|
|
||||||||
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 |
|---|
|
|
|---|

dimers, and the cytoplasmic linker between domains I and II of the CaV2
1 subunits binds G
dimers, whereas the intracellular N terminus of CaV2
1 subunits provides essential determinants for G protein modulation. Evidence suggests a key role for the
subunits of calcium channels in the process of G protein modulation, and the role of a class of proteins termed "regulators of G protein signaling" will also be described. | I. Introduction |
|---|
|
|
|---|
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 CaV2 subfamily of HVA calcium channels that shows classical modulation by G proteins, comprising CaV2.1 or
1A, the molecular counterpart of P/Q-type calcium channels (Mori et al., 1991
), CaV2.2 or
1B (Dubel et al., 1992
), the molecular counterpart of N-type calcium channels, and CaV2.3 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).
|
In the case of the N- and P/Q-type as well as the L-type HVA calcium channels, the CaV
1 subunit has been shown to copurify with an intracellular
subunit (CaV
) (Liu et al., 1996
; Scott et al., 1996
). Four
subunits have been cloned (
14), 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 CaV
2 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 (
28) (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
|
|---|
|
|
|---|
subunits have marked effects on the properties of HVA
1 subunits (CaV1 and CaV2 families), including trafficking of calcium channel complexes to the plasma membrane and modification of kinetic and voltage-dependent properties (Singer et al., 1991
subunits from native neurons results in a reduction of the amplitude of endogenous calcium currents and slowed kinetics of activation (Berrow et al., 1995
Most research indicates that all CaV
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 CaV
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 CaV
subunit was found to be both necessary and able to traffic at least some heterologously expressed CaV channels to the plasma membrane, since if endogenous
subunit expression was reduced or eliminated by injection of
3 antisense oligonucleotides, CaV expression was largely lost (Tareilus et al., 1997
; Canti et al., 2001
).
In COS-7 cells, small currents were observed when CaV2.1, CaV2.2, and CaV2.3 were expressed alone, but exogenous
subunits all increased CaV2.1, CaV2.2, and CaV2.3 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 CaV
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 CaV
subunits can be attributed to a number of effects on biophysical properties as well as the important influence on trafficking. All CaV
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 CaV
1 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 CaV
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 CaV
2a subunit expressed with CaV1.2 (Chien and Hosey, 1998
), CaV2.2 (Bogdanov et al., 2000
; Stephens et al., 2000
), or CaV2.3 (Qin et al., 1998
).
|
A. Binding of CaV
to the
1 I-II Linker
CaV
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 CaV2.2 is QQIERELNGYLEWIFKAE, and the consensus sequence present in both CaV1.x and CaV2.x subfamilies is QQxExxLxGYxxWIxxxE.
A 41-amino acid sequence (BID) on the CaV
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 KEPYDVVPSMRPLVGPSLKGYEVTDMMKQALFDF; the underlined serines are consensus protein kinase C (PKC) phosphorylation sites. The residues in bold have been identified as particularly important for binding to CaV
1 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 CaV
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 CaV2.1 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 CaV2.2 was about 20 nM, and the koff 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 CaV2.2 and CaV1.3 (Bell et al., 2001
).
|
We have studied the in vivo concentration dependence of the effects of CaV
subunits. Our evidence supports the hypothesis that there are two distinct binding processes for
subunits on CaV2.2 (Canti et al., 2001
). One is a high-affinity process related to the effect of CaV
on the maximum conductance of CaV2.2, 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 (KD
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 CaV
subunits once the CaV
1 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 CaV
subunit binding sites on each CaV2.2 molecule, but the binding of two CaV
subunits has not been demonstrated directly (Canti et al., 2001
). Whichever hypothesis is correct, it is highly likely that the CaV
subunit interacts with other domains on the CaV
1 subunit as well as the I-II linker.
B. Binding of CaV
Subunits to the N and C Termini of CaV
1 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 CaV
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 CaV2.3 appeared to involve binding to the BID domain of
2a, the same as that which binds to the CaV
1 I-II linker, making it an alternative, rather than an additional site, for an individual CaV
subunit (Qin et al., 1997
). Walker et al. (1999
) showed that the N terminus of CaV2.1 interacted with CaV
4 and
2a but not
1b or
3. The region of
4 involved was within its C terminus (amino acids 446482). The C terminus of CaV
4 also bound to the C terminus of CaV2.1. The N and C termini of CaV2.1 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 CaV
4 produced a smaller hyperpolarizing shift of CaV2.1 currents than did CaV
3, and that this differential was due to the CaV2.1 N terminus. Stephens et al. (2000
) showed that CaV2.2 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 CaV2.2.
| III. Modulation of Calcium Channels |
|---|
|
|
|---|
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
).
|
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 |
|---|
|
|
|---|
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 (Gi and Go 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) (GTP
S) (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 GTP
S (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, Gs- or Gq-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 G
s (Logothetis et al., 1987
; Kurachi et al., 1989
; Clapham and Neer, 1993
). Furthermore, most G
combinations tested except transducin (G
1
1) 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 CaV2.2 was coexpressed in COS-7 cells with
2a and G
1
2, 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 CaV2.2/
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
).
|
|
|
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 Go 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 CaV2.2 or CaV2.1 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 Go rather than Gi 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.
|
One G
subunit, G
5, 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 G
5 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 Gq 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 CaV
1 I-II Linker in G Protein Modulation of CaV2 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 CaV
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 CaV2.1, CaV2.2, and CaV2.3, intriguingly within the binding site described for the VGCC
subunit (QQIERELNGYWI-KAE) (Pragnell et al., 1994
). Furthermore, it is modified in the cognate region in L-type channels (QQLEEDL-GYWITQ-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 CaV2.2 of 62 nM (Fig. 3B) (Bell et al., 2001
). Furthermore, the I-II linker of the L-type channel CaV1.3 does not bind G
, although it will bind CaV
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 CaV
1 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 CaV2.2, 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 CaV2.1 and CaV2.2 by putting the I-II linker of CaV2.2 into CaV2.1, which resulted in an increased modulation, with CaV
1b 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 CaV2.1/
4 by GTP
S injection into oocytes, although in this study only a small amount of inhibition was observed with GTP
S even in the wild-type CaV2.1. However, Herlitze et al. (1997
) mutated the QQIER sequence in CaV2.1 to that in CaV1.2, which is QQLEE, and found a reduction of modulation of the channel coexpressed with
1b by GTP
S, but not an abolition. Interestingly, in this study, they observed that CaV2.1 with the sequence QQIEE showed increased, rather than decreased, modulation by GTP
S, 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 CaV2.1 nor that from CaV1.2 reduced modulation in a CaV2.2 backbone when coexpressed with
1. In agreement, the results of Page et al. (1997
) showed that insertion of the I-II linker of CaV2.2 into a CaV2.3 construct (RbEII) (Soong et al., 1993
), which had a truncated N terminus, did not restore the G protein modulation shown by CaV2.2/
1b. Furthermore, Canti et al. (1999
) showed that the I-II linker of CaV2.2 inserted into a CaV1.2 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 CaV
subunits used, because the different groups have coexpressed channels with a variety of different CaV
subunits, and the G
combination, where used, was G
1
2 or G
2
3.
|
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 CaV2.x channels by these G
dimers.
D. The Essential Role of the CaV
1 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 CaV2.2 and, to a lesser extent, for CaV2.1, but modulation of CaV2.3 was only observed by some groups (Yassin et al., 1996
; Meza and Adams, 1998
) but not by my group using a particular rat CaV2.3 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 CaV2.3 clone homologous to the rabbit and human clones, and this full-length CaV2.3 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 CaV2.3, my group further showed that partial truncation of the highly homologous N terminus of CaV2.2 abolished its ability to be G protein modulated (Page et al., 1998
). Furthermore, a chimeric channel consisting of only the cytoplasmic N terminus of CaV2.2 in a rat CaV1.2 backbone showed all the elements of classical G protein modulation, whereas CaV1.2 did not (Canti et al., 1999
) (Fig. 9). We then extended this study to show that an 11-amino acid motif YKQSIAQRART in CaV2.2 N terminus that is also highly conserved in CaV2.1 and CaV2.3 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 CaV
subunits, because deletion of this motif in CaV2.2 or mutation of certain residues countered the CaV
2a-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 Gq 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 G
5 in native tissues, and G
5 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 CaV2.2. 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 G
1 and not other G
subunits (Cooper et al., 2000
). Furthermore, the sequence is not completely conserved in rabbit CaV2.2, which has alanine in place of threonine at the equivalent residue 422 (KRAAAKKSR in rabbit CaV2.2), 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 CaV2 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 CaV2.2 and the entire final three domains and C terminus of CaV1.2 is strongly G protein-modulated (Stephens et al., 1998b
). Furthermore, truncation of the C terminus of CaV2.2 including the region homologous with that found to bind G
in CaV2.3 (Qin et al., 1997
) did not affect G protein modulation by GTP
S (Meza and Adams, 1998
), and a similar truncation of CaV2.2 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
|
|---|
|
|
|---|

dimers in other proteins, in the region of the
1 subunit I-II linker where the CaV
subunit binds, suggested that the CaV
subunit might be involved in G protein modulation.
A. Initial Evidence for the Role of CaV
Subunits in G Protein Modulation in Native Neurons
To investigate the involvement of CaV
subunits in G protein modulation, I first developed an antisense strategy to deplete dorsal root ganglion neurons of their CaV
subunits by microinjection of an antisense oligonucleotide complementary to a region common to all
subunits (Berrow et al., 1995
). Antisense knockdown of the CaV
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 CaV
Subunits in G Protein Inhibition of Heterologously Expressed Calcium Channels
The role of CaV
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 CaV
subunit. The result was interpreted in terms of a competition or displacement of CaV
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 CaV
subunits and G
dimers, but the interaction is dynamic and depends on the membrane voltage.
C. Does G
Displace CaV
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 CaV
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
:CaV
1-CaV
+ G
CaV
1-
-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 CaV
and G
compete for a single binding site on the
1 subunit, as in the reaction: CaV
1-CaV
+ G
CaV
1-G
+ CaV
as proposed by Bourinet et al. (1996
) and Qin et al. (1997
). Such a reaction would either require transient formation of an intermediate ternary CaV
1-
-G
complex or require that CaV
dissociates before G
binds, if they bind to the same site.
If G
binding either displaced CaV
or allosterically resulted in the physical dissociation of CaV
, 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 CaV
subunits on calcium channel properties. All CaV
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 CaV
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 CaV
subunits and prevent their interaction with the channel.
For the CaV2 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 CaV
subunits, since if G
unbinds during a prepulse, then CaV
might be expected to bind in its place. If so, the rate of facilitation would be directly dependent on CaV
concentration in the cytosol. Indeed, the rate of facilitation during a prepulse was markedly increased by the heterologous expression of all CaV
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 CaV
3 cDNA with a constant amount of CaV2.2 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 CaV
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 CaV
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 CaV
bound. The reason for this interpretation is that with these concentrations of
3 coexpressed, the steady-state inactivation of the CaV2.2 channels is fit by a single Boltzmann function, which is hyperpolarized compared with that for CaV2.2 expressed without a
subunit (Fig. 10C).
|
At intermediate CaV
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 CaV
concentration. The proportion of current showing the fast facilitation rate (Fig. 10D) shows exactly the same dependence on CaV
protein concentration as the proportion of current with a hyperpolarized steady-state inactivation (see the biphasic steady-state inactivation curves at intermediate CaV
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 CaV
bound. Reciprocally, the proportion of current showing a slow time constant of facilitation decreases as CaV
concentration is increased, as does the proportion of current with a depolarized steady-state inactivation (Fig. 10C). We interpret this component as representing CaV channels without a bound CaV
. Unlike the component with the fast time constant of facilitation, this slow component of facilitation has a time constant that does vary with CaV
concentration (Fig. 10D). We have interpreted this finding as representing CaV
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 CaV
subunits, but rather that under normal circumstances, G
dissociates at depolarized potentials from and rebinds at hyperpolarized potentials to channels that have CaV
bound. Only under circumstances when CaV
is limiting, which might rarely occur in native tissues, does CaV
bind with higher affinity during depolarization and thence induces G
unbinding.
Thus, CaV
must have a higher affinity for the channels in their depolarized state. This phenomenon of depolarization-dependent displacement of the
1-
equilibrium toward increased CaV
binding would only be observed under conditions where CaV
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 CaV2.2 channels expressed in oocytes without a CaV
subunit, in sharp contrast to the lack of tonic facilitation in the additional presence of exogenous
1b (Fig. 11) (Canti et al., 2000
).
|
It was found that the reinhibition rate following a prepulse (for the CaV2.2/
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 CaV
subunit together with CaV2.2, compared with the rate in the presence only of endogenous oocyte CaV
(Canti et al., 2000
). If there were direct competition between G
and CaV
for an overlapping binding site, then CaV
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 CaV
unbinding, rather than G
binding, and therefore should not be dependent on G
concentration. Elevation of CaV
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 CaV
1-
combination, and this species predominates when CaV
is overexpressed.
Our model for the functional interplay between G
dimers and CaV
subunits does not support the idea that there is a simple competition between CaV
and G
for binding to the channel, or that CaV
dissociates from the channel during G protein modulation, but rather that under normal conditions where the channels all have a CaV
bound, G
allosterically disrupts the effect of CaV
on CaV
1 channels. Conversely, depolarization, such as occurs during a prepulse, results in a state-dependent conformational change between CaV
1 and CaV
, 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 CaV
in Xenopus oocytes (Bourinet et al., 1996
), or following antisense depletion of CaV
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 CaV
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 CaV
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 CaV
reduces the inhibition observed. It should be noted that the direct effects of CaV
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 CaV
subunit protein was detected by immunocytochemistry (Meir et al., 2000
), coexpression of G
with CaV2.2 in the absence of CaV
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 CaV
was associated. The additional presence of heterologously expressed CaV
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 CaV
can be compared in the absence of the confounding effect of CaV
on the number of channels expressed. The main effect of coexpression CaV
2a on CaV2.2 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 CaV
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 CaV
, that is, the combination CaV2.2
1/G
. This is not the case as the main effect of G
in the presence of CaV
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 CaV2.2
1/
/G
combination is coexpressed, only G
should be bound; however, the channels do not show the same properties as the CaV2.2
1/G
combination (Meir et al., 2000
).
We concluded from that study that CaV
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 CaV
subunits (Fig. 7B), and reversal of this inhibition by a 100-ms prepulse was lost, implying that in the absence of CaV
subunits, G
dimers are able to bind and produce a small non-voltage-dependent inhibition of the CaV2.2 current, but their unbinding is not influenced by voltage (Meir et al., 2000
).
D. Potential Overlap of Determinants for CaV
Subunit and G
Subunit Function
There is overlap in the determinants for G protein modulation and CaV
binding or function for all three of the sites discussed above: the I-II linker, the C terminus, and the N terminus of CaV2 calcium channels. The G
binding site on the CaV
1 subunit intracellular I-II loop (De Waard et al., 1997
; Zamponi et al., 1997
) partially coincides with binding sites for auxiliary CaV
subunits (Pragnell et al., 1994
). However, the main amino acids that are critical for CaV
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 CaV2.2 between amino acids 45 and 55, four individual point mutations (S48A, I49A, R52A, and R54A) were isolated, which significantly compromised modulation of CaV2.2 by G proteins (Canti et al., 1999
). My group has subsequently shown that both the CaV2.2-R52,54A and CaV2.2-R52A constructs also exhibited compromised
2a-mediated retardation of inactivation as did CaV2.2-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 CaV2.2 amino terminus contributes determinants for both CaV
subunit and G
dimer function. However, the differentiating effect of CaV2.2-Q47A suggests that although the overall region involved may partially coincide, the determinants are not identical.
A partial overlap in CaV
subunit and G
binding sites has also been proposed for the CaV2.3 carboxyl-terminal site (Qin et al., 1997
). However, whereas deletion of the majority of this CaV2.3 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 |
|---|
|
|
|---|

binding to the CaV
1 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
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 CaV2 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 CaV2.2 was examined. This revealed that GTP
S 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 CaV2.2 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 CaV2.x channels on the actions of CaV
subunits, it might form part of a complex CaV
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 CaV
subunits or with the I-II linker of CaV2.2 (Canti et al., 2001
), although as described above (Section II.B.), the N terminus of CaV2.1 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 CaV2.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 CaV2.2, and amino acids N-terminal to this motif (144 in CaV2.2) are not required for G protein modulation (Canti et al., 1999
). The sequence of the N-terminal ball peptide in KV1.1 (Shaker B) is MAAVAGLYGLGEDRQHRKKQ, and there are no similarities with the N-terminal motif of CaV2.2 (YKQSIAQRART), apart from the presence of a number of essential positively charged residues and a KQ motif. In the case of KV 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 CaV2 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 CaV
1 and CaV
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 |
|---|
|
|
|---|

measured during the depolarizing prepulse (Zhou et al., 1997
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
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
o in neuronal membranes is blocked by an antibody against CaV
subunits (Campbell et al., 1995a
subunits. It will also be fascinating to examine whether GPCRs are included in such macromolecular signaling complexes. | VIII. Conclusion |
|---|
|
|
|---|
1 I-II linker is involved in the modulation of the CaV2 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
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
subunit plays a role in terminating the signal transduction process, which may be the case for GIRKs (Schreibmayer et al., 1996
subunits and G
dimers in the inhibition of the CaV2 family of calcium channels. | Acknowledgements |
|---|
| Footnotes |
|---|
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; GTP
S, 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 |
|---|
|
|
|---|
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.
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.
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, CaV1.3) voltage-dependent calcium currents. J Neurophysiol 85: 816-827.
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.
Bertaso F, Ward RJ, Viard P, Milligan G, and Dolphin AC (2003) Mechanism of Action of Gq to inhibit Gbeta gamma modulation of CaV2.2 calcium channels: probed by the use of receptor-Galpha tandems. Mol Pharmacol 63: 832-843.
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.
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.
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 alphao in rat frontal cortex. FEBS Lett 370: 135-140.[CrossRef][Medline]
Campbell V, Berrow N, and Dolphin AC (1993) GABAB receptor modulation of Ca2+ currents in rat sensory neurones by the G protein Go: antisense oligonucleotide studies. J Physiol (Lond) 470: 1-11.
Campbell V, Berrow NS, Fitzgerald EM, Brickley K, and Dolphin AC (1995b) Inhibition of the interaction of G protein Go with calcium channels by the calcium channel B-subunit in rat neurones. J Physiol (Lond) 485: 365-372.
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.
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 CaV2.2 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.
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.
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.
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.
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.
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.
Chien AJ, Carr KM, Shirokov RE, Rios E, and Hosey MM (1996) Identification of palmitoylation sites within the L type calcium channel B2a subunit and effects on channel function. J Biol Chem 271: 26465-26469.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 Gq
subunit. J Biol Chem 273: 636-644.
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.
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.
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.
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.
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.
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.
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.
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.
Hescheler J, Rosenthal W, Trautwein W, and Schultz G (1987) The GTP-binding protein, Go, 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.
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.
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.
Ikeda SR (1991) Double-pulse calcium channel current facilitation in adult rat sympathetic neurones. J Physiol (Lond) 439: 181-214.
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.
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.
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.
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.
Jones LP, Wei SK, and Yue DT (1998) Mechanism of auxiliary subunit modulation of neuronal
1E calcium channels. J Gen Physiol 112: 125-143.
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.
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.
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.
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.
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.
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.
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.
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.
MacKinnon R, Aldrich RW, and Lee AW (1993) Functional stoichiometry of Shaker potassium channel inactivation. Science (Wash DC) 262: 757-759.
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.
McFadzean I, Mullaney I, Brown DA, and Milligan G (1989) Antibodies to the GTP binding protein, Go, antagonize noradrenaline-induced calcium current inhibition in NG10815 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) Go transduces GABAB-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.
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 CaV2.2 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.
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.
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.
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.
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.
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.
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.
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.
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.
Qin N, Platano D, Olcese R, Stefani E, and Birnbaumer L (1997) Direct interaction of G
gamma with a C terminal G
gamma 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.
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.
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.
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 G
gamma. J Neurosci 18: 4883-4890.
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.
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.
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.
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.
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.
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 G
5 subunits. Proc Natl Acad Sci USA 95: 13307-13312.
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.
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.
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.
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 G
gamma subunits. J Physiol (Lond) 509: 15-27.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 Gk. Science (Wash DC) 235: 207-211.
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 G
gamma subunit. Proc Natl Acad Sci USA 95: 4035-4039.
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.
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.
Zhou J, Shapiro M, and Hille B (1997) Speed of calcium channel modulation by neurotransmitters in rat sympathetic neurons. J Neurophysiol 77: 2040-2048.
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.
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] |
||||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |