G protein inhibition of CaV2 calcium channels

Voltage-gated Ca²⁺ channels translate the electrical inputs of excitable cells into biochemical outputs by controlling influx of the ubiquitous second messenger Ca²⁺. As such the channels play pivotal roles in many cellular functions including the triggering of neurotransmitter and hormone release by Ca_V2.1 (P/Q-type) and Ca_V2.2 (N-type) channels. It is well established that G protein coupled receptors (GPCRs) orchestrate precise regulation neurotransmitter and hormone release through inhibition of Ca_V2 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 Ca_V2 channels. This article will review the basics of Ca²⁺-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.     

Functional properties and modulation of extracellular epitope - tagged CaV2.1 voltage-gated calcium channels

Depolarisation-induced Ca²⁺ influx into electrically excitable cells is determined by the density of voltage-gated Ca²⁺ channels at the cell surface. Surface expression is modulated by physiological stimuli as well as by drugs and can be altered under pathological conditions. Extracellular epitope tagging of channel subunits allows to quantify their surface expression and to distinguish surface channels from those in intracellular compartments. Here we report the first systematic characterisation of extracellularly epitope tagged Ca_V2.1 channels. We identified a permissive region in the pore-loop of repeat IV within the Ca_V2.1 α1 subunit which allowed integration of several different tags (hemagluttinine [HA], double HA; 6-histidine tag [His], 9-His, bungarotoxin-binding site) without compromising α1 subunit protein expression (in transfected tsA-201 cells) and function (after expression in X. laevis oocytes). Immunofluorescent studies revealed that the double-HA tagged construct (1722-HAGHA) was targeted to presynaptic sites in transfected cultured hippocampal neurons as expected for Ca_V2.1 channels. We also demonstrate that introduction of tags into this permissive position creates artifical sites for channel modulation. This was demonstrated by partial inhibition of 1722-HA channel currents with anti-HA antibodies and the concentration-dependent stimulation or partial inhibition by Ni-nitrilo triacetic acid (NTA) and novel bulkier derivatives (Ni-trisNTA, Ni-tetrakisNTA, Ni-nitro-o-phenyl-bisNTA, Ni-nitro-p-phenyl-bisNTA). Therefore our data also provide evidence for the concept that artificial modulatory sites for small ligands can be introduced into voltage-gated Ca²⁺ channel for their selective modulation.     

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.     

Cysteine string protein regulates G protein modulation of N-type calcium channels

Cysteine string proteins (CSPs) are secretory vesicle proteins bearing a "J domain" and a palmitoylated cysteine-rich "string" region that are critical for neurotransmitter release. The precise role of CSP in neurotransmission is controversial. Here, we demonstrate a novel interaction between CSP, receptor-coupled trimeric GTP binding proteins (G proteins), and N-type Ca2+ channels. G. subunits interact with the J domain of CSP in an ATP-dependent manner; in contrast, Gbetagamma subunits interact with the C terminus of CSP in both the presence and absence of ATP. The interaction of CSP with both G proteins and N-type Ca2+ channels results in a tonic G protein inhibition of the channels. In view of the crucial importance of N-type Ca2+ channels in presynaptic vesicle release, our data attribute a key role to CSP in the fine tuning of neurotransmission.     

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.     

Divergent Ca2+/calmodulin feedback regulation of CaV1 and CaV2 voltage-gated calcium channels evolved in the common ancestor of Placozoa and Bilateria

Ca_V1 and Ca_V2 voltage-gated calcium channels evolved from an ancestral Ca_V1/2 channel via gene duplication somewhere near the stem animal lineage. The divergence of these channel types led to distinguishing functional properties that are conserved among vertebrates and bilaterian invertebrates and contribute to their unique cellular roles. One key difference pertains to their regulation by calmodulin (CaM), wherein bilaterian Ca_V1 channels are uniquely subject to pronounced, buffer-resistant Ca²⁺/CaM-dependent inactivation, permitting negative feedback regulation of calcium influx in response to local cytoplasmic Ca²⁺ rises. Early diverging, nonbilaterian invertebrates also possess Ca_V1 and Ca_V2 channels, but it is unclear whether they share these conserved functional features. The most divergent animals to possess both Ca_V1 and Ca_V2 channels are placozoans such as Trichoplax adhaerens, which separated from other animals over 600 million years ago shortly after their emergence. Hence, placozoans can provide important insights into the early evolution of Ca_V1 and Ca_V2 channels. Here, we build upon previous characterization of Trichoplax Ca_V channels by determining the cellular expression and ion-conducting properties of the Ca_V1 channel orthologue, TCa_V1. We show that TCa_V1 is expressed in neuroendocrine-like gland cells and contractile dorsal epithelial cells. In vitro, this channel conducts dihydropyridine-insensitive, high-voltage–activated Ca²⁺ currents with kinetics resembling those of rat Ca_V1.2 but with left-shifted voltage sensitivity for activation and inactivation. Interestingly, TCa_V1, but not TCa_V2, exhibits buffer-resistant Ca²⁺/CaM-dependent inactivation, indicating that this functional divergence evolved prior to the emergence of bilaterian animals and may have contributed to their unique adaptation for cytoplasmic Ca²⁺ signaling within various cellular contexts.     

Signaling complexes of voltage-gated calcium channels and G protein-coupled receptors

Activation of opioid or opioid-receptor-like (ORL1 a.k.a. NOP or orphanin FQ) receptors mediates analgesia through inhibition of N-type calcium channels in dorsal root ganglion (DRG) neurons (1, 2). Unlike the three types of classical mu, delta, and kappa opioid receptors, ORL1 mediates an agonist-independent inhibition of N-type calcium channels. This is mediated via the formation of a physical protein complex between the receptor and the channel, which in turn allows the channel to effectively sense a low level of constitutive receptor activity (3). Further inhibition of N-type channel activity by activation of other G protein-coupled receptors is thus precluded. ORL1 receptors, however, also undergo agonist-induced internalization into lysosomes, and channels thereby become cointernalized in a complex with ORL1. This then results in removal of N-type channels from the plasma membrane and reduced calcium entry (4). Similar signaling complexes between N-type channels and GABA(B) receptors have been reported (5). Moreover, both L-type and P/Q-type channels appear to be able to associate with certain types of G protein-coupled receptors (6, 7). Hence, interactions between receptors and voltage-gated calcium channels may be a widely applicable means to optimize receptor channel coupling.     

Temperature-dependent modulation of CaV3 T-type calcium channels by protein kinases C and A in mammalian cells

Modulation of low voltage-activated Ca(V)3 T-type calcium channels remains poorly characterized compared with high voltage-activated Ca(V)1 and Ca(V)2 calcium channels. Notably, it is yet unresolved whether Ca(V)3 channels are modulated by protein kinases in mammalian cells. In this study, we demonstrate that protein kinase A (PKA) and PKC (but not PKG) activation induces a potent increase in Ca(V)3.1, Ca(V)3.2, and Ca(V)3.3 currents in various mammalian cell lines. Notably, we show that protein kinase effects occur at physiological temperature ( approximately 30-37 degrees C) but not at room temperature ( approximately 22-27 degrees C). This temperature dependence could involve kinase translocation, which is impaired at room temperature. A similar temperature dependence was observed for PKC-mediated increase in high voltage-activated Ca(V)2.3 currents. We also report that neither Ca(V)3 surface expression nor T-current macroscopic properties are modified upon kinase activation. In addition, we provide evidence for the direct phosphorylation of Ca(V)3.2 channels by PKA in in vitro assays. Overall, our results clearly establish the role of PKA and PKC in the modulation of Ca(V)3 T-channels and further highlight the key role of the physiological temperature in the effects described.     

Ni2+ block of CaV3.1 (alpha1G) T-type calcium channels

Ni(2+) inhibits current through calcium channels, in part by blocking the pore, but Ni(2+) may also allosterically affect channel activity via sites outside the permeation pathway. As a test for pore blockade, we examined whether the effect of Ni(2+) on Ca(V)3.1 is affected by permeant ions. We find two components to block by Ni(2+), a rapid block with little voltage dependence, and a slow block most visible as accelerated tail currents. Rapid block is weaker for outward vs. inward currents (apparent K(d) = 3 vs. 1 mM Ni(2+), with 2 mM Ca(2+) or Ba(2+)) and is reduced at high permeant ion concentration (110 vs. 2 mM Ca(2+) or Ba(2+)). Slow block depends both on the concentration and on the identity of the permeant ion (Ca(2+) vs. Ba(2+) vs. Na(+)). Slow block is 2-3x faster in Ba(2+) than in Ca(2+) (2 or 110 mM), and is approximately 10x faster with 2 vs. 110 mM Ca(2+) or Ba(2+). Slow block is orders of magnitude slower than the diffusion limit, except in the nominal absence of divalent cations ( approximately 3 muM Ca(2+)). We conclude that both fast and slow block of Ca(V)3.1 by Ni(2+) are most consistent with occlusion of the pore. The exit rate of Ni(2+) for slow block is reduced at high Ni(2+) concentrations, suggesting that the site responsible for fast block can "lock in" slow block by Ni(2+), at a site located deeper within the pore. In contrast to the complex pore block observed for Ca(V)3.1, inhibition of Ca(V)3.2 by Ni(2+) was essentially independent of voltage, and was similar in 2 mM Ca(2+) vs. Ba(2+), consistent with inhibition by a different mechanism, at a site outside the pore.     

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

Ca_V1.3 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 Ca_V1.3 inward calcium current (I_Ca) inactivates rapidly whereas in IHCs inactivation is slow. A candidate suggested in slowing Ca_V1.3 channel inactivation is the presynaptically located ribbon-synapse protein RIM that is expressed in immature IHCs in presynaptic compartments also expressing Ca_V1.3 channels. Ca_V1.3 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.     

Familial hemiplegic migraine type 1 mutations W1684R and V1696I alter G protein-mediated regulation of CaV2.1 voltage-gated calcium channels

Familial hemiplegic migraine type 1 (FHM-1) is a monogenic form of migraine with aura that is characterized by recurrent attacks of a typical migraine headache with transient hemiparesis during the aura phase. In a subset of patients, additional symptoms such as epilepsy and cerebellar ataxia are part of the clinical phenotype. FHM-1 is caused by missense mutations in the CACNA1A gene that encodes the pore-forming subunit of Ca_V2.1 voltage-gated Ca^2 + channels. Although the functional effects of an increasing number of FHM-1 mutations have been characterized, knowledge on the influence of most of these mutations on G protein regulation of channel function is lacking. Here, we explored the effects of G protein-dependent modulation on mutations W1684R and V1696I which cause FHM-1 with and without cerebellar ataxia, respectively. Both mutations were introduced into the human Ca_V2.1α₁ subunit and their functional consequences investigated after heterologous expression in human embryonic kidney 293 (HEK‐293) cells using patch-clamp recordings. When co-expressed along with the human μ-opioid receptor, application of the agonist [d‐Ala2, N‐MePhe4, Gly‐ol]‐enkephalin (DAMGO) inhibited currents through both wild-type (WT) and mutant Ca_V2.1 channels, which is consistent with the known modulation of these channels by G protein-coupled receptors. Prepulse facilitation, which is a way to characterize the relief of direct voltage-dependent G protein regulation, was reduced by both FHM-1 mutations. Moreover, the kinetic analysis of the onset and decay of facilitation showed that the W1684R and V1696I mutations affect the apparent dissociation and reassociation rates of the Gβγ dimer from the channel complex, suggesting that the G protein-Ca^2 + channel affinity may be altered by the mutations. These biophysical studies may shed new light on the pathophysiology underlying FHM-1.     

Conotoxin modulation of voltage-gated sodium channels

The rising phase of the action potential in excitable cells is mediated by voltage-gated sodium channels (VGSCs), of which there are nine mammalian subtypes with distinct tissue distribution and biophysical properties. The involvement of certain VGSC subtypes in disease states such as pain and epilepsy highlights the need for agents that modulate VGSCs in a subtype-specific manner. Conotoxins from marine snails of the Conus genus constitute a promising source of such modulators, since these peptide toxins have evolved to become selective for various membrane receptors, ion channels and transporters in excitable cells. This review covers the structure and function of three classes of conopeptides that modulate VGSCs: the pore-blocking mu-conotoxins, the delta-conotoxins which delay or inhibit VGSC inactivation, and the muO-conotoxins which inhibit VGSC Na(+) conductance independent of the tetrodotoxin binding site. Some of these toxins have potential therapeutic and research applications, in particular the muO-conotoxins, which may develop into potential drug leads for the treatment of pain states.     

Signaling complexes of voltage-gated calcium channels

Voltage gated calcium channels are key mediators of depolarization induced calcium entry into electrically excitable cells. There is increasing evidence that voltage gated calcium channels, like many other types of ionic channels, do not operate in isolation, but instead forms signaling complexes with signaling molecules, G protein coupled receptors, and other types of ion channels. Furthermore, there appears to be bidirectional signaling within these protein complexes, thus allowing not only for efficient translation of calcium signals into cellular responses, but also for tight control of calcium entry per se. In this review, we will focus predominantly on signaling complexes between G protein-coupled receptors and high voltage activated calcium channels, and on complexes of voltage-gated calcium channels and members of the potassium channel superfamily.     

Signaling complexes of voltage-gated calcium channels

Voltage gated calcium channels are key mediators of depolarization induced calcium entry into electrically excitable cells. There is increasing evidence that voltage gated calcium channels, like many other types of ionic channels, do not operate in isolation, but instead forms signaling complexes with signaling molecules, G protein coupled receptors, and other types of ion channels. Furthermore, there appears to be bidirectional signaling within these protein complexes, thus allowing not only for efficient translation of calcium signals into cellular responses, but also for tight control of calcium entry per se. In this review, we will focus predominantly on signaling complexes between G protein-coupled receptors and high voltage activated calcium channels, and on complexes of voltage-gated calcium channels and members of the potassium channel superfamily.     

RGK regulation of voltage-gated calcium channels

Voltage-gated calcium channels(VGCCs) play critical roles in cardiac and skeletal muscle contractions,hormone and neurotransmitter release,as well as slower processes such as cell proliferation,differentiation,migration and death.Mutations in VGCCs lead to numerous cardiac,muscle and neurological disease,and their physiological function is tightly regulated by kinases,phosphatases,G-proteins,calmodulin and many other proteins.Fifteen years ago,RGK proteins were discovered as the most potent endogenous regulators of VGCCs.They are a family of monomeric GTPases(Rad,Rem,Rem2,and Gem/Kir),in the superfamily of Ras GTPases,and they have two known functions: regulation of cytoskeletal dynamics including dendritic arborization and inhibition of VGCCs.Here we review the mechanisms and molecular determinants of RGK-mediated VGCC inhibition,the physiological impact of this inhibition,and recent evidence linking the two known RGK functions.     

Grina/TMBIM3 modulates voltage-gated CaV2.2 Ca2+ channels in a G-protein-like manner

Grina/TMBIM3 is a poorly characterized transmembrane protein with a broad expression pattern in mammals and with a very ancient origin within eukaryotes. Although initially characterized as an NMDA-receptor associated subunit, there is increasing evidence that Grina/TMBIM3 is involved in the unfolded protein response and controls apoptosis via regulation of Ca²⁺ homeostasis. Here, we investigate a putative direct interaction of Grina/TMBIM3 with voltage gated Ca²⁺ channels, in particular with the Ca_V2.2 α1-subunit and describe its modulatory effects on the current through Ca_V2.2 N-type channels. Direct interaction was confirmed by co-immunoprecipitation studies and membrane localization was proven. Co-expression of Grina/TMBIM3 with Ca_V2.2 channels resulted in a significant decrease of the current amplitude and in a slowing of the kinetics of current activation. This effect was accompanied by a significant shift of the voltage dependencies of activation time constants towards more depolarized voltages. Application of a stimulus protocol including a strong depolarizing pulse relieved inhibition of current amplitude by Grina/TMBIM3. When Grina/TMBIM3 was present, inactivation by an action potential-like train of pulses was diminished. Both observations resemble mechanisms that are well-studied modulatory effects of G-protein βγ subunits on Ca_V2 channels. The impact of Grina/TMBIM3 and G-protein βγ subunits are rather comparable with respect to suppression of current amplitude and slowing of activation kinetics. Furthermore, both modulators had the same effect on current inactivation when evoked by an action potential-like train of pulses.     

Transthyretin oligomers induce calcium influx via voltage-gated calcium channels

The deposition of transthyretin (TTR) amyloid in the PNS is a major pathological feature of familial amyloidotic polyneuropathy. The aim of the present study was to examine whether TTR could disrupt cytoplasmic Ca(2+) homeostasis and to determine the role of TTR aggregation in this process. The aggregation of amyloidogenic TTR was examined by solution turbidity, dynamic light scattering and atomic force microscopy. A nucleation-dependent polymerization process was observed in which TTR formed low molecular weight aggregates (oligomers < 100 nm in diameter) before the appearance of mature fibrils. TTR rapidly induced an increase in the concentration of intracellular Ca(2+) ([Ca(2+)](i)) when applied to SH-SY5Y human neuroblastoma cells. The greatest effect on [Ca(2+)](i) was induced by a preparation that contained the highest concentration of TTR oligomers. The TTR-induced increase in [Ca(2+)](i) was due to an influx of extracellular Ca(2+), mainly via L- and N-type voltage-gated calcium channels (VGCCs). These results suggest that increasing [Ca(2+)](i) via VGCCs may be an important early event which contributes to TTR-induced cytotoxicity, and that TTR oligomers, rather than mature fibrils, may be the major cytotoxic form of TTR.