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Hotchkiss Brain Institute, Department of Physiology and Biophysics, University of Calgary, Calgary, Canada
Abstract
Abstract I. Introduction II. Molecular Structure and Distributions of Voltage-Gated Calcium Channels A. alpha1 Subunit B. Ancillary Calcium Channel Subunits III. G Protein-Coupled Receptor Signaling—A Brief Overview A. Activation of G Proteins via G Protein-Coupled Receptors B. Subtypes of G Protein Subunits C. Regulation of G Protein Activity D. Receptor Desensitization and Internalization IV. Discovery and Characterization of Direct G Protein Inhibition of Cav2 Calcium Channels A. Electrophysiological Hallmarks of Direct G Protein Inhibition B. Does the Nature of the Galpha Subunit Affect Voltage-Dependent Modulation? C. Voltage-Independent G Protein Inhibition D. Regulator of G Protein Signaling and Activator of G Protein Signaling Proteins and Calcium Channel Inhibition V. Calcium Channel Structural Determinants of G Protein Modulation A. Calcium Channel alpha1 Subunit Structural Determinants B. Role of the Calcium Channel beta Subunit VI. Modulation of G Protein Modulation A. Cross-Talk between G Protein Inhibition and Protein Kinase C Modulation B. Synaptic Proteins VII. G Protein Structural Determinants of N-Type Channel Modulation A. Gbeta Subtype Dependence B. Gbeta Structural Determinants C. Ggamma Subtype Dependence VIII. Signaling Complexes Involving N-Type Channels and G Protein-Coupled Receptors IX. Concluding Remarks
The regulation of presynaptic, voltage-gated calcium channels by activation of heptahelical G protein-coupled receptors exerts a crucial influence on presynaptic calcium entry and hence on neurotransmitter release. Receptor activation subjects presynaptic N- and P/Q-type calcium channels to a rapid, membrane-delimited inhibition—mediated by direct, voltage-dependent interactions between G protein 
subunits and the channels—and to a slower, voltage-independent modulation involving soluble second messenger molecules. In turn, the direct inhibition of the channels is regulated as a function of many factors, including channel subtype, ancillary calcium channel subunits, and the types of G proteins and G protein regulatory factors involved. Twenty-five years after this mode of physiological regulation was first described, we review the investigations that have led to our current understanding of its molecular mechanisms.
Depolarization-mediated calcium influx via voltage-gated calcium channels elicits a range of cytoplasmic responses, including the contraction of cardiac muscle, the initiation of calcium-dependent gene transcription, cellular proliferation, the activation of calcium-dependent enzymes, and the release of hormones and neurotransmitter molecules (Tsien et al., 1988
; Wheeler et al., 1994; Dunlap et al., 1995; Martin-Moutot et al., 1996; Sutton et al., 1999; Dolmetsch et al., 2001; Reid et al., 2004). However, excessive calcium entry produces deleterious effects and may result in cell death. So it is essential for cells to carefully buffer intracellular calcium and to precisely regulate calcium entry via calcium-permeant membrane proteins such as voltage-gated calcium channels.
Multiple subtypes of voltage-gated calcium channels have been identified in mammalian tissues and classified, on the basis of their pharmacological and electrophysiological properties, into T-, L-, N-, P-, Q-, and R-types (Tsien et al., 1988, 1991; Snutch et al., 2005). Based on their thresholds of activation, these channel subtypes can be more grossly divided into low-voltageand high-voltage-activated (LVA1 and HVA, respectively) channels (Catterall et al., 2005). However, it is important to note that this criterion is not absolute, as the activation ranges of most calcium channels are modulated by alternate splicing, subunit composition, and interactions with regulatory elements. LVA channels comprise the family of T-type channels, which typically require only small membrane depolarizations to open. They activate and inactivate rapidly and are partially inactivated at normal neuronal resting potentials (Perez-Reyes, 2003). HVA calcium channels comprise all other channel subtypes named above. Relative to LVA channels they require stronger membrane depolarizations for activation and inactivation, hence showing a greater availability for opening at normal resting potentials.
Members of the HVA class are well distinguished by their pharmacological profiles. L-type channels are sensitive to dihydropyridine agonists and antagonists, although some L-type channel isoforms are less effectively inhibited by dihydropyridines than others (Fox et al., 1987; Xu and Lipscombe, 2001). N-type calcium channels are potently and selectively blocked by 
-conotoxins GVIA, MVIIA, and CVID, peptides isolated from various fish hunting cone snails (Olivera et al., 1984; Reynolds et al., 1986; Mintz et al., 1991; Feng et al., 2003). Both P- and Q-type channels are inhibited by -agatoxin IVA, a toxin isolated from the North American funnel-web spider, Agelenopsis aperta (Mintz et al., 1992; Adams et al., 1993). R-type channels were defined as such because they represent an HVA current that is resistant to the above blockers (Randall and Tsien, 1995). However, SNX-482, a peptide toxin isolated from a species of giant tarantula is now considered a potent and semiselective inhibitor of these channels (Newcomb et al., 1998; Bourinet et al., 2001).
Why are there so many subtypes of calcium channels, if they only function to allow passage of calcium ions into excitable cells? As suggested by their differences in biophysical properties, cellular expression pattern, and subcellular distribution, the channel subtypes also differ in the cellular functions they support (see below). Furthermore, different subtypes of voltage-gated calcium channels are subject to differential regulation by cytoplasmic messenger molecules, including protein kinases and G proteins.
The modulation of voltage-gated calcium channels is a vast field with many fascinating details, too extensive to review comprehensively in one article. Herein, we focus on the modulation of presynaptic calcium channels by G proteins. As notably illustrated by the action of morphine—a µ-opioid receptor agonist, which mediates potent analgesia by virtue of inhibition of N-type calcium channels and activation of potassium channels (Altier and Zamponi, 2004)—this type of regulation has important physiological implications.
II. Molecular Structure and Distributions of Voltage-Gated Calcium Channels
The core of every functional voltage-gated calcium channel and main determinant of channel subtype is the 
1 subunit (Fig. 1A). Each calcium channel contains a single 1 subunit, which in turn consists of homologous domains (I, II, III, and IV, linked by cytoplasmic loops referred to as the I-II, II-III, and III-IV loops) and cytoplasmic N- and C-terminal regions (Fig. 1A) (Catterall, 1993, 2000). As outlined below, these cytoplasmic regions are key sites for second messenger modulation and for association with regulatory and adaptor proteins. Each homologous domain contains six transmembrane spanning helices (termed S1 through S6), plus a reentrant p-loop structure between S5 and S6 that is believed to form the ion-selective pore region of the channels. The S4 helices have characteristic positively charged amino acid residues at every third sequence position, which allows the channel to sense membrane depolarization and respond with channel opening (Catterall, 2000).
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1 subunits in higher mammals have been identified and functionally characterized (Fig. 1B). These genes fall into three homologous families, termed Cav1, Cav2, and Cav3 (Snutch et al., 2005). The Cav1 family has four genes that encode L-type calcium channels with various physiological functions: Cav1.1, expressed exclusively in skeletal muscle, forms the voltage-sensor for excitation-contraction coupling (Tanabe et al., 1987); Cav1.2 and Cav1.3 are widely expressed, including in the pancreas, the heart, and the brain (Mikami et al., 1989; Williams et al., 1992b; Tomlinson et al., 1993); and Cav1.4 is expressed in retina and is functionally linked to photo-transduction (Bech-Hansen et al., 1998; Koschak et al., 2003; McRory et al., 2004). The Cav2 family is widely expressed in neurons (further discussed below) and includes Cav2.2 [encoding the N-type channel (Dubel et al., 1992; Williams et al., 1992a)], Cav2.3 [encoding the R-type channel (Soong et al., 1993; Williams et al., 1994; Randall and Tsien, 1995)], and Cav2.1, with alternate splice isoforms of the latter giving rise to P- and Q-type channels (Bourinet et al., 1999). The three members of the Cav3 family encode different isoforms of T-type channels, with Cav3.1 and Cav3.2 expressed across numerous tissues and Cav3.3 confined to neuronal tissue (Cribbs et al., 1998; Perez-Reyes et al., 1998; Lee et al., 1999; Monteil et al., 2000a,b; McRory et al., 2001; Chemin et al., 2002).
The specialized roles of individual calcium channel 1 subunits are apparent from the phenotypes of knockout mice deficient in these genes (Miller, 2001). Cav1.1-/- mice die at birth of asphyxiation caused by lack of skeletal muscle contraction and, thus, the inability to move their diaphragms (Strube et al., 1996). Cav1.2-/- mice die before birth because of an inability to contract cardiac muscle (Seisenberger et al., 2000). Cav1.3-/- and Cav1.4-/- mice are viable, but lack key aspects of sensory signal transduction, such that Cav1.3-/- mice are deaf (Platzer et al., 2000) and Cav1.4-/- mice display blindness due to compromised rod photoreceptor function (Mansergh et al., 2005). Cav1.3-/- mice also display cardiac arrhythmias. Mice lacking the Cav2.1 gene are severely ataxic and show absence seizures (Jun et al., 1999), whereas Cav2.2-/- mice are viable and show hyposensitivity to pain, as well as reduced anxiety and alcohol withdrawal symptoms (Hatakeyama et al., 2001; Kim et al., 2001; Saegusa et al., 2001; Newton et al., 2004). Cav2.3-/- mice are also viable, and show reduced response to certain pain stimuli, as well as reduced seizure activity in certain types of seizure models (Saegusa et al., 2000; Weiergräber et al., 2006). Cav3.1-/- mice are resistant to baclofen-induced seizures (Kim et al., 2001), and finally Cav3.2-/- mice show compromised vascular function (Chen et al., 2003).
The physiological consequences of gene knockout are consistent with the cellular and subcellular distributions of these channels in the central nervous system. For example, Cav3.1 channels are expressed on cell bodies and dendrites where they contribute to regulate cellular excitability (Molineux et al., 2006), which is consistent with their involvement in spike wave discharges. By contrast, Cav2.3 channels are localized to proximal dendrites and presynaptic nerve termini (Wu and Saggau, 1995; Yokoyama et al., 1995; Wu et al., 1998). Cav2.1 and Cav2.2 channels are also located at presynaptic nerve terminals, where they contribute to the release of neurotransmitters (Westenbroek et al., 1992, 1995, 1998). Yet, as noted above, individual knockouts of the Cav2.1 and Cav2.2 channels yield very different phenotypes, suggesting that the channels are not created equally in terms of coupling to the neurotransmitter release machinery. Indeed, Cav2.1 channels seem to preferentially contribute to the release of excitatory neurotransmitters, whereas Cav2.2 channels are more frequently linked to inhibitory synaptic transmission (although this linkage is by no means absolute) (Burke et al., 1993; Potier et al., 1993; Doroshenko et al., 1997; Caddick et al., 1999; Leenders et al., 2002). It should also be noted that these channels may serve functions other than simply triggering neurotransmitter release, e.g., regulation of gene transcription, as has been suggested for Cav2.1 (Sutton et al., 1999). Given the above arguments and evidence for a clear contrast in the physiological roles of Cav2.1 and Cav2.2 channels, the existence of second messenger/signaling mechanisms that would allow differential modulation of these channels seems essential.
B. Ancillary Calcium Channel Subunits
Since the first purification of the Cav1.1 calcium channel from skeletal muscle (Curtis and Catterall, 1984), it has been evident that these channels are complexes of multiple subunits (Fig. 1C). In skeletal muscle, the Cav1.1 1 subunit copurified with ancillary , 2-
, and subunits (Catterall, 2000). We now know that all subtypes of HVA calcium channels contain at least one and one 2- subunit, but it remains unclear whether subunits associate with nonskeletal muscle HVA channels (Dolphin, 2003). There is recent evidence that certain types of subunits can bind to Cav3 channels directly, but these investigations are ongoing (Best et al., 2006). In contrast, members of the LVA calcium channel family do not seem to associate physically with 2- and subunits; however, coexpression of these subunits with members of the Cav3 family does seem to regulate channel density (Lambert et al., 1997; Leuranguer et al., 1998; Dolphin et al., 1999; Hobom et al., 2000; Dubel et al., 2004). How this occurs mechanistically remains unclear.
Vertebrates express four genes that encode different types of calcium channel subunits (1, 2, 3, and 4), with further heterogeneity arising from alternate splicing (Dolphin, 2003; Richards et al., 2004). With one exception—the 2a subunit, which is palmitoylated and thereby plasma membrane-anchored (Qin et al., 1998)— these subunits are cytoplasmic proteins. Their overall architecture encompasses two highly conserved regions, flanked and separated by more variable domains (Stotz et al., 2004). Recent crystal structure data indicate that these subunits contain guanylate kinase and SH3 domains, which interact with each other to form functional subunits (Chen et al., 2004; Opatowsky et al., 2004; Van Petegem et al., 2004). Biochemical studies have revealed that the subunits bind to a highly conserved region within the I-II loop of the HVA calcium channel subunit (Pragnell et al., 1994). Termed the -interaction domain (AID) and not found in the LVA channels, this region fits into a hydrophobic groove on the surface of the subunit. There is also evidence of a second calcium channel subunit-binding domain, localized to the C-terminal region of certain subtypes of HVA calcium channels, but its functional role is unclear (Qin et al., 1997). Whereas the 1 subunit contains the minimal machinery to form a functional channel, the coexpression of a subunit modulates a number of functional properties of the 1 subunit, resulting in a massive up-regulation in current densities, changes in the midpoint of the current voltage relations and steady-state inactivation curves, and altered activation and inactivation kinetics (Pragnell et al., 1994; Chien et al., 1995; Bichet et al., 2000a,b; Yasuda et al., 2004).
Four vertebrate genes encoding 2- subunits (termed 2-1 through 2-4) have been identified and characterized (Klugbauer et al., 1999; Arikkath and Campbell, 2003). Each 2- isoform is encoded by a single gene, translated as a single peptide, and post-translationally cleaved into 2 (extracellular) and (single transmembrane helix) portions, which are then reconnected via a disulfide bond (De Jongh et al., 1990). The consequences of 2- coexpression include an enhancement of peak current amplitude, altered channel pharmacology, and slightly altered channel gating (Klugbauer et al., 2003; Yasuda et al., 2004). The physiological role of 2- subunits is exemplified by mouse models of absence epilepsy in which the 2-2 subunit is truncated; in these mice, in addition to the epileptic phenotype, cerebellar Purkinje cell morphology is altered, and P/Q-type channel activity is diminished (Barclay et al., 2001).
To date, eight different subunits (termed 1 through 8) have been isolated, all of which comprise four transmembrane helices with intracellular N and C termini (Arikkath and Campbell, 2003; Black, 2003). The 1 subunit is specific to skeletal muscle, but the other subunits can all be detected in the brain. With the exception of the 7 subunit, which drastically reduces the activity of Cav2.2 channels in heterologous expression systems (Moss et al., 2002), coexpression of the remaining subunits with any of the other neuronal voltage-gated calcium channel 1 subunits mediates only small effects on channel kinetics (Rousset et al., 2001). That said, premature truncation of the 2 subunit (also known as stargazin) results in absence seizures in mice, consistent with an important role in modulation of calcium channel function (Letts et al., 1998). However, this subunit has also been shown to associate with and regulate -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (Chen et al., 2000; Tomita et al., 2005), and, therefore, the in vivo role of the 2 subunit in the calcium channel complex, if any, remains unclear.
The multiple subtypes of calcium channel 1 subunits, the splice isoforms thereof, and the many potential combinations of ancillary subunits collectively imply a vast diversity in terms of the calcium channels that can be generated. This diversity is an important consideration for issues of channel function and for second messenger regulation of channel function, as described below.
III. G Protein-Coupled Receptor Signaling—A Brief Overview
To fully appreciate regulation of calcium channels by G proteins, we will briefly review some key aspects of G protein-coupled receptor (GPCR) signaling. GPCR signaling is a very extensive topic, too much so for comprehensive review herein. Thus, we give a brief synopsis and refer the reader elsewhere for further detail (Ferguson, 2001; McCudden et al., 2005; Perez and Karnik, 2005).
A. Activation of G Proteins via G Protein-Coupled Receptors
GPCRs are a family of seven transmembrane helix receptors that are activated by a variety of physiological stimuli, in most cases extracellular neurotransmitters and hormones. Over 350 different types of GPCRs have been identified; for many of these, cellular roles have not yet been defined (Landry et al., 2006). GPCRs have a common transmembrane topology: an extracellular N terminus, three cytoplasmic loops (connecting transmembrane helices I and II, III and IV, and V and VI), three extracellular loops (connecting helices II and III, IV and V, and VI and VII), and a cytoplasmically localized C-terminal region (Fig. 2A). GPCRs also have a common mechanism of signal transduction: when activated by agonists, GPCRs interact with heterotrimeric complexes of G protein subunits and stimulate exchange of G-bound GDP for cytoplasmic GTP. Nucleotide exchange in turn favors dissociation of the heterotrimeric complexes into G-GTP and heterodimers of G, each of the latter being active signaling entities that modulate various downstream effector systems, often with crucial consequences for cellular function (Fig. 2A). The intrinsic GTPase activity of the G subunit hydrolyzes bound GTP back into GDP + Pi, thus terminating G activity and promoting the reassembly of the inactive G-GDPG complex (Fig. 2B). In the continued presence of agonist, each receptor is able to activate multiple G subunits, which in turn may modulate multiple effector molecules, resulting in signal amplification.
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; Perez and Karnik, 2005) and will not be reviewed here. By contrast, although mutagenesis- and chimera-based studies have defined many molecular determinants of the interactions between activated receptors and inactive G protein heterotrimers, the molecular and thermodynamic details of these interactions remain largely unclear, an obvious issue to address in future investigations (Gether, 2000). B. Subtypes of G Protein Subunits
In the human genome, 16 genes encode different types of G subunits (McCudden et al., 2005); the products of these genes are classified into 5 groups based on their abilities to activate various cell signaling systems (Table 1). Gs proteins activate adenylyl cyclase; the Gi proteins, which include Go and Gz, generally inhibit adenylyl cyclase; Gt proteins (a group which includes Ggust) are found in sensory transduction pathways and activate cyclic GMP phosphodiesterase activity; Gq proteins are activators of phospholipase C; and, finally, G12 proteins are regulators of sodium-proton exchange. Most known G subunits have molecular masses of 39 to 52 kDa and are post-translationally modified with palmitoyl lipid functionalities; Gi proteins often carry myristoyl functionalities as well, and both of these lipid functionalities are thought to contribute to proper subcellular localization of G subunits (Chen and Manning, 2001).
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There are also five known subtypes of G protein subunits [plus potential alternate splice isoforms, with molecular masses of 35-39 kDa (Clapham and Neer, 1997; Fletcher et al., 1998)], and 12 different subtypes of G subunits (Ray et al., 1995; Clapham and Neer, 1997; Huang et al., 1999). G subunits contain tryptophanaspartate repeats (WD repeats) and an N-terminal amphipathic helix, which is known to interact with the G subunit (Wall et al., 1995; Lambright et al., 1996). The G subunits (G1-5,7-13) are smaller molecules (molecular mass of 
6-8 kDa) that are isoprenylated at the C terminus, which results in the plasma membrane anchoring of this subunit. Because G and G subunits typically exist as a complex, this results in membrane localization of the G subunit.
One could propose >1000 unique, potential combinations of G, G, and G subunits. However, because of specific cellular and subcellular expression patterns and unfavorable thermodynamics of binding, one may expect many of these to be rare or not to exist at all as functional, heterotromeric complexes (further discussed below in section VII.). Furthermore, GPCR-G protein coupling is preferential according to G subunit type (Schoneberg et al., 1999; Cabrera-Vera et al., 2003) and also according to G subtypes (Taylor et al., 1994; Kisselev et al., 1995a,b; Yasuda et al., 1996; McIntire et al., 2001; Cabrera-Vera et al., 2003; Jones et al., 2004). And, finally, many types of GPCRs are known to homo- and heterodimerize, which can alter the specificity of GPCR-G coupling (George et al., 2000) or affect receptor internalization (AbdAlla et al., 2000). Collectively, this process provides a tremendous potential for linking GPCR activity to specific signaling pathways, as illustrated by the requirement for specific G subunit compositions for direct regulation of N- and P/Q-type calcium channels (described below).
C. Regulation of G Protein Activity
Various endogenous modulators can regulate the activities of G subunits independently of GPCR activation. Activator of G protein signaling (AGS) proteins can directly stimulate guanine nucleotide exchange of the G subunit (Fig. 2A), resulting in receptor-independent activation of G (Blumer et al., 2005). Regulator of G protein signaling (RGS) proteins comprise a large family of proteins with >20 different members. A primary function of RGS proteins is to stimulate the intrinsic GTPase activity of G, thus accelerating inactivation of these subunits (Berman et al., 1996) (Fig. 2B). In turn, this promotes the reformation of the inactive G protein heterotrimer and the consequent termination of G action on effector molecules (Doupnik et al., 1997). There is also evidence that some types of RGS proteins can directly interfere with the interactions between activated G subunits and their effectors (Berman and Gilman, 1998). We also note that certain RGS proteins contain a G protein -like domain and associate with G subunits by replacing the G subunit (Snow et al., 1998).
G protein activity can also be regulated by pharmacological means: intracellular application of the nonhydrolyzable GTP analog GTPS results in permanent activation of all known types of G subunits and, at the same time, a massive increase in free G subunits. In contrast, guanosine 5'-O-(2-thio)diphosphate, once bound to G subunits, renders the subunits permanently inactive: they cannot exchange this GDP analog for GTP and thus remain permanently associated with G. Aluminum fluoride produces a permanently active G-GDP subunit by mimicking the effects of the phosphate group of GTP. Cholera toxin permanently activates Gs subunits via ADP ribosylation, which drastically reduces their ability to hydrolyze GTP. In contrast, pertussis toxin (PTX) permanently inhibits Gi subunits (with the exception of Gz) by blocking their abilities to interact with GPCRs, again via ADP ribosylation (Fields and Casey, 1997). Gt is sensitive to both toxins; Ggust is presumed to be sensitive to both on the basis of amino acid sequence, but has only been experimentally demonstrated to be sensitive to PTX (Spielman et al., 1994; Fields and Casey, 1997; Ming et al., 1998).
D. Receptor Desensitization and Internalization
Many GPCRs display intrinsic desensitization mechanisms that allow them to terminate their activities during the sustained presence of receptor agonist. There are multiple mechanisms by which GPCRs desensitize. Heterologous desensitization involves protein kinase A- and/or protein kinase C-dependent phosphorylation of the third intracellular loop of the receptors, irrespective of whether agonist is bound. These phosphorylation events block the interaction of the receptor with the G subunit, effectively terminating GPCR-mediated signaling, and serving as a general mechanism by which receptors are desensitized. Homologous desensitization involves the phosphorylation of specific residues in the C terminus of the receptor (in its agonist bound state) by specific G protein-coupled receptor kinases (GRK) (Diverse-Pierluissi et al., 1996). These GRKs are activated by G subunits and translocate from the cytoplasm to the plasma membrane. Once phosphorylated, the C terminus becomes available for binding to arrestins, which then block receptor-G protein coupling. Another means of terminating GPCR activity in the presence of agonist is internalization of GPCRs into cytoplasmic vesicular compartments (Fig. 2C). This process is enhanced by GRK-dependent phosphorylation and the binding of arrestins (Ferguson, 2001); internalization can either be reversible, allowing the reinsertion of the receptors into the plasma membrane, or irreversible, leading to receptor degradation in lysosomal compartments. The net result in the above cases is termination of GPCR-mediated signaling and, hence, the return of effectors such as voltage-gated calcium channels to their basal, nonmodulated activity. In the context of voltage-gated calcium channels, these desensitization and internalization mechanisms are important means for restoring normal calcium channel function in the continued presence of receptor agonists.
IV. Discovery and Characterization of Direct G Protein Inhibition of Cav2 Calcium Channels
G protein inhibition of voltage-gated calcium currents was first described 25 years ago in two seminal articles by Dunlap and Fischbach (1978, 1981). These authors showed that the contribution of calcium channels to somatic action potentials in chick dorsal root ganglion (DRG) neurons was reduced in response to activation of GABAB, serotonin, or adrenergic receptors (see Fig. 3A, top), thus shortening action potential duration (Dunlap and Fischbach, 1978). These authors subsequently showed that this effect was due to a robust, receptor-mediated inhibition of HVA calcium currents in chick DRG neurons, currents now known to be carried almost entirely by N-type channels (Dunlap and Fischbach, 1981) (Fig. 3A, bottom). Numerous studies followed, revealing that many types of GPCRs—muscarinic, opioid, somatostatin, and dopamine receptors among them— have the propensity to inhibit native calcium currents (Forscher et al., 1986; Holz et al., 1986b; Bean, 1989; Ikeda and Schofield, 1989; Kasai and Aosaki, 1989; Lipscombe et al., 1989; Beech et al., 1992; Bernheim et al., 1992; Ikeda, 1992; Golard and Siegelbaum, 1993; Mintz and Bean, 1993; Shapiro and Hille, 1993; Zhu and Ikeda, 1993; Caulfield et al., 1994). A unifying property of this GPCR-mediated inhibition of calcium currents was its sensitivity to pertussis toxin, thus implicating Gi and/or Go proteins (Holz et al., 1986a). The receptor-mediated inhibition was found to be blocked by the application of guanosine 5'-O-(2-thio)diphosphate (Holz et al., 1986a), hence further implicating G subunits. A role for G subunits in this mode of inhibition was thus established, yet not well understood.
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The inhibition was then found to be membrane-delimited, i.e., to involve a second messenger molecule that remained associated with the plasma membrane, rather than diffusing to the channel via a cytoplasmic pathway (Forscher et al., 1986; Hille, 1994). Because the G protein subunit is anchored to the plasma membrane (described above), these findings were consistent with direct, inhibitory physical interaction between calcium channels and membrane-tethered G subunits. This paradigm was left untested at first, because the dominant view of G subunit function was that it served only to capture GDP-bound G subunits. But it has since become clear that the physiological role of G subunits is much more complex and includes direct modulatory interaction with many target effectors, e.g., the G protein-coupled inwardly rectifying potassium channel, the first such effector identified (Logothetis et al., 1987; Ford et al., 1998; Albsoul-Younes et al., 2001; Mirshahi et al., 2002b). A direct role for G in the inhibition of voltage-gated calcium channels was first proposed by Bourinet et al. (1996) and later demonstrated in experiments testing the effects of overexpressed G subunits (Herlitze et al., 1996; Ikeda, 1996). The results of these experiments were fully consistent with the notion of a "membrane-delimited" pathway and were reported in back-to-back publications by the Ikeda and Hille groups (Herlitze et al., 1996; Ikeda, 1996).
The direct G protein inhibition of voltage-gated calcium channels occurs in a calcium channel subtype-dependent manner. N-type channels have long been considered a prime target for direct G protein inhibition (see above), and it is now established that P/Q-type calcium channels are also inhibited upon activation of Gi- or Go-coupled receptors. However, P/Q-type channels typically undergo a smaller degree of inhibition relative to N-type channels (Currie and Fox, 1997). In contrast, other calcium channel subtypes expressed in native cells do not seem to be subject to direct G protein inhibition, suggesting that this type of modulation is confined to the two main presynaptic calcium channel isoforms. Similar findings have been obtained in expression systems (Bourinet et al., 1996; Toth et al., 1996; Page et al., 1997; Stephens et al., 1998; Beedle et al., 2004), although there have been reports of G-mediated inhibition of Cav2.3 (i.e., R-type) channels, particularly in the absence of the calcium channel subunit (Mehrke et al., 1997; Qin et al., 1997; Shekter et al., 1997). More recently, a putative direct modulation of Cav3.2 calcium channels by G22 has been reported (Wolfe et al., 2003). However, the hallmarks of this modulation differ from those of the classic G protein inhibition described for HVA calcium channels (see below). Taken together, these considerations indicate that all members of the Cav2 channel family have at least some ability to undergo direct G protein inhibition, whereas other calcium channel subtypes typically do not.
A. Electrophysiological Hallmarks of Direct G Protein Inhibition
The membrane-delimited inhibition of voltage-gated calcium channels bears a number of distinct hallmarks. At the whole-cell level, peak current amplitudes are reduced in a voltage-dependent manner, with inhibition being stronger at more hyperpolarized potentials (Bean, 1989; Kasai and Aosaki, 1989; Lipscombe et al., 1989). This finding is reflected in a depolarizing shift in the midpoint of the activation curve of the channel. In addition, the time courses of current activation and inactivation can be slowed after receptor activation. All of the above effects are reversed after strong membrane depolarization (+100 mV) (Fig. 3B), hence the term "prepulse facilitation" or "prepulse relief" to describe the current increase that occurs when a depolarizing voltage pulse is applied before a test depolarization (Bean, 1989; Hille, 1994; Zamponi and Snutch, 1998a,b; Arnot et al., 2000). Such strong membrane depolarizations do not occur in normal mammalian physiology. However, rapid trains of action potentials (as well as increases in action potential duration) can lead to a similar recovery from G protein inhibition (Fig. 3C), leading to the suggestion that voltage-dependent G protein disinhibition may be important for synaptic function (Brody et al., 1997; Williams et al., 1997; Park and Dunlap, 1998). It has even been suggested that this phenomenon contributes to a novel form of paired-pulse facilitation observed in autaptic hippocampal cultures in the presence of GABAB or adenosine A1 receptor agonists (Brody and Yue, 2000). Moreover, in hippocampal slices, carbachol-induced inhibition of postsynaptic responses is relieved by application of paired presynaptic depolarizations (de Sevilla et al., 2002). A potential role of voltage-dependent disinhibition of G protein regulation in synaptic function is also supported by modeling studies (Bertram and Behan, 1999; Bertram et al., 2002, 2003).
Voltage-dependent G protein inhibition of N-type calcium channels has also been examined at the single channel level (Carabelli et al., 1996; Patil et al., 1996). In these studies, inhibited N-type channels show an increased first latency to opening, giving rise to the slowed activation kinetics observed in whole-cell N-type currents. Inactivation kinetics of the whole-cell currents also seem to be slowed, again due to the occurrence of delayed channel openings. Hence, the altered first latency to opening can account for the hallmark features of voltage-dependent G protein inhibition observed in whole-cell recordings. Mechanistically, the delay in opening can be explained by a stabilization of the closed state of G-bound channels, and this G-induced stabilization of a closed channel conformation is consistent with previous suggestions of "willing" (i.e., G protein free) and "reluctant" (i.e., G protein-bound) gating modes of the channel. (Bean, 1989; Kasai and Aosaki, 1989; Elmslie, 1992; Boland and Bean, 1993; Golard and Siegelbaum, 1993). Kinetic modeling (Patil et al., 1996) suggests that the transition from the reluctant to the willing gating mode involves the dissociation of the G complex from the channel (Fig. 3D), although reluctant N-type channel openings can also occur, albeit with very low probability (Colecraft et al., 2000; Lee and Elmslie, 2000). The significance of dissociation of G subunits from the N-type calcium channel for transition to a willing gating mode is also supported by experiments in which the free G concentration was varied (Zamponi and Snutch, 1998a). The kinetics of G protein reinhibition that follow a strong depolarizing prepulse become faster at increasing concentrations of free G, implying that G subunits must physically dissociate from the G protein complex during the prepulse and consistent with modeling work of Bertram and Behan (1999). These kinetic data are also consistent with a bimolecular interaction between G and the channel, thus resolving the extensively discussed issue of G protein-calcium channel stoichiometry (Kasai and Aosaki, 1989; Boland and Bean, 1993; Golard and Siegelbaum, 1993).
B. Does the Nature of the G Subunit Affect Voltage-Dependent Modulation?
As mentioned above, in the majority of early studies, the voltage-dependent modulation of N-type calcium channels seemed to be sensitive to PTX, thus implicating Gi and/or Go subunits. However, there is evidence that receptors coupling to other types of G subunits can also mediate voltage-dependent modulation. For example, vasoactive intestinal peptide (VIP) mediates voltage-dependent inhibition of N-type calcium channels in sympathetic neurons via activation of Gs and independently of protein kinase (PK) A (Zhu and Ikeda, 1994). Similarly, work from our own laboratory indicates that dopamine D1 receptors, despite coupling to Gs, can mediate voltage-dependent modulation of heterologously expressed Cav2.2 calcium channels (Kisilevsky et al., 2006). Likewise, overexpression of Gz in rat sympathetic neurons effectively rescues the loss of voltage-dependent modulation of N-type channels by adrenergic, adenosine, prostaglandin E2, and somatostatin receptors that occurs after incubation with PTX (Jeong and Ikeda, 1998). The ability of many types of G protein subunits to couple N-type channels to G modulation is also supported by overexpression studies in which a wide range of G subunit subtypes (including G11 and Gt, but interestingly not Gz) were found to interfere with norepinephrine- or VIP-mediated inhibition of N-type channels in rat sympathetic neurons (Jeong and Ikeda, 1999). Together, the results described above show that many subtypes of G subunits can form heterotrimers with the same types of G subunits that are involved N-type channel modulation.
Because all GPCR types activate G irrespective of the types of G subunits to which they (the GPCRs) couple, then in principle any type of GPCR should be able to promote G-mediated inhibition of N-type channels. However, there are several additional considerations: First, the receptors and channels need to be localized in close proximity to allow for effective diffusion and binding of G subunits to the channel. Second, although G subunits may be able to biochemically interact with a wide range of G dimers, structural features of the GPCR itself can prevent coupling to certain combinations of G subunits—a crucial point, since the isoform of the G protein subunit is a key determinant of voltage-dependent calcium channel modulation (as we discuss below). Finally, the activation of other intracellular signaling pathways by particular types of G subunits may interfere with the ability of G to inhibit N-type channel activity (also described below). Hence, control of the specificity of GPCR signaling to N-type calcium channels goes beyond the coupling of the receptor to a particular G subunit.
C. Voltage-Independent G Protein Inhibition
The free G heterodimers resulting from GPCR activation do not signal exclusively to voltage-gated calcium channels: they also modulate components of other cytoplasmic messenger systems such as phospholipase C and adenylyl cyclase (Gao and Gilman, 1991; Tang and Gilman, 1991; Camps et al., 1992). Moreover, activated G subunits trigger various intracellular responses, which may converge on voltage-gated calcium channels to either up-regulate or inhibit their activities. Thus, in addition to mediating voltage-dependent inhibition of Cav2 calcium channels via G, a number of GPCRs trigger a concomitant inhibition of the channels via soluble second messenger pathways (Beech et al., 1991, 1992; Bernheim et al., 1991, 1992; Luebke and Dunlap, 1994; Surmeier et al., 1995; Delmas et al., 1998a,b; Shapiro et al., 1999; Kammermeier et al., 2000; Schiff et al., 2000; Beedle et al., 2004). Because this type of inhibition cannot be reversed by strong membrane depolarizations, it is referred to as voltage-independent.
The precise molecular mechanisms by which voltage-independent modulation occurs are incompletely understood, but evidence suggests it can be elicited by a number of distinct signaling pathways that may be tailored to particular types of GPCRs. For example, dopamine D1 receptors couple to Gs and thereby activate PKA, which in turn phosphorylates protein phosphatase 1. This phosphatase has been shown to dephosphorylate residues on N- and P/Q-type calcium channels, resulting in voltage-independent current inhibition (Surmeier et al., 1995). In contrast, robust voltage-independent modulation of N-type channels in chick DRG neurons has been attributed to tyrosine kinase-dependent phosphorylation of the N-type calcium channel 1 subunit (Schiff et al., 2000) and involves classes of G proteins different from those involved in voltage-dependent modulation (Diverse-Pierluissi et al., 1995). Finally, muscarinic M1 and neurokinin 1 receptors both trigger voltage-independent inhibition of N-type channels via a Gq, but also require the action of G subunits as part of the signaling cascade (Kammermeier et al., 2000). The regulation of voltage-gated calcium channels by kinases and phosphatases is a vast area of research that has been reviewed recently (Bannister et al., 2005) and will not be further described herein. However, the above examples serve to illustrate the fact that G protein-coupled receptors mediate more than just voltage-dependent, membrane-delimited inhibition of Cav2 calcium channels. And, as described below, there may be cross-talk between voltage-dependent and voltage-independent pathways that contributes to the overall complexity of GPCR signaling to voltage-gated calcium channels.
D. Regulator of G Protein Signaling and Activator of G Protein Signaling Proteins and Calcium Channel Inhibition
RGS proteins have recently emerged as important factors in the voltage-dependent modulation of N- and P/Q-type calcium channels (Jeong and Ikeda, 1998, 2000; Diverse-Pierluissi et al., 1999; Melliti et al., 1999, 2001; Mark et al., 2000). RGS2 proteins accelerate the recovery from inhibition of heterologously expressed P/Q-type calcium channels by M2 muscarinic receptors and alter the availability of G subunits for producing voltage-dependent inhibition of these channels (Mark et al., 2000). Likewise, by stimulating the GTPase activity of G (and thus reducing the amount of free G that is available to modulate the channels), overexpression of RGS3 and RGS8 proteins attenuates the muscarinic inhibition of heterologously expressed N-type calcium channels (Melliti et al., 1999). Interestingly, RGS2 and RGS12 also alter voltage-independent inhibition of N-type channels in a manner that seems to be independent of altered GTPase activity (Schiff et al., 2000; Melliti et al., 2001), suggesting that RGS proteins can act directly on effector molecules such as N-type channels (Richman et al., 2005). RGS proteins have also been shown to reduce adrenergic inhibition of native N-type currents (Jeong and Ikeda, 2000). When RGS4 proteins are depleted by intracellular dialysis of an RGS4 specific antibody, adrenergic modulation of these channels becomes enhanced (Diverse-Pierluissi et al., 1999). Both RGS4 and RGS10 accelerate the deactivation of Gz-mediated noradrenergic modulation of N-type channels in rat sympathetic neurons (Jeong and Ikeda, 1998). Finally, RGS9 antagonizes dopamine D2 receptor-mediated modulation of N-type currents in rat striatal cholinergic interneurons (Cabrera-Vera et al., 2004). Hence, a host of different RGS proteins have been linked to regulation of voltage-dependent modulation of N- and P/Q-type calcium channels, presumably via their stimulation of GTPase activity. The fact that many endogenous types of RGS proteins can affect receptor signaling to voltage-gated calcium channels indicates that caution should be exercised when attempting to reconstitute receptor-channel signaling in transient expression systems.
A number of RGS proteins have been shown to regulate calcium channel activity independently of an action on the G subunit. For example, both RGS6 and RGS11 antagonize the modulation of human Cav2.2 calcium channels by coexpressed G5 subunits (Zhou et al., 2000). Both of these RGS protein types have been shown to interact directly with G5 by competing with the G protein subunit (Snow et al., 1999), presumably resulting in an RGS-G5 complex that is incapable of inhibiting the channel. RGS6 does not seem to interact with any other G protein subunit subtype, but both RGS7 and RGS9 also have G protein -like domains (Snow et al., 1998), raising the possibility that some of the RGS proteins could directly modulate the actions of other subtypes of G subunits on voltage-gated calcium channels. Such a scenario needs to be investigated experimentally in expression systems.
AGS proteins serve as receptor-independent means of activating G protein signaling (Blumer et al., 2005). Hence, up-regulation of AGS protein expression may trigger receptor-independent inhibition of voltage-gated calcium channels, although such an action has yet to be demonstrated. The synaptic vesicle release protein cysteine string protein (CSP), although not an AGS protein, does contain a region capable of stimulating exchange of G-bound GDP for GTP in purified G subunits (Natochin et al., 2005), which may account for voltage-dependent modulation of N-type channels that results from overexpression of this region of CSP (Miller et al., 2003). Collectively, these findings indicate that G protein inhibition of Cav2 calcium channels, be it indirect or mediated by direct interaction between the channel and G, is subject to a number of regulatory mechanisms that occur upstream of the channel itself.
V. Calcium Channel Structural Determinants of G Protein Modulation
A. Calcium Channel 1 Subunit Structural Determinants
The calcium channel structural determinants that underlie G
