Modulation of inactivation properties of CaV2.2 channels by 14-3-3 proteins

Inactivation of presynaptic Ca(V)2.2 channels may play a role in regulating short-term synaptic plasticity. Here, we report a direct modulation of Ca(V)2.2 channel inactivation properties by 14-3-3, a family of signaling proteins involved in a wide range of biological processes. The structural elements critical for 14-3-3 binding and channel modulation lie in the carboxyl tail of the pore-forming alpha(1B) subunit, where we have identified two putative 14-3-3 interaction sites, including a phosphoserine-containing motif that directly binds to 14-3-3 and a second region near the EF hand and IQ domain. In transfected tsA 201 cells, 14-3-3 coexpression dramatically slows open-state inactivation and reduces cumulative inactivation of Ca(V)2.2 channels. In hippocampal neurons, interference with 14-3-3 binding accelerates Ca(V)2.2 channel inactivation and enhances short-term synaptic depression. These results demonstrate that 14-3-3 proteins are important regulators of Ca(V)2.2 channel activities and through this mechanism may contribute to their regulation of synaptic transmission and plasticity.     

Resistance of presynaptic CaV2.2 channels to voltage-dependent inactivation: dynamic palmitoylation and voltage sensitivity

Presynaptic CaV2.2 (N type) calcium channels gate the influx of calcium ions to trigger transmitter release. We have previously demonstrated at the chick ciliary ganglion presynaptic calyx terminal that the bulk of these channels are highly resistant to voltage dependent inactivation [E.F. Stanley, G. Goping, Characterization of a calcium current in a vertebrate cholinergic presynaptic nerve terminal, J. Neurosci. 11 (1991) 985-993; E.F. Stanley, Syntaxin I modulation of presynaptic calcium channel inactivation revealed by botulinum toxin C1, Eur. J. Neurosci. 17 (2003) 1303-1305; E.F. Stanley, R.R. Mirotznik, Cleavage of syntaxin prevents G-protein regulation of presynaptic calcium channels, Nature (Lond.) 385 (1997) 340-343]. Recent studies have suggested that CaV2.2 can be rendered inactivation resistant when expressed with the palmitoylated beta2A subunit and that this effect can be eliminated by tunicamycin, a general inhibitor of dynamic palmitoylation [J.H. Hurley, A.L. Cahill, K.P. Currie, A.P. Fox, The role of dynamic palmitoylation in Ca(2+) channel inactivation, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 9293-9298]. We find that while tunicamycin treatment had no effect on CaV2.2 current in the inactivation-sensitive isolated chick dorsal root ganglion (DRG) neuron, it caused a 10mV hyperpolarized shift in the profile of the inactivation-resistant presynaptic CaV2.2 population. This shift occurred without any effect on the voltage sensitivity of the inactivation process, as measured by a Boltzmann slope factor. Our findings suggest that dynamic palmitoylation contributes to the hyperpolarized steady inactivation profile of presynaptic CaV2.2. However, some other factor must also contribute since its inhibition does is not restore the inactivation profile to that of channels in the cell soma.     

Unified mechanisms of Ca2+ regulation across the Ca2+ channel family

L-type (CaV1.2) and P/Q-type (CaV2.1) calcium channels possess lobe-specific CaM regulation, where Ca2+ binding to one or the other lobe of CaM triggers regulation, even with inverted polarity of modulation between channels. Other major members of the CaV1-2 channel family, R-type (CaV2.3) and N-type (CaV2.2), have appeared to lack such CaM regulation. We report here that R- and N-type channels undergo Ca(2+)-dependent inactivation, which is mediated by the CaM N-terminal lobe and present only with mild Ca2+ buffering (0.5 mM EGTA) characteristic of many neurons. These features, together with the CaM regulatory profiles of L- and P/Q-type channels, are consistent with a simplifying principle for CaM signal detection in CaV1-2 channels-independent of channel context, the N- and C-terminal lobes of CaM appear invariably specialized for decoding local versus global Ca2+ activity, respectively.     

Functional expression and characterization of a voltage-gated CaV1.3 (alpha1D) calcium channel subunit from an insulin-secreting cell line.

L-type calcium channels mediate depolarization-induced calcium influx in insulin-secreting cells and are thought to be modulated by G protein-coupled receptors (GPCRs). The major fraction of L-type alpha1-subunits in pancreatic beta-cells is of the neuroendocrine subtype (CaV1.3 or alpha1D). Here we studied the biophysical properties and receptor regulation of a CaV1.3 subunit previously cloned from HIT-T15 cells. In doing so, we compared this neuroendocrine CaV1.3 channel with the cardiac L-type channel CaV1.2a (or alpha1C-a) after expression together with alpha2delta- and beta3-subunits in Xenopus oocytes. Both the current voltage relation and voltage dependence of inactivation for the neuroendocrine CaV1.3 channel were shifted to more negative potentials compared with the cardiac CaV1.2 channel. In addition, the CaV1.3 channel activated and inactivated more rapidly than the CaV1.2a channel. Both subtypes showed a similar sensitivity to the dihydropyridine (+)isradipine. More interestingly, the CaV1.3 channels were found to be stimulated by ligand-bound G(i)/G(o)-coupled GPCRs whereas a neuronal CaV2.2 (or alpha1B) channel was inhibited. The observed receptor-induced stimulation of CaV1.3 channels could be mimicked by phorbol-12-myristate-13-acetate and was sensitive to inhibitors of protein kinases, but not to the phosphoinositol-3-kinase-inhibitor wortmannin, pointing to serine/threonine kinase-dependent regulation. Taken together, we describe a neuroendocrine L-type CaV1.3 calcium channel that is stimulated by G(i)/G(o)-coupled GPCRs and differs significantly in distinct biophysical characteristics from the cardiac subtype (CaV1.2a), suggesting that the channels have different roles in native cells.     

Beta-cell CaV channel regulation in physiology and pathophysiology

The beta-cell is equipped with at least six voltage-gated Ca2+ (CaV) channel alpha1-subunits designated CaV1.2, CaV1.3, CaV2.1, CaV2.2, CaV2.3, and CaV3.1. These principal subunits, together with certain auxiliary subunits, assemble into different types of CaV channels conducting L-, P/Q-, N-, R-, and T-type Ca2+ currents, respectively. The beta-cell shares customary mechanisms of CaV channel regulation with other excitable cells, such as protein phosphorylation, Ca2+-dependent inactivation, and G protein modulation. However, the beta-cell displays some characteristic features to bring these mechanisms into play. In islet beta-cells, CaV channels can be highly phosphorylated under basal conditions and thus marginally respond to further phosphorylation. In beta-cell lines, CaV channels can be surrounded by tonically activated protein phosphatases dominating over protein kinases; thus their activity is dramatically enhanced by inhibition of protein phosphatases. During the last 10 years, we have revealed some novel mechanisms of beta-cell CaV channel regulation under physiological and pathophysiological conditions, including the involvement of exocytotic proteins, inositol hexakisphosphate, and type 1 diabetic serum. This minireview highlights characteristic features of customary mechanisms of CaV channel regulation in beta-cells and also reviews our studies on newly identified mechanisms of beta-cell CaV channel regulation.     

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

The single channel gating properties of human CaV2.1 (P/Q-type) calcium channels and their modulation by the auxiliary beta1b, beta2e, beta3a, and beta4a subunits were investigated with cell-attached patch-clamp recordings on HEK293 cells stably expressing human CaV2.1 channels. These calcium channels showed a complex modal gating, which is described in this and the following paper (Fellin, T., S. Luvisetto, M. Spagnolo, and D. Pietrobon. 2004. J. Gen. Physiol. 124:463-474). Here, we report the characterization of two modes of gating of human CaV2.1 channels, the slow mode and the fast mode. A channel in the two gating modes differs in mean closed times and latency to first opening (both longer in the slow mode), in voltage dependence of the open probability (larger depolarizations are necessary to open the channel in the slow mode), in kinetics of inactivation (slower in the slow mode), and voltage dependence of steady-state inactivation (occurring at less negative voltages in the slow mode). CaV2.1 channels containing any of the four beta subtypes can gate in either the slow or the fast mode, with only minor differences in the rate constants of the transitions between closed and open states within each mode. In both modes, CaV2.1 channels display different rates of inactivation and different steady-state inactivation depending on the beta subtype. The type of beta subunit also modulates the relative occurrence of the slow and the fast gating mode of CaV2.1 channels; beta3a promotes the fast mode, whereas beta4a promotes the slow mode. The prevailing mode of gating of CaV2.1 channels lacking a beta subunit is a gating mode in which the channel shows shorter mean open times, longer mean closed times, longer first latency, a much larger fraction of nulls, and activates at more positive voltages than in either the fast or slow mode.     

The separation of antagonist from agonist effects of trisubstituted purines on CaV2.2 (N-type) channels

Dihydropyridines can affect L-type calcium channels (CaV1) as either agonists or antagonists. Seliciclib or R-roscovitine, a 2,6,9-trisubstituted purine, is a potent cyclin-dependent kinase inhibitor that induces both agonist and antagonist effects on CaV2 channels (N-, P/Q- and R-type). We studied the effects induced by various trisubstituted purines on CaV2.2 (N-type) channels to learn about chemical structure–function relationships. We found that S-roscovitine and R-roscovitine showed similar potency to inhibit, but agonist activity of S-roscovitine required at least a 20-fold higher concentration, suggesting stereospecificity of the agonist-binding site. The testing of other trisubstituted purines showed a correlation between CaV2.2 inhibition and cyclin-dependent kinase affinity that broke down after determining that a chemically unrelated inhibitor, kenpaullone, was a poor CaV2.2 inhibitor, and a kinase inactive analog (dimethylamino-olomoucine; DMAO) was a strong inhibitor, which together support a kinase independent effect. In fact, like dihydropyridine-induced L-channel inhibition, R-roscovitine left-shifted the closed-state inactivation versus voltage relationship, which suggests that inhibition results from CaV2 channels moving into the inactivated state. Trisubstituted purine antagonists could become clinically important drugs to treat diseases, such as heart failure and neuropathic pain that result from elevated CaV2 channel activity.     

Biophysics and structure-function relationship of T-type Ca2+ channels

T-type channels are distinguished among voltage-gated Ca2+ channels by their low voltage thresholds for activation and inactivation, fast inactivation and small single channel conductance in isotonic Ba2+. Detailed biophysical and pharmacological characterization of native T-type channels indicated that these channels represent a heterogeneous family. Cloning of three family members (CaV3.1-3.3) confirmed these observations and allowed the study of the structure-function relationship of these channels. T-type channels are likely heterotetrameric structures consisting of a single polypeptide of four homologous domains (I-IV), each one containing six transmembrane spans (S1-S6), and cytoplasmic N- and C-termini. Structure-function studies have revealed that fast macroscopic inactivation of CaV3.1 is modulated by specific residues in the proximal C-terminus and in the transmembrane domain IIIS6. The particular gating properties within the T-type channel subfamily are determined by several parts of the protein, whereas differences with respect to high-voltage-activated Ca2+ channels are mostly determined by domains I, II and III. Several gating properties are affected by alternative splicing, C-terminal truncations and mutations associated to idiopathic epilepsy. Intriguingly, the aspartate residues of the EEDD locus of the selectivity filter not only determine the permeation properties and the block by Cd2+ and protons, but also activation and deactivation. Mutagenesis has also revealed that the outermost arginines of the S4 segment of domain IV influence the activation of CaV3.2, though no specific voltage-sensing amino acid has yet been properly identified. The selective modulation of CaV3.2 by G-proteins, CaMKII and PKA is determined by the II-III linker and the high-affinity inhibition of CaV3.2 by Ni2+ relies on a histidine residue in the IS3-S4 linker. Certainly, more structure-function studies are needed for a better understanding of T-type channel physiology and the rational design of treatments against T-type channel-related pathologies.     

Impact of gating modulation in CaV1.3 L-type calcium channels

CaV1.3 L-type channels control inner hair cell (IHC) sensory and sinoatrial node (SAN) function, and excitability in central neurons by means of their low-voltage activation and inactivation properties. In SAN cells CaV1.3 inward calcium current (ICa) inactivates rapidly whereas in IHCs inactivation is slow. A candidate suggested in slowing CaV1.3 channel inactivation is the presynaptically located ribbon-synapse protein RIM that is expressed in immature IHCs in presynaptic compartments also expressing CaV1.3 channels. CaV1.3 channel gating is also modulated by an intramolecular C-terminal mechanism. This mechanism was elicited during analysis of human C-terminal splice variants that differ in the length of their C-terminus and that modulates the channel's negative activation range and slows calcium-dependent inactivation.     

Several structural domains contribute to the regulation of N-type calcium channel inactivation by the beta 3 subunit

Calcium channel beta subunits are essential regulatory elements of the gating properties of high voltage-activated calcium channels. Co-expression with beta(3) subunits typically accelerates inactivation, whereas co-expression with beta(4) subunits results in a slowly inactivating phenotype. Here, we have examined the molecular basis of the differential effect of these two subunits on the inactivation characteristics of Ca(v)2.2 + alpha(2)-delta(1) N-type calcium channels by creating a series of 22 chimeric beta subunits that are based on various combinations of variable and conserved regions of the parent beta subunit isoforms. Our data show that replacement of the N terminus region of beta(4) with a corresponding 14-amino acid stretch of beta(3) sequence accelerates the inactivation kinetics to levels seen with wild type beta(3). A similar kinetic speeding is observed by a concomitant substitution of the second conserved and variable regions, but not when these regions are substituted individually, suggesting that 1) the second variable and conserved regions cooperatively regulate N-type calcium channel inactivation and 2) that there are two redundant mechanisms that allow the beta(3) subunit to accelerate N-type channel inactivation. In contrast with previous reports in Ca(v)2.1 calcium channels, deletion of the C-terminal region of Ca(v)2.2 did not alter the regulation of the channel by wild type and chimeric beta subunits. Hence, the molecular underpinnings of beta subunit regulation of voltage-gated calcium channels appear to vary with calcium channel subtype.     

Zn2+ sensitivity of high- and low-voltage activated calcium channels

The essential cation zinc (Zn2+) blocks voltage-dependent calcium channels in several cell types, which exhibit different sensitivities to Zn2+. The specificity of the Zn2+ effect on voltage-dependent calcium channel subtypes has not been systematically investigated. In this study, we used a transient protein expression system to determine the Zn2+ effect on low- and high-voltage activated channels. We found that in Ba2+, the IC50 value of Zn2+ was alpha1-subunit-dependent with lowest value for CaV1.2, and highest for CaV3.1; the sensitivity of the channels to Zn2+ was approximately ranked as CaV1.2>CaV3.2>CaV2.3>CaV2.2=CaV 2.1>or=CaV3.3=CaV3.1. Although the CaV2.2 and CaV3.1 channels had similar IC50 for Zn2+ in Ba2+, the CaV2.2, but not CaV3.1 channels, had approximately 10-fold higher IC50 to Zn2+ in Ca2+. The reduced sensitivity of CaV2.2 channels to Zn2+ in Ca2+ was partially reversed by disrupting a putative EF-hand motif located external to the selectivity filter EEEE locus. Thus, our findings support the notion that the Zn2+ block, mediated by multiple mechanisms, may depend on conformational changes surrounding the alpha1 pore regions. These findings provide fundamental insights into the mechanism underlying the inhibitory effect of zinc on various Ca2+ channel subtypes.     

Rab3a interacting molecule (RIM) and the tethering of pre‐synaptic transmitter release site‐associated CaV2.2 calcium channels

Biochemical and physiological evidence suggest that pre‐synaptic calcium channels are attached to the transmitter release site within the active zone by a molecular tether. A recent study has proposed that ‘Rab3a Interacting Molecule’ (RIM) serves as the tether for CaV2.1 channels in mouse brain, based in part on biochemical co‐immunoprecipitation (co‐IP) using a monoclonal antibody, mRIM. We previously argued against this idea for CaV2.2 calcium channel at chick synapses based on experiments using a different anti‐RIM antibody, pRIM1,2: while staining for the two proteins co‐localized and co‐varied at the transmitter release face, consistent with an association, they failed to co‐IP from a synaptosome membrane lysate. RIM is, however, a family of proteins and we tested the possibility that the mRIM antibody used in the more recent study identifies a particular channel‐tethering variant. We find that co‐immunostaining with mRIM and anti‐CaV2.2 antibody neither co‐localized nor co‐varied at the transmitter release face and the two proteins did not co‐IP, arguing against a common protein complex and a key CaV2.2 scaffolding role for RIM at the active zone. The differing results might be reconciled, however, in a model where a RIM family member contributes to a protein bridge that anchors the pre‐fusion secretory vesicle to the calcium channel protein complex.     

Modified sympathetic nerve system activity with overexpression of the voltage-dependent calcium channel beta3 subunit

N-type voltage-dependent calcium channels (VDCCs) play determining roles in calcium entry at sympathetic nerve terminals and trigger the release of the neurotransmitter norepinephrine. The accessory beta3 subunit of these channels preferentially forms N-type channels with a pore-forming CaV2.2 subunit. To examine its role in sympathetic nerve regulation, we established a beta3-overexpressing transgenic (beta3-Tg) mouse line. In these mice, we analyzed cardiovascular functions such as electrocardiography, blood pressure, echocardiography, and isovolumic contraction of the left ventricle with a Langendorff apparatus. Furthermore, we compared the cardiac function with that of beta3-null and CaV2.2 (alpha1B)-null mice. The beta3-Tg mice showed increased expression of the beta3 subunit, resulting in increased amounts of CaV2.2 in supracervical ganglion (SCG) neurons. The beta3-Tg mice had increased heart rate and enhanced sensitivity to N-type channel-specific blockers in electrocardiography, blood pressure, and echocardiography. In contrast, cardiac atria of the beta3-Tg mice revealed normal contractility to isoproterenol. Furthermore, their cardiac myocytes showed normal calcium channel currents, indicating unchanged calcium influx through VDCCs. Langendorff heart perfusion analysis revealed enhanced sensitivity to electric field stimulation in the beta3-Tg mice, whereas beta3-null and Cav2.2-null showed decreased responsiveness. The plasma epinephrine and norepinephrine levels in the beta3-Tg mice were significantly increased in the basal state, indicating enhanced sympathetic tone. Electrophysiological analysis in SCG neurons of beta3-Tg mice revealed increased calcium channel currents, especially N- and L-type currents. These results identify a determining role for the beta3 subunit in the N-type channel population in SCG and a major role in sympathetic nerve regulation.     

Molecular determinants of modulation of CaV2.1 channels by visinin-like protein 2

CaV2.1 channels, which conduct P/Q-type Ca2+ currents, initiate synaptic transmission at most synapses in the central nervous system. Ca2+/calmodulin-dependent facilitation and inactivation of these channels contributes to short-term facilitation and depression of synaptic transmission, respectively. Other calcium sensor proteins displace calmodulin (CaM) from its binding site, differentially regulate CaV2.1 channels, and contribute to the diversity of short-term synaptic plasticity. The neuronal calcium sensor protein visinin-like protein 2 (VILIP-2) inhibits inactivation and enhances facilitation of CaV2.1 channels. Here we examine the molecular determinants for differential regulation of CaV2.1 channels by VILIP-2 and CaM by construction and functional analysis of chimeras in which the functional domains of VILIP-2 are substituted in CaM. Our results show that the N-terminal domain, including its myristoylation site, the central α-helix, and the C-terminal lobe containing EF-hands 3 and 4 of VILIP-2 are sufficient to transfer its regulatory properties to CaM. This regulation by VILIP-2 requires binding to the IQ-like domain of CaV2.1 channels. Our results identify the essential molecular determinants of differential regulation of CaV2.1 channels by VILIP-2 and define the molecular code that these proteins use to control short-term synaptic plasticity.     

Modal gating of human CaV2.1 (P/Q-type) calcium channels: II. the b mode and reversible uncoupling of inactivation

The single channel gating properties of human CaV2.1 (P/Q-type) calcium channels were investigated with cell-attached patch-clamp recordings on HEK293 cells stably expressing these calcium channels. Human CaV2.1 channels showed a complex modal gating, which is described in this and the preceding paper (Luvisetto, S., T. Fellin, M. Spagnolo, B. Hivert, P.F. Brust, M.M. Harpold, K.A. Stauderman, M.E. Williams, and D. Pietrobon. 2004. J. Gen. Physiol. 124:445-461). Here, we report the characterization of the so-called b gating mode. A CaV2.1 channel in the b gating mode shows a bell-shaped voltage dependence of the open probability, and a characteristic low open probability at high positive voltages, that decreases with increasing voltage, as a consequence of both shorter mean open time and longer mean closed time. Reversible transitions of single human CaV2.1 channels between the b gating mode and the mode of gating in which the channel shows the usual voltage dependence of the open probability (nb gating mode) were much more frequent (time scale of seconds) than those between the slow and fast gating modes (time scale of minutes; Luvisetto et al., 2004), and occurred independently of whether the channel was in the fast or slow mode. We show that the b gating mode produces reversible uncoupling of inactivation in human CaV2.1 channels. In fact, a CaV2.1 channel in the b gating mode does not inactivate during long pulses at high positive voltages, where the same channel in both fast-nb and slow-nb gating modes inactivates relatively rapidly. Moreover, a CaV2.1 channel in the b gating mode shows a larger availability to open than in the nb gating modes. Regulation of the complex modal gating of human CaV2.1 channels could be a potent and versatile mechanism for the modulation of synaptic strength and plasticity as well as of neuronal excitability and other postsynaptic Ca2+-dependent processes.     

CaMKII-independent effects of KN93 and its inactive analog KN92: reversible inhibition of L-type calcium channels

Widely regarded as a specific and potent inhibitor of CaM kinases, especially CaMKII, KN93 has long been used to investigate the possible roles of CaMKII in a wide range of biological functions and systems, such as cultured cells, primary neurons, and brain slices. However, here we present evidence showing that KN93 and its structural analog KN92, which does not inhibit CaMKII, exert an unexpected, reversible, and specific reduction of currents of L-type calcium channels (CaV1.3 and CaV1.2), as compared to N-type calcium channels (CaV2.2). This effect is dependent not only on incubation time, but also on the dose of KN93 or KN92. Moreover, the effect appears to be independent of endocytosis, exocytosis, and proteasome activity. Washout and return to normal media rescues the L channel currents. Conversely, the structurally unrelated CaMKII inhibitor, AIP, fails to mimic the KN93/KN92 effect on L channel currents. Together, our data suggest that, in addition to inhibiting CaMKII, KN93 also affects CaV1.3 and CaV1.2 calcium channels in a CaMKII-independent manner.     

Further characterization of regulation of Ca(V)2.2 by stargazin

Stargazin, a transmembrane protein expressed in the nervous system, shares similarities with the γ? subunit of skeletal muscle calcium channels. It was thus termed γ? subunit of neuronal calcium channels. Stargazin downregulates the expression of Ca(V)2 channels, however, its functional modulation of these channels remains debated. We have reported that Stargazin modulates Ca(V)2.2 channel by a Gβγ-dependent mechanism and suggested that Stargazin is not a true subunit of this channel, since all its effects on channel function are dependent on the presence of Gβγ. Moreover, Stargazin also modulated the GIRK channel in a Gβγ-dependent fashion. Here we report that Gβγ-dependent modulation by Stargazin of the biophysical properties of Ca(V)2.2 is unrelated to its negative effect on channel expression and current amplitude. Finally, we suggest that this Gβγ dependent modulation of Stargazin may have physiological relevance, since it was still present when we used Ca2(+) as charge carrier, instead of Ba2(+).     

Analysis of the complex between Ca2+ channel beta-subunit and the Rem GTPase

Voltage-gated calcium channels are multiprotein complexes that regulate calcium influx and are important contributors to cardiac excitability and contractility. The auxiliary beta-subunit (CaV beta) binds a conserved domain (the alpha-interaction domain (AID)) of the pore-forming CaV alpha1 subunit to modulate channel gating properties and promote cell surface trafficking. Recently, members of the RGK family of small GTPases (Rem, Rem2, Rad, Gem/Kir) have been identified as novel contributors to the regulation of L-type calcium channel activity. Here, we describe the Rem-association domain within CaV beta2a. The Rem interaction module is located in a approximately 130-residue region within the highly conserved guanylate kinase domain that also directs AID binding. Importantly, CaV beta mutants were identified that lost the ability to bind AID but retained their association with Rem, indicating that the AID and Rem association sites of CaV beta2a are structurally distinct. In vitro binding studies indicate that the affinity of Rem for CaV beta2a interaction is lower than that of AID for CaV beta2a. Furthermore, in vitro binding studies indicate that Rem association does not inhibit the interaction of CaV beta2a with AID. Instead, CaV beta can simultaneously associate with both Rem and CaV alpha1-AID. Previous studies had suggested that RGK proteins may regulate Ca2+ channel activity by blocking the association of CaV beta subunits with CaV alpha1 to inhibit plasma membrane trafficking. However, surface biotinylation studies in HIT-T15 cells indicate that Rem can acutely modulate channel function without decreasing the density of L-type channels at the plasma membrane. Together these data suggest that Rem-dependent Ca2+ channel modulation involves formation of a Rem x CaV beta x AID regulatory complex without the need to disrupt CaV alpha1 x CaV beta association or alter CaV alpha1 expression at the plasma membrane.     

G protein modulation of CaV2 voltage-gated calcium channels

Voltage-gated Ca(2+) channels translate the electrical inputs of excitable cells into biochemical outputs by controlling influx of the ubiquitous second messenger Ca(2+) . As such the channels play pivotal roles in many cellular functions including the triggering of neurotransmitter and hormone release by CaV2.1 (P/Q-type) and CaV2.2 (N-type) channels. It is well established that G protein coupled receptors (GPCRs) orchestrate precise regulation neurotransmitter and hormone release through inhibition of CaV2 channels. Although the GPCRs recruit a number of different pathways, perhaps the most prominent, and certainly most studied among these is the so-called voltage-dependent inhibition mediated by direct binding of Gβγ to the α1 subunit of CaV2 channels. This article will review the basics of Ca(2+) -channels and G protein signaling, and the functional impact of this now classical inhibitory mechanism on channel function. It will also provide an update on more recent developments in the field, both related to functional effects and crosstalk with other signaling pathways, and advances made toward understanding the molecular interactions that underlie binding of Gβγ to the channel and the voltage-dependence that is a signature characteristic of this mechanism.     

G protein-gated inhibitory module of N-type (ca(v)2.2) ca2+ channels

Voltage-dependent G protein (Gbetagamma) inhibition of N-type (CaV2.2) channels supports presynaptic inhibition and represents a central paradigm of channel modulation. Still controversial are the proposed determinants for such modulation, which reside on the principal alpha1B channel subunit. These include the interdomain I-II loop (I-II), the carboxy tail (CT), and the amino terminus (NT). Here, we probed these determinants and related mechanisms, utilizing compound-state analysis with yeast two-hybrid and mammalian cell FRET assays of binding among channel segments and G proteins. Chimeric channels confirmed the unique importance of NT. Binding assays revealed selective interaction between NT and I-II elements. Coexpressing NT peptide with Gbetagamma induced constitutive channel inhibition, suggesting that the NT domain constitutes a G protein-gated inhibitory module. Such inhibition was limited to NT regions interacting with I-II, and G-protein inhibition was abolished within alpha1B channels lacking these NT regions. Thus, an NT module, acting via interactions with the I-II loop, appears fundamental to such modulation.