berrow NS, Fitzgerald EM, Brickley K, and Dolphin AC (1995b) Inhibition of the interaction of G protein G_o with calcium channels by the calcium channel B-subunit in rat neurones. J Physiol (Lond) 485: 365-372.[Abstract/Free Full Text]

Canti C, Bogdanov Y, and Dolphin AC (2000) Interaction between G proteins and accessory subunits in the regulation of 1B calcium channels in Xenopus oocytes. J Physiol (Lond) 527: 419-432.[Abstract/Free Full Text]

Canti C, Davies A, Berrow NS, Butcher AJ, Page KM, and Dolphin AC (2001) Evidence for two concentration-dependent processes for subunit effects on 1B calcium channels. Biophys J 81: 1439-1451.[Medline]

Canti C and Dolphin AC (2003) CaV subunit-mediated up-regulation of Ca_V2.2 currents triggered by D2 dopamine receptor activation. Neuropharmacology, 45: 814-827.[CrossRef][Medline]

Canti C, Page KM, Stephens GJ, and Dolphin AC (1999) Identification of residues in the N-terminus of 1B critical for inhibition of the voltage-dependent calcium channel by G. J Neurosci 19: 6855-6864.[Abstract/Free Full Text]

Carbone E and Lux HD (1984) A low voltage-activated, fully inactivating Ca channel in vertebrate sensory neurones. Nature (Lond) 310: 501-502.[Medline]

Castellano A, Wei X, Birnbaumer L, and Perez-Reyes E (1993a) Cloning and expression of a neuronal calcium channel subunit. J Biol Chem 268: 12359-12366.[Abstract/Free Full Text]

Castellano A, Wei X, Birnbaumer L, and Perez-Reyes E (1993b) Cloning and expression of a third calcium channel subunit. J Biol Chem 268: 3450-3455.[Abstract/Free Full Text]

Catterall WA (2000) Structure and regulation of voltage-gated Ca²⁺ channels. Annu Rev Cell Dev Biol 16: 521-555.[CrossRef][Medline]

Cens T, Restituito S, Galas S, and Charnet P (1999) Voltage and calcium use the same molecular determinants to inactivate calcium channels. J Biol Chem 274: 5483-5490.[Abstract/Free Full Text]

Chang FC and Hosey MM (1988) Dihydropyridine and phenylalkylamine receptors associated with cardiac and skeletal muscle calcium channels are structurally different. J Biol Chem 263: 18929-18937.[Abstract/Free Full Text]

Chen J, DeVivo M, Dingus J, Harry A, Li J, Sui J, Carty DJ, Blank JL, Exton JH, Stoffel RH, et al. (1995) A region of adenylyl cyclase 2 critical for regulation by G protein Bgamma subunits. Science (Wash DC) 268: 1166-1169.[Abstract/Free Full Text]

Chien AJ, Carr KM, Shirokov RE, Rios E, and Hosey MM (1996) Identification of palmitoylation sites within the L type calcium channel B_2a subunit and effects on channel function. J Biol Chem 271: 26465-26469.[Abstract/Free Full Text]

Chien AJ and Hosey MM (1998) Post-translational modifications of subunits of voltage-dependent calcium channels. J Bioenerg Biomembr 30: 377-386.[CrossRef][Medline]

Chien AJ, Zhao XL, Shirokov RE, Puri TS, Chang CF, Sun D, Rios E, and Hosey MM (1995) Roles of a membrane-localized subunit in the formation and targeting of functional L-type Ca²⁺ channels. J Biol Chem 270: 30036-30044.[Abstract/Free Full Text]

Clapham DE and Neer EJ (1993) New roles for G-protein gamma dimers in transmembrane signalling. Nature (Lond) 365: 403-406.[CrossRef][Medline]

Cooper CB, Arnot MI, Feng ZP, Jarvis SE, Hamid J, and Zamponi GW (2000) Cross-talk between G-protein and protein kinase C modulation of N-type calcium channels is dependent on the G-protein subunit isoform. J Biol Chem 275: 40777-40781.[Abstract/Free Full Text]

Degtiar VE, Wittig B, Schultz G, and Kalkbrenner F (1996) A specific G₀ heterotrimer couples somatostatin receptors to voltage-gated calcium channels in RINm5F cells. FEBS Lett 380: 137-141.[CrossRef][Medline]

Delcour AH, Lipscombe D, and Tsien RW (1993) Multiple modes of N-type calcium channel activity distinguished by differences in gating kinetics. J Neurosci 13: 181-194.[Abstract]

Delcour AH and Tsien RW (1993) Altered prevalence of gating modes in neurotransmitter inhibition of N-type calcium channels. Science (Wash DC) 259: 980-984.[Abstract]

Delmas P, Abogadie FC, Buckley NJ, and Brown DA (2000) Calcium channel gating and modulation by transmitters depend on cellular compartmentalization. Nat Neurosci 3: 670-678.[CrossRef][Medline]

Delmas P, Abogadie FC, Milligan G, Buckley NJ, and Brown DA (1999) Beta gamma dimers derived from G(o) and G(i) proteins contribute different components of adrenergic inhibition of Ca2+ channels in rat sympathetic neurones. J Physiol (Lond) 518: 23-36.[Abstract/Free Full Text]

De Waard M, Liu HY, Walker D, Scott VES, Gurnett CA, and Campbell KP (1997) Direct binding of G-protein Bgamma complex to voltage-dependent calcium channels. Nature (Lond) 385: 446-450.[CrossRef][Medline]

De Waard M, Pragnell M, and Campbell KP (1994) Ca²⁺ channel regulation by a conserved subunit domain. Neuron 13: 495-503.[CrossRef][Medline]

De Waard M, Scott VES, Pragnell M, and Campbell KP (1996) Identification of critical amino acids involved in ₁- interaction in voltage-dependent Ca²⁺ channels. FEBS Lett 380: 272-276.[CrossRef][Medline]

De Waard M, Witcher DR, Pragnell M, Liu H, and Campbell KP (1995) Properties of the ₁- anchoring site in voltage-dependent Ca²⁺ channels. J Biol Chem 270: 12056-12064.[Abstract/Free Full Text]

Diversé-Pierluissi M and Dunlap K (1993) Distinct, convergent second messenger pathways modulate neuronal calcium currents. Neuron 10: 753-760.[CrossRef][Medline]

Diversé-Pierluissi M, Goldsmith PK, and Dunlap K (1995) Transmitter-mediated inhibition of N-type calcium channels in sensory neurons involves multiple GTP-binding proteins and subunits. Neuron 14: 191-200.[CrossRef][Medline]

Dohlman HG and Thorner J (1997) RGS proteins and signaling by heterotrimeric G proteins. J Biol Chem 272: 3871-3874.[Free Full Text]

Dolphin AC (1991) Regulation of calcium channel activity by GTP binding proteins and second messengers. Biochim Biophys Acta 1091: 68-80.[Medline]

Dolphin AC (1998) Mechanisms of modulation of voltage-dependent calcium channels by G proteins. J Physiol (Lond) 506: 3-11.[Free Full Text]

Dolphin AC (1999) L-type calcium channel modulation. Adv Second Messenger Phosphoprotein Res 33: 153-177.[Medline]

Dolphin AC (2003) subunits of voltage-gated calcium channels. J Bioenerg Biomembr, in press.

Dolphin AC, Forda SR, and Scott RH (1986) Calcium-dependent currents in cultured rat dorsal root ganglion neurones are inhibited by an adenosine analogue. J Physiol (Lond) 373: 47-61.[Abstract/Free Full Text]

Dolphin AC and Prestwich SA (1985) Pertussis toxin reverses adenosine inhibition of neuronal glutamate release. Nature (Lond) 316: 148-150.[CrossRef][Medline]

Dolphin AC and Scott RH (1987) Calcium channel currents and their inhibition by (-)-baclofen in rat sensory neurones: modulation by guanine nucleotides. J Physiol (Lond) 386: 1-17.[Abstract/Free Full Text]

Dolphin AC and Scott RH (1990) Modulation of neuronal calcium currents by G protein activation, in G Proteins and Signal Transduction (Nathanson N ed) pp 12-27, Rockefeller University Press, New York, NY.

Dolphin AC, Wootton JF, Scott RH, and Trentham DR (1988) Photoactivation of intracellular guanosine triphosphate analogues reduces the amplitude and slows the kinetics of voltage-activated calcium channel currents in sensory neurones. Pflueg Arch Eur J Physiol 411: 628-636.[CrossRef][Medline]

Dubel SJ, Starr TVB, Hell J, Ahlijanian MK, Enyeart JJ, Catterall WA, and Snutch TP (1992) Molecular cloning of the -1 subunit of an -conotoxin-sensitive calcium channel. Proc Natl Acad Sci USA 89: 5058-5062.[Abstract/Free Full Text]

Dunlap K and Fischbach GD (1978) Neurotransmitters decrease the calcium component of sensory neurone action potentials. Nature (Lond) 276: 837-839.[CrossRef][Medline]

Dunlap K and Fischbach GD (1981) Neurotransmitters decrease the calcium conductance activated by depolarization of embryonic chick sensory neurones. J Physiol (Lond) 317: 519-535.[Abstract/Free Full Text]

Dunlap KL and Ikeda SR (1998) Receptor-mediated pathways that modulate calcium channels. Semin Neurosci 9: 198-208.

Ellis SB, Williams ME, Ways NR, Brenner R, Sharp AH, Leung AT, Campbell KP, McKenna E, Koch WJ, Hui A, et al. (1988) Sequence and expression of MRNAs encoding the ₁ and ₂ subunits of a DHP-sensitive calcium channel. Science (Wash DC) 241: 1661-1664.[Abstract/Free Full Text]

Elmslie KS and Jones SW (1994) Concentration dependence of neurotransmitter effects on calcium current kinetics in frog sympathetic neurones. J Physiol (Lond) 481: 35-46.[Abstract/Free Full Text]

Elmslie KS, Zhou W, and Jones SW (1990) LHRH and GTPyS modify calcium current activation in bullfrog sympathetic neurons. Neuron 5: 75-80.[CrossRef][Medline]

Ertel EA, Campbell KP, Harpold MM, Hofmann F, Mori Y, Perez-Reyes E, Schwartz A, Snutch TP, Tanabe T, Birnbaumer L, et al. (2000) Nomenclature of voltagegated calcium channels. Neuron 25: 533-535.[CrossRef][Medline]

Ewald DA, Pang I-H, Sternweis PC, and Miller RJ (1989) Differential G protein-mediated coupling of neurotransmitter receptors to Ca²⁺ channels in rat dorsal root ganglion neurons in vitro. Neuron 2: 1185-1193.[CrossRef][Medline]

Fatt P and Katz B (1953) The electrical properties of crustacean muscle fibres. J Physiol (Lond) 120: 171-204.

Fletcher JE, Lindorfer MA, DeFilippo JM, Yasuda H, Guilmard M, and Garrison JC (1998) The G protein ₅ subunit interacts selectively with the G_q subunit. J Biol Chem 273: 636-644.[Abstract/Free Full Text]

Flockerzi V, Oeken H-J, Hofmann F, Pelzer D, Cavalié A, and Trautwein W (1986) Purified dihydropyridine-binding site from skeletal muscle T-tubules is a functional calcium channel. Nature (Lond) 323: 66-68.[CrossRef][Medline]

Forscher P, Oxford GS, and Schulz D (1986) Noradrenaline modulates calcium channels in avian dorsal root ganglion cells through tight receptor-channel coupling. J Physiol (Lond) 379: 131-144.[Abstract/Free Full Text]

Furukawa T, Miura R, Mori Y, Strobeck M, Suzuki K, Ogihara Y, Asano T, Morishita R, Hashii M, Higashida H, et al. (1998a) Differential interactions of the C terminus and the cytoplasmic I-II loop of neuronal Ca2+ channels with G-protein alpha and beta gamma subunits. II. Evidence for direct binding. J Biol Chem 273: 17595-17603.[Abstract/Free Full Text]

Furukawa T, Nukada T, Mori Y, Wakamori M, Fujita Y, Ishida H, Fukuda K, Kato S, and Yoshii M (1998b) Differential interactions of the C terminus and the cytoplasmic I-II loop of neuronal Ca2+ channels with G-protein alpha and beta gamma subunits. I. Molecular determination. J Biol Chem 273: 17585-17594.[Abstract/Free Full Text]

Garcia DE, Li B, Garcia-Ferreiro RE, Hernandez-Ochoa EO, Yan K, Gautam N, Catterall WA, Mackie K, and Hille B (1998) G protein subunit specificity in the fast membrane delimited inhibition of Ca²⁺ channels. J Neurosci 18: 9163-9170.[Abstract/Free Full Text]

García-Palomero E, Renart J, Andrés-Mateos E, Solís-Garrido L, Matute C, Herrero CJ, García AG, and Montiel C (2001) Differential expression of calcium channel subtypes in the bovine adrenal medulla. Neuroendocrinology 74: 251-261.[CrossRef][Medline]

Golard A, Role L, and Siegelbaum SA (1994) Substance P potentiates calcium channel modulation by somatostatin in chick sympathetic ganglia. J Neurophysiol 72: 2683-2690.[Abstract/Free Full Text]

Hagiwara S and Takahashi K (1967) Surface density of calcium ions and calcium spikes in the barnacle muscle fiber membrane. J Gen Physiol 50: 583-601.[Abstract/Free Full Text]

Hamid J, Nelson D, Spaetgens R, Dubel SJ, Snutch TP, and Zamponi GW (1999) Identification of an integration center for cross-talk between protein kinase C and G protein modulation of N-type calcium channels. J Biol Chem 274: 6195-6202.[Abstract/Free Full Text]

Hanlon MR, Berrow NS, Dolphin AC, and Wallace BA (1999) Modelling of a voltage-dependent Ca²⁺ channel subunit as a basis for understanding its functional properties. FEBS Lett 445: 366-370.[CrossRef][Medline]

Herlitze S, Garcia DE, Mackie K, Hille B, Scheuer T, and Catterall WA (1996) Modulation of Ca²⁺ channels by G-protein gamma subunits. Nature (Lond) 380: 258-262.[CrossRef][Medline]

Herlitze S, Hockerman GH, Scheuer T, and Catterall WA (1997) Molecular determinants of inactivation and G protein modulation in the intracellular loop connecting domains I and II of the calcium channel _1A subunit. Proc Natl Acad Sci USA 94: 1512-1516.[Abstract/Free Full Text]

Hescheler J, Rosenthal W, Trautwein W, and Schultz G (1987) The GTP-binding protein, G_o, regulates neuronal calcium channels. Nature (Lond) 325: 445-447.[CrossRef][Medline]

Hess P, Lansman JB, and Tsien RW (1984) Different modes of Ca channel gating behaviour favoured by dihydropyridine Ca agonists and antagonists. Nature (Lond) 311: 538-544.[CrossRef][Medline]

Hibino H, Pironkova R, Onwumere O, Rousset M, Charnet P, Hudspeth AJ, and Lesage F (2003) Direct interaction with a nuclear protein and regulation of gene silencing by a variant of the Ca²⁺ channel 4 subunit. Proc Natl Acad Sci USA 100: 307-312.[Abstract/Free Full Text]

Hille B (1992) G protein-coupled mechanisms and nervous signaling. Neuron 9: 187-195.[Medline]

Hille B (2001) Ion Channels of Excitable Membranes, Sinauer Associates Inc, Sunderland, MA.

Hillman D, Chen S, Aung TT, Cherksey B, Sugimori M, and Llinás R (1991) Localization of P-type calcium channels in the central nervous system. Proc Natl Acad Sci USA 88: 7076-7080.[Abstract/Free Full Text]

Holz GGI, Rane SG, and Dunlap K (1986) GTP-binding proteins mediate transmitter inhibition of voltage-dependent calcium channels. Nature (Lond) 319: 670-672.[CrossRef][Medline]

Hosey MM, Barhanin J, Schmid A, Vandaele S, Ptasienski J, O'Callahan C, Cooper C, and Lazdunski M (1987) Photoaffinity labelling and phosphorylation of a 165 kilodalton peptide associated with dihydropyridine and phenylalkylamine-sensitive calcium channels. Biochem Biophys Res Commun 147: 1137-1145.[CrossRef][Medline]

Huang CL, Feng S, and Hilgemann DW (1998) Direct activation of inward rectifier potassium channels by PIP2 and its stabilization by Gbetagamma. Nature (Lond) 391: 803-806.[CrossRef][Medline]

Hullin R, Khan IFY, Wirtz S, Mohacsi P, Varadi G, Schwartz A, and Herzig S (2003) Cardiac L-type calcium channel -subunits expressed in human heart have differential effects on single channel characteristics. J Biol Chem 2003: 21623-21630.

Hymel L, Striessnig J, Glossmann H, and Schindler H (1988) Purified skeletal muscle 1,4-dihydropyridine receptor forms phosphorylation-dependent oligomeric calcium channels in planar bilayers. Proc Natl Acad Sci USA 85: 4290-4294.[Abstract/Free Full Text]

Ikeda SR (1991) Double-pulse calcium channel current facilitation in adult rat sympathetic neurones. J Physiol (Lond) 439: 181-214.[Abstract/Free Full Text]

Ikeda SR (1996) Voltage-dependent modulation of N-type calcium channels by G protein gamma subunits. Nature (Lond) 380: 255-258.[CrossRef][Medline]

Ikeda SR and Schofield GG (1989) Somatostatin blocks a calcium current in rat sympathetic ganglion neurones. J Physiol (Lond) 409: 221-240.[Abstract/Free Full Text]

Jeong SW and Ikeda SR (1999) Sequestration of G-protein beta gamma subunits by different G-protein alpha subunits blocks voltage-dependent modulation of Ca2+ channels in rat sympathetic neurons. J Neurosci 19: 4755-4761.[Abstract/Free Full Text]

Jeong SW and Ikeda SR (2000) Endogenous regulator of G-protein signaling proteins modify N-type calcium channel modulation in rat sympathetic neurons. J Neurosci 20: 4489-4496.[Abstract/Free Full Text]

Jones LP, Patil PG, Snutch TP, and Yue DT (1997) G-protein modulation of N-type calcium channel gating current in human embryonic kidney cells (HEK 293). J Physiol (Lond) 498: 601-610.[Abstract/Free Full Text]

Jones LP, Wei SK, and Yue DT (1998) Mechanism of auxiliary subunit modulation of neuronal _1E calcium channels. J Gen Physiol 112: 125-143.[Abstract/Free Full Text]

Jones SW and Elmslie KS (1997) Transmitter modulation of neuronal calcium channels. J Membr Biol 155: 1-10.[CrossRef][Medline]

Josephson IR and Varadi G (1996) The subunit increases Ca²⁺ currents and gating charge movements of human cardiac L-type Ca²⁺ channels. Biophys J 70: 1285-1293.[Medline]

Kammermeier PJ, Ruiz-Velasco V, and Ikeda SR (2000) A voltage-independent calcium current inhibitory pathway activated by muscarinic agonists in rat sympathetic neurons requires both Galpha Q/11 and Gbeta gamma. J Neurosci 20: 5623-5629.[Abstract/Free Full Text]

Kang MG, Chen CC, Felix R, Letts VA, Frankel WN, Mori Y, and Campbell KP (2001) Biochemical and biophysical evidence for gamma₂ subunit association with neuronal voltage-activated Ca²⁺ channels. J Biol Chem 276: 32917-32924.[Abstract/Free Full Text]

Kasai H and Aosaki T (1989) Modulation of Ca²⁺ channel current by an adenosine analog mediated by a GTP-binding protein in chick sensory neurons. Pflueg Arch Eur J Physiol 414: 145-149.[CrossRef][Medline]

Kinoshita M, Nukada T, Asano T, Mori Y, Akaike A, Satoh M, and Kaneko S (2001) Binding of Galpha(o) N terminus is responsible for the voltage-resistant inhibition of alpha (1A) (P/Q-type, Ca(v)2.1) Ca(2+) channels. J Biol Chem 276: 28731-28738.[Abstract/Free Full Text]

Kleuss C, Hescheler J, Ewel C, Rosenthal W, Schultz G, and Wittig B (1991) Assignment of G-protein subtypes to specific receptors inducing inhibition of calcium currents. Nature (Lond) 353: 43-48.[CrossRef][Medline]

Klugbauer N, Dai SP, Specht V, Lacinová L, Marais E, Bohn G, and Hofmann F (2000) A family of gamma-like calcium channel subunits. FEBS Lett 470: 189-197.[CrossRef][Medline]

Klugbauer N, Lacinova L, Marais E, Hobom M, and Hofmann F (1999) Molecular diversity of the calcium channel 2- subunit. J Neurosci 19: 684-691.[Abstract/Free Full Text]

Koh DS and Hille B (1997) Modulation by neurotransmitters of catecholamine secretion from sympathetic ganglion neurons detected by amperometry. Proc Natl Acad Sci USA 94: 1506-1511.[Abstract/Free Full Text]

Kurachi Y, Ito H, Sugimoto T, Katada T, and Ui M (1989) Activation of atrial muscarinic K⁺ channels by low concentrations of subunits of rat brain G protein. Pflueg Arch Eur J Physiol 413: 325-327.[CrossRef][Medline]

Lee HK and Elmslie KS (2000) Reluctant gating of single N-type calcium channels during neurotransmitter-induced inhibition in bullfrog sympathetic neurons. J Neurosci 20: 3115-3128.[Abstract/Free Full Text]

Letts VA, Felix R, Biddlecome GH, Arikkath J, Mahaffey CL, Valenzuela A, Bartlett FS, Mori Y, Campbell KP, and Frankel WN (1998) The mouse stargazer gene encodes a neuronal Ca²⁺ channel gamma subunit. Nat Genet 19: 340-347.[CrossRef][Medline]

Liu H, De Waard M, Scott VES, Gurnett CA, Lennon VA, and Campbell KP (1996) Identification of three subunits of the high affinity -conotoxin MVIIC-sensitive Ca²⁺ channel. J Biol Chem 271: 13804-13810.[Abstract/Free Full Text]

Logothetis DE, Kurachi Y, Galper J, Neer EJ, and Clapham DE (1987) The gamma subunits of GTP-binding proteins activate the muscarinic K⁺ channel in heart. Nature (Lond) 325: 321-326.[CrossRef][Medline]

Logothetis DE and Zhang HL (1999) Gating of G protein-sensitive inwardly rectifying K⁺ channels through phosphatidylinositol 4,5-bisphosphate. J Physiol (Lond) 520: 630.[Free Full Text]

MacKinnon R, Aldrich RW, and Lee AW (1993) Functional stoichiometry of Shaker potassium channel inactivation. Science (Wash DC) 262: 757-759.[Abstract/Free Full Text]

Man-Son-Hing H, Zoran MJ, Lukowiak K, and Haydon PG (1989) A neuromodulator of synaptic transmission acts on the secretory apparatus as well as on ion channels. Nature (Lond) 341: 237-239.[CrossRef][Medline]

McCleskey EW, Fox AP, Feldman DH, Cruz LJ, Olivera BM, Tsien RW, and Yoshickami D (1987) Omega-conotoxin: direct and persistent blockade of specific types of calcium channels in neurons but not muscle. Proc Natl Acad Sci USA 84: 4327-4331.[Abstract/Free Full Text]

McFadzean I, Mullaney I, Brown DA, and Milligan G (1989) Antibodies to the GTP binding protein, G_o, antagonize noradrenaline-induced calcium current inhibition in NG108–15 hybrid cells. Neuron 3: 177-182.[CrossRef][Medline]

Meir A, Bell DC, Stephens GJ, Page KM, and Dolphin AC (2000) Calcium channel subunit promotes voltage-dependent modulation of 1B by G. Biophys J 79: 731-746.[Medline]

Meir A and Dolphin AC (1998) Known calcium channel 1 subunits can form low threshold, small conductance channels, with similarities to native T type channels. Neuron 20: 341-351.[CrossRef][Medline]

Meir A and Dolphin AC (2002) Kinetics and Gbetagamma modulation of Ca(v)2.2 channels with different auxiliary beta subunits. Pflueg Arch Eur J Physiol 444: 263-275.[CrossRef][Medline]

Menon-Johansson AS, Berrow N, and Dolphin AC (1993) G_o transduces GABA_B-receptor modulation of N-type calcium channels in cultured dorsal root ganglion nNeurons. Pflueg Arch Eur J Physiol 425: 335-343.[CrossRef][Medline]

Meza U and Adams B (1998) G-protein-dependent facilitation of neuronal alpha1A, alpha1B and alpha1E Ca channels. J Neurosci 18: 5240-5252.[Abstract/Free Full Text]

Mikami A, Imoto K, Tanabe T, Niidome T, Mori Y, Takeshima H, Narumiya S, and Numa S (1989) Primary structure and functional expression of the cardiac dihydropyridine-sensitive calcium channel. Nature (Lond) 340: 230-233.[CrossRef][Medline]

Mintz IM, Venema VJ, Swiderek KM, Lee TD, Bean BP, and Adams ME (1992) P-type calcium channels blocked by the spider toxin -Aga-IVA. Nature (Lond) 355: 827-829.[CrossRef][Medline]

Mochida S, Sheng ZH, Baker C, Kobayashi H, and Catterall WA (1996) Inhibition of neurotransmission by peptides containing the synaptic protein interaction site of N-type Ca²⁺ channels. Neuron 17: 781-788.[CrossRef][Medline]

Mori Y, Friedrich T, Kim M-S, Mikami A, Nakai J, Ruth P, Bosse E, Hofmann F, Flockerzi V, Furuichi T, et al. (1991) Primary structure and functional expression from complementary DNA of a brain calcium channel. Nature (Lond) 350: 398-402.[CrossRef][Medline]

Moss FJ, Viard P, Davies A, Bertaso F, Page KM, Graham A, Canti C, Plumpton M, Plumpton M, Clare JJ, and Dolphin AC (2002) The novel product of a five-exon stargazin-related gene abolishes Ca_V2.2 calcium channel expression. EMBO J 21: 1514-1523.[CrossRef][Medline]

Nasuhoglu C, Feng S, Mao Y, Shammat I, Yamamato M, Earnest S, Lemmon M, and Hilgemann DW (2002) Modulation of cardiac PIP2 by cardioactive hormones and other physiologically relevant interventions. Am J Physiol Cell Physiol 283: C223-C234.[Abstract/Free Full Text]

Neely A, Wei X, Olcese R, Birnbaumer L, and Stefani E (1993) Potentiation by the subunit of the ratio of the ionic current to the charge movement in the cardiac calcium channel. Science (Wash DC) 262: 575-578.[Abstract/Free Full Text]

Nowycky MC, Fox AP, and Tsien RW (1985a) Long-opening mode of gating of neuronal calcium channels and its promotion by the dihydropyridine calcium agonist Bay K 8644. Proc Natl Acad Sci USA 82: 2178-2182.[Abstract/Free Full Text]

Nowycky MC, Fox AP, and Tsien RW (1985b) Three types of neuronal calcium channel with different calcium agonist sensitivity. Nature (Lond) 316: 440-446.[CrossRef][Medline]

Nunoki K, Florio V, and Catterall WA (1989) Activation of purified calcium channels by stoichiometric protein phosphorylation. Proc Natl Acad Sci USA 86: 6816-6820.[Abstract/Free Full Text]

Olcese R, Qin N, Schneider T, Neely A, Wei X, Stefani E, and Birnbaumer L (1994) The amino terminus of a calcium channel subunit sets rates of channel inactivation independently of the subunit's effect on activation. Neuron 13: 1433-1438.[CrossRef][Medline]

Page KM, Canti C, Stephens GJ, Berrow NS, and Dolphin AC (1998) Identification of the amino terminus of neuronal Ca²⁺ channel 1 subunits 1B and 1E as an essential determinant of G protein modulation. J Neurosci 18: 4815-4824.[Abstract/Free Full Text]

Page KM, Stephens GJ, Berrow NS, and Dolphin AC (1997) The intracellular loop between domains I and II of the B type calcium channel confers aspects of G protein sensitivity to the E type calcium channel. J Neurosci 17: 1330-1338.[Abstract/Free Full Text]

Patil PG, De Leon M, Reed RR, Dubel S, Snutch TP, and Yue DT (1996) Elementary events underlying voltage-dependent G-protein inhibition of N-type calcium channels. Biophys J 71: 2509-2521.[Medline]

Perez-Reyes E, Castellano A, Kim HS, Bertrand P, Baggstrom E, Lacerda AE, Wei X, and Birnbaumer L (1992) Cloning and expression of a cardiac/brain subunit of the L-type calcium channel. J Biol Chem 267: 1792-1797.[Abstract/Free Full Text]

Piedras-Rentería ES and Tsien RW (1998) Antisense oligonucleotides against alpha1E reduce R-type calcium currents in cerebellar granule cells. Proc Natl Acad Sci USA 95: 7760-7765.[Abstract/Free Full Text]

Pragnell M, De Waard M, Mori Y, Tanabe T, Snutch TP, and Campbell KP (1994) Calcium channel -subunit binds to a conserved motif in the I-II cytoplasmic linker of the ₁-subunit. Nature (Lond) 368: 67-70.[CrossRef][Medline]

Qin N, Platano D, Olcese R, Costantin JL, Stefani E, and Birnbaumer L (1998) Unique regulatory properties of the type 2a Ca²⁺ channel subunit caused by palmitoylation. Proc Natl Acad Sci USA 95: 4690-4695.[Abstract/Free Full Text]

Qin N, Platano D, Olcese R, Stefani E, and Birnbaumer L (1997) Direct interaction of Ggamma with a C terminal Ggamma binding domain of the calcium channel 1 subunit is responsible for channel inhibition by G protein coupled receptors. Proc Natl Acad Sci USA 94: 8866-8871.[Abstract/Free Full Text]

Qin N, Yagel S, Momplaisir ML, Codd EE, and D'Andrea MR (2002) Molecular cloning and characterization of the human voltage-gated calcium channel ₂-4 subunit. Mol Pharmacol 62: 485-496.[Abstract/Free Full Text]

Randall A and Tsien RW (1995) Pharmacological dissection of multiple types of Ca²⁺ channel currents in rat cerebellar granule neurons. J Neurosci 15: 2995-3012.[Abstract]

Restituito S, Cens T, Barrere C, Geib S, Galas S, De Waard M, and Charnet P (2000) The beta(2a) subunit is a molecular groom for the Ca2+ channel inactivation gate. J Neurosci 20: 9046-9052.[Abstract/Free Full Text]

Reuter H (1987) Calcium channel modulation by -adrenergic neurotransmitters in the heart. Experientia (Basel)43: 1173-1175.[CrossRef]

Roche JP and Treistman SN (1998) The calcium channel 3 subunit enhances voltage-dependent relief of G protein inhibition induced by muscarinic receptor activation and Ggamma. J Neurosci 18: 4883-4890.[Abstract/Free Full Text]

Ruiz-Velasco V and Ikeda SR (2000) Multiple G-protein gamma combinations produce voltage-dependent inhibition of N-type calcium channels in rat superior cervical ganglion neurons. J Neurosci 20: 2183-2191.[Abstract/Free Full Text]

Ruth P, Röhrkasten A, Biel M, Bosse E, Regulla S, Meyer HE, Flockerzi V, and Hofmann F (1989) Primary structure of the subunit of the DHP-sensitive calcium channel from skeletal muscle. Science (Wash DC) 245: 1115-1118.[Abstract/Free Full Text]

Schreibmayer W, Dessauer CW, Vorobiov D, Gilman AG, Lester HA, Davidson N, and Dascal N (1996) Inhibition of an inwardly rectifying K⁺ channel by G-protein -subunits. Nature (Lond) 380: 624-627.[CrossRef][Medline]

Scott RH and Dolphin AC (1986) Regulation of calcium currents by a GTP analogue: potentiation of (-)-baclofen-mediated inhibition. Neurosci Lett 69: 59-64.[CrossRef][Medline]

Scott VES, De Waard M, Liu HY, Gurnett CA, Venzke DP, Lennon VA, and Campbell KP (1996) subunit heterogeneity in N-type Ca²⁺ channels. J Biol Chem 271: 3207-3212.[Abstract/Free Full Text]

Shapiro MS and Hille B (1993) Substance P and somatostatin inhibit calcium channels in rat sympathetic neurons via different G protein pathways. Neuron 10: 11-20.[CrossRef][Medline]

Simen AA, Lee CC, Simen BB, Bindokas VP, and Miller RJ (2001) The C terminus of the Ca channel alpha1B subunit mediates selective inhibition by G-protein-coupled receptors. J Neurosci 21: 7587-7597.[Abstract/Free Full Text]

Singer D, Biel M, Lotan I, Flockerzi V, Hofmann F, and Dascal N (1991) The roles of the subunits in the function of the calcium channel. Science (Wash DC) 253: 1553-1557.[Abstract/Free Full Text]

Snow BE, Krumins AM, Brothers GM, Lee SF, Wall MA, Chung S, Mangion J, Arya S, Gilman AG, and Siderovski DP (1998) A G protein gamma subunit-like domain shared between RGS11 and other RGS proteins specifies binding to G₅ subunits. Proc Natl Acad Sci USA 95: 13307-13312.[Abstract/Free Full Text]

Soong TW, Stea A, Hodson CD, Dubel SJ, Vincent SR, and Snutch TP (1993) Structure and functional expression of a member of the low voltage-activated calcium channel family. Science (Wash DC) 260: 1133-1136.[Abstract/Free Full Text]

Spaetgens RL and Zamponi GW (1999) Multiple structural domains contribute to voltage-dependent inactivation of rat brain _1E calcium channels. J Biol Chem 274: 22428-22436.[Abstract/Free Full Text]

Stanley EF and Atrakchi AH (1990) Calcium currents recorded from a vertebrate presynaptic nerve terminal are resistant to the dihydropyridine nifedipine. Proc Natl Acad Sci USA 87: 9683-9687.[Abstract/Free Full Text]

Stea A, Soong TW, and Snutch TP (1995) Determinants of PKC-dependent modulation of a family of neuronal calcium channels. Neuron 15: 929-940.[CrossRef][Medline]

Stephens GJ, Brice NL, Berrow NS, and Dolphin AC (1998a) Facilitation of rabbit 1B calcium channels: involvement of endogenous Ggamma subunits. J Physiol (Lond) 509: 15-27.[Abstract/Free Full Text]

Stephens GJ, Canti C, Page KM, and Dolphin AC (1998b) Role of domain I of neuronal Ca²⁺ channel 1 subunits in G protein modulation. J Physiol (Lond) 509: 163-169.[Abstract/Free Full Text]

Stephens GJ, Page KM, Bogdanov Y, and Dolphin AC (2000) The 1B calcium channel amino terminus contributes determinants for subunit mediated voltage-dependent inactivation properties. J Physiol (Lond) 525: 377-390.[Abstract/Free Full Text]

Stephens GJ, Page KM, Burley JR, Berrow NS, and Dolphin AC (1997) Functional expression of rat brain cloned 1E calcium channels in COS-7 cells. Pflueg Arch Eur J Physiol 433: 523-532.[CrossRef][Medline]

Stotz SC, Hamid J, Spaetgens RL, Jarvis SE, and Zamponi GW (2000) Fast inactivation of voltage-dependent calcium channels. A hinged-lid mechanism? J Biol Chem 275: 24575-24582.[Abstract/Free Full Text]

Surprenant A, Shen K-Z, North RA, and Tatsumi H (1990) Inhibition of calcium currents by noradrenaline, somatostatin and opioids in guinea-pig submucosal neurones. J Physiol (Lond) 431: 585-608.[Abstract/Free Full Text]

Takahashi M, Seager MJ, Jones JF, Reber BFX, and Catterall WA (1987) Subunit structure of dihydropyridine-sensitive calcium channels from skeletal muscle. Proc Natl Acad Sci USA 84: 5478-5482.[Abstract/Free Full Text]

Takahashi T, Kajikawa Y, and Tsujimoto T (1998) G-protein-coupled modulation of presynaptic calcium currents and transmitter release by a GABAB receptor. J Neurosci 18: 3138-3146.[Abstract/Free Full Text]

Takahashi T and Momiyama A (1993) Different types of calcium channels mediate central synaptic transmission. Nature (Lond) 366: 156-158.[CrossRef][Medline]

Tanabe T, Takeshima H, Mikami A, Flockerzi V, Takahashi H, Kangawa K, Kojima M, Matsuo H, Hirose T, and Numa S (1987) Primary structure of the receptor for calcium channel blockers from skeletal muscle. Nature (Lond) 328: 313-318.[CrossRef][Medline]

Tareilus E, Roux M, Qin N, Olcese R, Zhou JM, Stefani E, and Birnbaumer L (1997) A Xenopus oocyte subunit: evidence for a role in the assembly/expression of voltage-gated calcium channels that is separate from its role as a regulatory subunit. Proc Natl Acad Sci USA 94: 1703-1708.[Abstract/Free Full Text]

Tomita S, Chen L, Kawasaki Y, Petralia RS, Wenthold RJ, Nicoll RA, and Bredt DS (2003) Functional studies and distribution define a family of transmembrane AMPA receptor regulatory proteins. J Cell Biol 161: 805-816.[Abstract/Free Full Text]

Toth PT, Bindokas VP, Bleakman D, Colmers WF, and Miller RJ (1993) Mechanism of presynaptic inhibition by neuropeptide Y at sympathetic nerve terminals. Nature (Lond) 364: 635-639.[CrossRef][Medline]

Wakamori M, Mikala G, and Mori Y (1999) Auxiliary subunits operate as a molecular switch in determining gating behaviour of the unitary N-type Ca2+ channel current in Xenopus oocytes. J Physiol (Lond) 517: 659-672.[Abstract/Free Full Text]

Walker D, Bichet D, Campbell KP, and De Waard M (1998) A ₄ isoform-specific interaction site in the carboxyl-terminal region of the voltage-dependent Ca²⁺ channel _1A subunit. J Biol Chem 273: 2361-2367.[Abstract/Free Full Text]

Walker D, Bichet D, Geib S, Mori E, Cornet V, Snutch TP, Mori Y, and De Waard M (1999) A new subtype-specific interaction in 1A subunit controls P/Q type Ca²⁺ channel activation. J Biol Chem 274: 12383-12390.[Abstract/Free Full Text]

Walker D and De Waard M (1998) Subunit interaction sites in voltage-dependent Ca2+ channels: role in channel function. Trends Neurosci 21: 148-154.[CrossRef][Medline]

Watson N, Linder ME, Druey KM, Kehrl JH, and Blumer KJ (1996) RGS family members: GTPase-activating proteins for heterotrimeric G-protein -subunits. Nature (Lond) 383: 172-175.[CrossRef][Medline]

Wickman K, Iniguez-Lluhl JA, Davenport PA, Taussig R, Krapivinsky GB, Linder ME, Gilman AG, and Clapham DE (1994) Recombinant G protein gamma subunits activate the muscarinic-gated atrial potassium channel. Nature (Lond) 368: 255-257.[CrossRef][Medline]

Williams S, Serafin M, Mühlethaler M, and Bernheim L (1997) Facilitation of N-type calcium current is dependent on the frequency of action potential-like depolarizations in dissociated cholinergic basal forebrain neurons of the guinea pig. J Neurosci 17: 1625-1632.[Abstract/Free Full Text]

Witcher DR, De Waard M, Sakamoto J, Franzini-Armstrong C, Pragnell M, Kahl SD, and Campbell KP (1993) Subunit identification and reconstitution of the N-type Ca²⁺ channel complex purified from brain. Science (Wash DC) 261: 486-489.[Abstract/Free Full Text]

Wolfe JT, Wang H, Howard J, Garrison JC, and Barrett PQ (2003) T-type calcium channel regulation by specific G-protein betagamma subunits. Nature (Lond) 424: 209-213.[CrossRef][Medline]

Wu L, Bauer CS, Zhen XG, Xie C, and Yang J (2002) Dual regulation of voltage-gated calcium channels by PtdIns(4,5)P₂. Nature (Lond) 419: 947-952.[CrossRef][Medline]

Yamada M, Ho Y-K, Lee RH, Kontani K, Takahashi T, Katada T, and Kurachi Y (1997) Muscarinic K⁺ channels are activated by gamma subunits and inhibited by the GDP bound form of the subunit of transducin. Biochem Biophys Res Commun 200: 1484-1490.

Yassin M, Zong SQ, and Tanabe T (1996) G-protein modulation of neuronal class E (_1E) calcium channel expressed in GH₃ cells. Biochem Biophys Res Commun 220: 453-458.[CrossRef][Medline]

Yatani A, Codina J, Brown AM, and Birnbaumer L (1987) Direct activation of mammalian atrial muscarinic potassium channels by GTP regulatory protein G_k. Science (Wash DC) 235: 207-211.[Abstract/Free Full Text]

Zamponi GW, Bourinet E, Nelson D, Nargeot J, and Snutch TP (1997) Crosstalk between G proteins and protein kinase C mediated by the calcium channel ₁ subunit. Nature (Lond) 385: 442-446.[CrossRef][Medline]

Zamponi GW and Snutch TP (1998) Decay of prepulse facilitation of N type calcium channels during G protein inhibition is consistent with binding of a single Ggamma subunit. Proc Natl Acad Sci USA 95: 4035-4039.[Abstract/Free Full Text]

Zhang H, He C, Yan X, Mirshahi T, and Logothetis DE (1999) Activation of inwardly rectifying K+ channels by distinct PtdIns(4,5)P2 interactions. Nat Cell Biol 1: 183-188.[CrossRef][Medline]

Zhang J-F, Ellinor PT, Aldrich RW, and Tsien RW (1994) Molecular determinants of voltage-dependent inactivation in calcium channels. Nature (Lond) 372: 97-100.[CrossRef][Medline]

Zhang JF, Ellinor PT, Aldrich RW, and Tsien RW (1996) Multiple structural elements in voltage-dependent Ca²⁺ channels support their inhibition by G proteins. Neuron 17: 991-1003.[CrossRef][Medline]

Zhao X, Varnai P, Tuymetova G, Balla A, Toth ZE, Oker-Blom C, Roder J, Jeromin A, and Balla T (2001) Interaction of neuronal calcium sensor-1 (NCS-1) with phosphatidylinositol 4-kinase beta stimulates lipid kinase activity and affects membrane trafficking in COS-7 cells. J Biol Chem 276: 40183-40189.[Abstract/Free Full Text]

Zhong H, Li B, Scheuer T, and Catterall WA (2001) Control of gating mode by a single amino acid residue in transmembrane segment IS3 of the N-type Ca2+ channel. Proc Natl Acad Sci USA 98: 4705-4709.[Abstract/Free Full Text]

Zhou J, Shapiro M, and Hille B (1997) Speed of calcium channel modulation by neurotransmitters in rat sympathetic neurons. J Neurophysiol 77: 2040-2048.[Abstract/Free Full Text]

Zhou JY, Siderovski DP, and Miller RJ (2000) Selective regulation of N-type Ca channels by different combinations of G-protein /gamma subunits and RGS proteins. J Neurosci 20: 7143-7148.[Abstract/Free Full Text]

Zhu Y and Ikeda SR (1994) VIP inhibits N-type Ca²⁺ channels of sympathetic neurons via a pertussis toxin-insensitive but cholera toxin-sensitive pathway. Neuron 13: 657-669.[CrossRef][Medline]

This article has been cited by other articles:

				S. Link, M. Meissner, B. Held, A. Beck, P. Weissgerber, M. Freichel, and V. Flockerzi Diversity and Developmental Expression of L-type Calcium Channel {beta}2 Proteins and Their Influence on Calcium Current in Murine Heart J. Biol. Chem., October 30, 2009; 284(44): 30129 - 30137. [Abstract] [Full Text] [PDF]

				J. Striessnig An oily competition: role of {beta} subunit palmitoylation for Ca2+ channel modulation by fatty acids J. Gen. Physiol., October 26, 2009; 134(5): 363 - 367. [Full Text] [PDF]

				C. P. Walsh, A. Davies, A. J. Butcher, A. C. Dolphin, and A. Kitmitto Three-dimensional Structure of CaV3.1: COMPARISON WITH THE CARDIAC L-TYPE VOLTAGE-GATED CALCIUM CHANNEL MONOMER ARCHITECTURE J. Biol. Chem., August 14, 2009; 284(33): 22310 - 22321. [Abstract] [Full Text] [PDF]

				S. Dai, D. D. Hall, and J. W. Hell Supramolecular Assemblies and Localized Regulation of Voltage-Gated Ion Channels Physiol Rev, April 1, 2009; 89(2): 411 - 452. [Abstract] [Full Text] [PDF]

				C. Hu, S. D. DePuy, J. Yao, W. E. McIntire, and P. Q. Barrett Protein Kinase A Activity Controls the Regulation of T-type CaV3.2 Channels by G{beta}{gamma} Dimers J. Biol. Chem., March 20, 2009; 284(12): 7465 - 7473. [Abstract] [Full Text] [PDF]

				Y. Zhang, Y.-h. Chen, S. D. Bangaru, L. He, K. Abele, S. Tanabe, T. Kozasa, and J. Yang Origin of the Voltage Dependence of G-Protein Regulation of P/Q-type Ca2+ Channels J. Neurosci., December 24, 2008; 28(52): 14176 - 14188. [Abstract] [Full Text] [PDF]

				L. Ferron, A. Davies, K. M. Page, D. J. Cox, J. Leroy, D. Waithe, A. J. Butcher, P. Sellaturay, S. Bolsover, W. S. Pratt, et al. The Stargazin-Related Protein {gamma}7 Interacts with the mRNA-Binding Protein Heterogeneous Nuclear Ribonucleoprotein A2 and Regulates the Stability of Specific mRNAs, Including CaV2.2 J. Neurosci., October 15, 2008; 28(42): 10604 - 10617. [Abstract] [Full Text] [PDF]

				M. Murakami, T. Ohba, F. Xu, E. Satoh, I. Miyoshi, T. Suzuki, Y. Takahashi, E. Takahashi, H. Watanabe, K. Ono, et al. Modified Sympathetic Nerve System Activity with Overexpression of the Voltage-dependent Calcium Channel {beta}3 Subunit J. Biol. Chem., September 5, 2008; 283(36): 24554 - 24560. [Abstract] [Full Text] [PDF]

				D. Heitzmann and R. Warth Physiology and Pathophysiology of Potassium Channels in Gastrointestinal Epithelia Physiol Rev, July 1, 2008; 88(3): 1119 - 1182. [Abstract] [Full Text] [PDF]

				E. M. Silinsky Selective disruption of the mammalian secretory apparatus enhances or eliminates calcium current modulation in nerve endings PNAS, April 29, 2008; 105(17): 6427 - 6432. [Abstract] [Full Text] [PDF]

				M. Zhu, A. A. Gach, G. Liu, X. Xu, C. C. Lim, J. X. Zhang, L. Mao, K. Chuprun, W. J. Koch, R. Liao, et al. Enhanced calcium cycling and contractile function in transgenic hearts expressing constitutively active G{alpha}o* protein Am J Physiol Heart Circ Physiol, March 1, 2008; 294(3): H1335 - H1347. [Abstract] [Full Text] [PDF]

				S. Ramanathan, T. Tkatch, J. F. Atherton, C. J. Wilson, and M. D. Bevan D2-Like Dopamine Receptors Modulate SKCa Channel Function in Subthalamic Nucleus Neurons Through Inhibition of Cav2.2 Channels J Neurophysiol, February 1, 2008; 99(2): 442 - 459. [Abstract] [Full Text] [PDF]

				M. R. Kasten, B. Rudy, and M. P. Anderson Differential regulation of action potential firing in adult murine thalamocortical neurons by Kv3.2, Kv1, and SK potassium and N-type calcium channels J. Physiol., October 15, 2007; 584(2): 565 - 582. [Abstract] [Full Text] [PDF]

				M. E. Hildebrand, L. S. David, J. Hamid, K. Mulatz, E. Garcia, G. W. Zamponi, and T. P. Snutch Selective Inhibition of Cav3.3 T-type Calcium Channels by G{alpha}q/11-coupled Muscarinic Acetylcholine Receptors J. Biol. Chem., July 20, 2007; 282(29): 21043 - 21055. [Abstract] [Full Text] [PDF]

				R. C. E. Wykes, C. S. Bauer, S. U. Khan, J. L. Weiss, and E. P. Seward Differential Regulation of Endogenous N- and P/Q-Type Ca2+ Channel Inactivation by Ca2+/Calmodulin Impacts on Their Ability to Support Exocytosis in Chromaffin Cells J. Neurosci., May 9, 2007; 27(19): 5236 - 5248. [Abstract] [Full Text] [PDF]

				T. Mustafa, M. Grimaldi, and L. E. Eiden The Hop Cassette of the PAC1 Receptor Confers Coupling to Ca2+ Elevation Required for Pituitary Adenylate Cyclase-activating Polypeptide-evoked Neurosecretion J. Biol. Chem., March 16, 2007; 282(11): 8079 - 8091. [Abstract] [Full Text] [PDF]

				C. S. Bauer, R. J. Woolley, A. G. Teschemacher, and E. P. Seward Potentiation of Exocytosis by Phospholipase C-Coupled G-Protein-Coupled Receptors Requires the Priming Protein Munc13-1 J. Neurosci., January 3, 2007; 27(1): 212 - 219. [Abstract] [Full Text] [PDF]

				G. E. Yevenes, G. Moraga-Cid, L. Guzman, S. Haeger, L. Oliveira, J. Olate, G. Schmalzing, and L. G. Aguayo Molecular Determinants for G Protein beta{gamma} Modulation of Ionotropic Glycine Receptors J. Biol. Chem., December 22, 2006; 281(51): 39300 - 39307. [Abstract] [Full Text] [PDF]

				S. McDavid and K. P. M. Currie G-Proteins Modulate Cumulative Inactivation of N-Type (CaV2.2) Calcium Channels J. Neurosci., December 20, 2006; 26(51): 13373 - 13383. [Abstract] [Full Text] [PDF]

				A. Puckerin, L. Liu, N. Permaul, P. Carman, J. Lee, and M. A. Diverse-Pierluissi Arrestin Is Required for Agonist-induced Trafficking of Voltage-dependent Calcium Channels J. Biol. Chem., October 13, 2006; 281(41): 31131 - 31141. [Abstract] [Full Text] [PDF]

				S.-N. Yang and P.-O. Berggren The Role of Voltage-Gated Calcium Channels in Pancreatic {beta}-Cell Physiology and Pathophysiology Endocr. Rev., October 1, 2006; 27(6): 621 - 676. [Abstract] [Full Text] [PDF]

				S. D. DePuy, J. Yao, C. Hu, W. McIntire, I. Bidaud, P. Lory, F. Rastinejad, C. Gonzalez, J. C. Garrison, and P. Q. Barrett The molecular basis for T-type Ca2+ channel inhibition by G protein beta2{gamma}2 subunits PNAS, September 26, 2006; 103(39): 14590 - 14595. [Abstract] [Full Text] [PDF]

				P. Hidalgo, G. Gonzalez-Gutierrez, J. Garcia-Olivares, and A. Neely The {alpha}1-beta-Subunit Interaction That Modulates Calcium Channel Activity Is Reversible and Requires a Competent {alpha}-Interaction Domain J. Biol. Chem., August 25, 2006; 281(34): 24104 - 24110. [Abstract] [Full Text] [PDF]

				N. Wettschureck, M. van der Stelt, H. Tsubokawa, H. Krestel, A. Moers, S. Petrosino, G. Schutz, V. Di Marzo, and S. Offermanns Forebrain-Specific Inactivation of Gq/G11 Family G Proteins Results in Age-Dependent Epilepsy and Impaired Endocannabinoid Formation Mol. Cell. Biol., August 1, 2006; 26(15): 5888 - 5894. [Abstract] [Full Text] [PDF]

				H. W. Tedford, A. E. Kisilevsky, J. B. Peloquin, and G. W. Zamponi Scanning Mutagenesis Reveals a Role for Serine 189 of the Heterotrimeric G-Protein Beta 1 Subunit in the Inhibition of N-Type Calcium Channels J Neurophysiol, July 1, 2006; 96(1): 465 - 470. [Abstract] [Full Text] [PDF]

				H. Khosravani and G. W. Zamponi Voltage-gated calcium channels and idiopathic generalized epilepsies. Physiol Rev, July 1, 2006; 86(3): 941 - 966. [Abstract] [Full Text] [PDF]

				M. Kawaguchi, K. Minami, K. Nagashima, and S. Seino Essential Role of Ubiquitin-Proteasome System in Normal Regulation of Insulin Secretion J. Biol. Chem., May 12, 2006; 281(19): 13015 - 13020. [Abstract] [Full Text] [PDF]

				E. Tombler, N. J. Cabanilla, P. Carman, N. Permaul, J. J. Hall, R. W. Richman, J. Lee, J. Rodriguez, D. P. Felsenfeld, R. F. Hennigan, et al. G Protein-induced Trafficking of Voltage-dependent Calcium Channels J. Biol. Chem., January 20, 2006; 281(3): 1827 - 1839. [Abstract] [Full Text] [PDF]

				Z. Lu, Y.-P. Jiang, L. M. Ballou, I. S. Cohen, and R. Z. Lin G{alpha}q Inhibits Cardiac L-type Ca2+ Channels through Phosphatidylinositol 3-Kinase J. Biol. Chem., December 2, 2005; 280(48): 40347 - 40354. [Abstract] [Full Text] [PDF]

				H. Photowala, R. Freed, and S. Alford Location and function of vesicle clusters, active zones and Ca2+ channels in the lamprey presynaptic terminal J. Physiol., November 15, 2005; 569(1): 119 - 135. [Abstract] [Full Text] [PDF]

				C. Acuna-Goycolea and A. N. van den Pol Peptide YY3-36 Inhibits Both Anorexigenic Proopiomelanocortin and Orexigenic Neuropeptide Y Neurons: Implications for Hypothalamic Regulation of Energy Homeostasis J. Neurosci., November 9, 2005; 25(45): 10510 - 10519. [Abstract] [Full Text] [PDF]

				N. Wettschureck and S. Offermanns Mammalian G Proteins and Their Cell Type Specific Functions Physiol Rev, October 1, 2005; 85(4): 1159 - 1204. [Abstract] [Full Text] [PDF]

				C. Acuna-Goycolea, N. Tamamaki, Y. Yanagawa, K. Obata, and A. N. van den Pol Mechanisms of Neuropeptide Y, Peptide YY, and Pancreatic Polypeptide Inhibition of Identified Green Fluorescent Protein-Expressing GABA Neurons in the Hypothalamic Neuroendocrine Arcuate Nucleus J. Neurosci., August 10, 2005; 25(32): 7406 - 7419. [Abstract] [Full Text] [PDF]

				E. M Silinsky Modulation of calcium currents is eliminated after cleavage of a strategic component of the mammalian secretory apparatus J. Physiol., August 1, 2005; 566(3): 681 - 688. [Abstract] [Full Text] [PDF]

				J. Leroy, M. S. Richards, A. J. Butcher, M. Nieto-Rostro, W. S. Pratt, A. Davies, and A. C. Dolphin Interaction via a Key Tryptophan in the I-II Linker of N-Type Calcium Channels Is Required for {beta}1 But Not for Palmitoylated {beta}2, Implicating an Additional Binding Site in the Regulation of Channel Voltage-Dependent Properties J. Neurosci., July 27, 2005; 25(30): 6984 - 6996. [Abstract] [Full Text] [PDF]

				N. Hosoi, I. Arai, and M. Tachibana Group III Metabotropic Glutamate Receptors and Exocytosed Protons Inhibit L-Type Calcium Currents in Cones But Not in Rods J. Neurosci., April 20, 2005; 25(16): 4062 - 4072. [Abstract] [Full Text] [PDF]

				S.-N. Yang and P.-O. Berggren {beta}-Cell CaV channel regulation in physiology and pathophysiology Am J Physiol Endocrinol Metab, January 1, 2005; 288(1): E16 - E28. [Abstract] [Full Text] [PDF]

				S. Luvisetto, T. Fellin, M. Spagnolo, B. Hivert, P. F. Brust, M. M. Harpold, K. A. Stauderman, M. E. Williams, and D. Pietrobon Modal Gating of Human CaV2.1 (P/Q-type) Calcium Channels: I. The Slow and the Fast Gating Modes and their Modulation by {beta} Subunits J. Gen. Physiol., October 25, 2004; 124(5): 445 - 461. [Abstract] [Full Text] [PDF]

				T. Fellin, S. Luvisetto, M. Spagnolo, and D. Pietrobon Modal Gating of Human CaV2.1 (P/Q-type) Calcium Channels: II. The b Mode and Reversible Uncoupling of Inactivation J. Gen. Physiol., October 25, 2004; 124(5): 463 - 474. [Abstract] [Full Text] [PDF]

				L.-Y. Fu, C. Acuna-Goycolea, and A. N. van den Pol Neuropeptide Y Inhibits Hypocretin/Orexin Neurons by Multiple Presynaptic and Postsynaptic Mechanisms: Tonic Depression of the Hypothalamic Arousal System J. Neurosci., October 6, 2004; 24(40): 8741 - 8751. [Abstract] [Full Text] [PDF]

				S. J. Ennion, A. D. Powell, and E. P. Seward Identification of the P2Y12 Receptor in Nucleotide Inhibition of Exocytosis from Bovine Chromaffin Cells Mol. Pharmacol., September 1, 2004; 66(3): 601 - 611. [Abstract] [Full Text] [PDF]

				H. Chen and S. R. Ikeda Modulation of Ion Channels and Synaptic Transmission by a Human Sensory Neuron-Specific G-Protein-Coupled Receptor, SNSR4/mrgX1, Heterologously Expressed in Cultured Rat Neurons J. Neurosci., May 26, 2004; 24(21): 5044 - 5053. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Dolphin, A. C. Search for Related Content PubMed PubMed Citation Articles by Dolphin, A. C.