Cavβ Subunit Research Articles

A membrane-associated phosphoswitch in Rad controls adrenergic regulation of cardiac calcium channels.

The ability to fight or flee from a threat relies on an acute adrenergic surge that augments cardiac output, which is dependent on increased cardiac contractility and heart rate. This cardiac response depends on β-adrenergic-initiated reversal of the small RGK G protein Rad-mediated inhibition of voltage-gated calcium channels (CaV) acting through the Cavβ subunit. Here, we investigate how Rad couples phosphorylation to augmented Ca2+ influx and increased cardiac contraction. We show that reversal required phosphorylation of Ser272 and Ser300 within Rad's polybasic, hydrophobic C-terminal domain (CTD). Phosphorylation of Ser25 and Ser38 in Rad's N-terminal domain (NTD) alone was ineffective. Phosphorylation of Ser272 and Ser300 or the addition of 4 Asp residues to the CTD reduced Rad's association with the negatively charged, cytoplasmic plasmalemmal surface and with CaVβ, even in the absence of CaVα, measured here by FRET. Addition of a posttranslationally prenylated CAAX motif to Rad's C-terminus, which constitutively tethers Rad to the membrane, prevented the physiological and biochemical effects of both phosphorylation and Asp substitution. Thus, dissociation of Rad from the sarcolemma, and consequently from CaVβ, is sufficient for sympathetic upregulation of Ca2+ currents.

Molecular tuning of sea anemone stinging.

Jellyfish and sea anemones fire single-use, venom-covered barbs to immobilize prey or predators. We previously showed that the anemone Nematostella vectensis uses a specialized voltage-gated calcium (CaV) channel to trigger stinging in response to synergistic prey-derived chemicals and touch (Weir et al., 2020). Here, we use experiments and theory to find that stinging behavior is suited to distinct ecological niches. We find that the burrowing anemone Nematostella uses uniquely strong CaV inactivation for precise control of predatory stinging. In contrast, the related anemone Exaiptasia diaphana inhabits exposed environments to support photosynthetic endosymbionts. Consistent with its niche, Exaiptasia indiscriminately stings for defense and expresses a CaV splice variant that confers weak inactivation. Chimeric analyses reveal that CaVβ subunit adaptations regulate inactivation, suggesting an evolutionary tuning mechanism for stinging behavior. These findings demonstrate how functional specialization of ion channel structure contributes to distinct organismal behavior.

Molecular tuning of sea anemone stinging

Jellyfish and sea anemones fire single-use, venom-covered barbs to immobilize prey or predators. We previously showed that the anemone Nematostella vectensis uses a specialized voltage-gated calcium (CaV) channel to trigger stinging in response to synergistic prey-derived chemicals and touch (Weir et al., 2020). Here, we use experiments and theory to find that stinging behavior is suited to distinct ecological niches. We find that the burrowing anemone Nematostella uses uniquely strong CaV inactivation for precise control of predatory stinging. In contrast, the related anemone Exaiptasia diaphana inhabits exposed environments to support photosynthetic endosymbionts. Consistent with its niche, Exaiptasia indiscriminately stings for defense and expresses a CaV splice variant that confers weak inactivation. Chimeric analyses reveal that CaVβ subunit adaptations regulate inactivation, suggesting an evolutionary tuning mechanism for stinging behavior. These findings demonstrate how functional specialization of ion channel structure contributes to distinct organismal behavior.

Molecular Mechanisms of Nuclear Transport of the Neuronal Voltage-gated Ca2+ Channel β3 Auxiliary Subunit

Previous studies have shown that in addition to its role within the voltage-gated calcium channel complex in the plasma membrane, the neuronal CaVβ subunit can translocate to the cell nucleus. However, little is known regarding the role this protein could play in the nucleus, nor the molecular mechanism used by CaVβ to enter this cell compartment. This report shows evidence that CaVβ3 has nuclear localization signals (NLS) that are not functional, suggesting that the protein does not use a classical nuclear import pathway. Instead, its entry into the nucleus could be associated with another protein that would function as a carrier, using a mechanism known as a piggyback. Mass spectrometry assays and bioinformatic analysis allowed the identification of proteins that could be participating in the entry of CaVβ3 into the nucleus. Likewise, through proximity ligation assays (PLA), it was found that members of the heterogeneous nuclear ribonucleoproteins (hnRNPs) and B56δ, a regulatory subunit of the protein phosphatase 2A (PP2A), could function as proteins that regulate this piggyback mechanism. On the other hand, bioinformatics and site-directed mutagenesis assays allowed the identification of a functional nuclear export signal (NES) that controls the exit of CaVβ3 from the nucleus, which would allow the completion of the nuclear transport cycle of the protein. These results reveal a novel mechanism for the nuclear transport cycle of the neuronal CaVβ3 subunit.

STAC proteins inhibit the voltage dependent inactivation of l-type calcium channels.

The inactivation of voltage-gated calcium channels is governed by two distinct processes: voltage dependent inactivation (VDI) and calcium-dependent inactivation (CDI). The STAC family of adaptor proteins inhibits the CDI of L-type calcium channels (LTCC). In fact, all LTCC (CaV1.2, CaV1.3 and CaV1.4) showed reduced CDI in the presence of any STAC isoform. However, whether STAC proteins also modulate the CDI of the skeletal muscle channel CaV1.1 was initially not investigated, as CaV1.1 requires STAC3 for its functional expression. Interestingly, we could recently demonstrate that STAC3 does not inhibit the CDI, but the VDI of CaV1.1. Therefore, we hypothesized that STAC proteins inhibit also the VDI of LTCC. Indeed, when co-expressed in HEK cells, all STAC isoforms reduced the VDI of CaV1.2 and CaV1.3 currents. As for CDI, also the VDI inhibition is specific for LTCC, as the VDI of CaV2.1 and CaV2.3 is not affected by STAC proteins. As the predominant contribution to VDI is known to arise from the I-II loop interaction with the CaVβ subunit, and that STAC proteins were shown to interact with distinct cytoplasmic domains (II-III loop and C-terminus), we hypothesized that STAC proteins and membrane tethered β subunits utilize different mechanism to inhibit VDI. Indeed, co-expression of STAC3 with CaV1.3/β2a revealed additional deceleration of VDI in HEK cells. Ongoing experiments, involving STAC and channels mutants and chimeras, will reveal the molecular determinants of the VDI inhibition by STAC proteins.

Co-Silencing of the Voltage-Gated Calcium Channel β Subunit and High-Voltage Activated α1 Subunit by dsRNA Soaking Resulted in Enhanced Defects in Locomotion, Stylet Thrusting, Chemotaxis, Protein Secretion, and Reproduction in Ditylenchus destructor.

The voltage-gated calcium channel (VGCC) β subunit (Cavβ) protein is a kind of cytosolic auxiliary subunit that plays an important role in regulating the surface expression and gating characteristics of high-voltage-activated (HVA) calcium channels. Ditylenchus destructor is an important plant-parasitic nematode. In the present study, the putative Cavβ subunit gene of D. destructor, namely, DdCavβ, was subjected to molecular characterization. In situ hybridization assays showed that DdCavβ was expressed in all nematode tissues. Transcriptional analyses showed that DdCavβ was expressed during each developmental stage of D. destructor, and the highest expression level was recorded in the third-stage juveniles. The crucial role of DdCavβ was verified by dsRNA soaking-mediated RNA interference (RNAi). Silencing of DdCavβ or HVA Cavα1 alone and co-silencing of the DdCavβ and HVA Cavα1 genes resulted in defective locomotion, stylet thrusting, chemotaxis, protein secretion and reproduction in D. destructor. Co-silencing of the HVA Cavα1 and Cavβ subunits showed stronger interference effects than single-gene silencing. This study provides insights for further study of VGCCs in plant-parasitic nematodes.

Circulating microRNAs predispose to takotsubo syndrome following high-dose adrenaline exposure.

Aims Takotsubo syndrome (TTS) is an acute heart failure, typically triggered by high adrenaline during physical or emotional stress. It is distinguished from myocardial infarction (MI) by a characteristic pattern of ventricular basal hypercontractility with hypokinesis of apical segments, and in the absence of culprit coronary occlusion. We aimed to understand whether recently discovered circulating biomarkers miR-16 and miR-26a, which differentiate TTS from MI at presentation, were mechanistically involved in the pathophysiology of TTS.Methods and results miR-16 and miR-26a were co-overexpressed in rats with AAV and TTS induced with an adrenaline bolus. Untreated isolated rat cardiomyocytes were transfected with pre-/anti-miRs and functionally assessed. Ventricular basal hypercontraction and apical depression were accentuated in miR-transfected animals after induction of TTS. In vitro miR-16 and/or miR-26a overexpression in isolated apical (but not basal), cardiomyocytes produced strong depression of contraction, with loss of adrenaline sensitivity. They also enhanced the initial positive inotropic effect of adrenaline in basal cells. Decreased contractility after TTS-miRs was reproduced in non-failing human apical cardiomyocytes. Bioinformatic profiling of miR targets, followed by expression assays and functional experiments, identified reductions of CACNB1 (L-type calcium channel Cavβ subunit), RGS4 (regulator of G-protein signalling 4), and G-protein subunit Gβ (GNB1) as underlying these effects.ConclusionmiR-16 and miR-26a sensitize the heart to TTS-like changes produced by adrenaline. Since these miRs have been associated with anxiety and depression, they could provide a mechanism whereby priming of the heart by previous stress causes an increased likelihood of TTS in the future.

Molecular Mechanism of Voltage-Gated Ca2+ Channel Regulation by Membrane PIP2

Voltage-gated calcium (CaV) channels play essential roles in adjusting calcium influx upon membrane depolarization. CaV2 (N-, P/Q- and R-type) channels are concentrated in the presynaptic nerve terminals and important for the neurotransmitter release. Adjusting the presynaptic calcium channel gating exerts potent influence on synaptic plasticity. CaV channels need auxiliary subunits for proper trafficking to the plasma membrane and the channel gating. Especially CaVβ subunit plays crucial roles in the surface expression of CaV channels and fine-tuning of channel gating. It has been known that CaV channels are modulated by membrane phosphatidylinositol 4,5-bisphosphate (PIP2). The binding affinity between ion channel and PIP2 is important for the channel gating normally, but molecular mechanism of PIP2 regulation remains unclear. It was recently reported that subcellular localization of β subunit is a key factor for the control of PIP2 sensitivity of CaV channels. Here we found that the intracellular movement of I-II linker in a1 subunit is important for determining the PIP2 sensitivity of CaV channels. When the I-II linker was shifted to the plasma membrane, current inhibition by PIP2 depletion significantly decreased as like the responses triggered by membrane-tethered β subunit. Consistently we also found that inserting a flexible linker between membrane-tethered Lyn and GK domain of β subunit increased the PIP2 sensitivity of CaV channels. Polybasic motif at the C-terminal end of the I-II linker of CaV channels is a potential PIP2 interaction site. Neutralization of the polybasic motif of I-II linker abolished PIP2 sensitivity of CaV channels. Together, our results indicate that the conformational shift of I-II linker to the plasma membrane is the key mechanism for decreasing the PIP2 sensitivity of CaV channels and this shift is mainly regulated by auxiliary CaVβ subunit in physiological condition.

Regulation of Blood Pressure by Targeting CaV1.2-Galectin-1 Protein Interaction.

L-type CaV1.2 channels play crucial roles in the regulation of blood pressure. Galectin-1 (Gal-1) has been reported to bind to the I-II loop of CaV1.2 channels to reduce their current density. However, the mechanistic understanding for the downregulation of CaV1.2 channels by Gal-1 and whether Gal-1 plays a direct role in blood pressure regulation remain unclear. In vitro experiments involving coimmunoprecipitation, Western blot, patch-clamp recordings, immunohistochemistry, and pressure myography were used to evaluate the molecular mechanisms by which Gal-1 downregulates CaV1.2 channel in transfected, human embryonic kidney 293 cells, smooth muscle cells, arteries from Lgasl1-/- mice, rat, and human patients. In vivo experiments involving the delivery of Tat-e9c peptide and AAV5-Gal-1 into rats were performed to investigate the effect of targeting CaV1.2-Gal-1 interaction on blood pressure monitored by tail-cuff or telemetry methods. Our study reveals that Gal-1 is a key regulator for proteasomal degradation of CaV1.2 channels. Gal-1 competed allosterically with the CaVβ subunit for binding to the I-II loop of the CaV1.2 channel. This competitive disruption of CaVβ binding led to CaV1.2 degradation by exposing the channels to polyubiquitination. It is notable that we demonstrated that the inverse relationship of reduced Gal-1 and increased CaV1.2 protein levels in arteries was associated with hypertension in hypertensive rats and patients, and Gal-1 deficiency induces higher blood pressure in mice because of the upregulated CaV1.2 protein level in arteries. To directly regulate blood pressure by targeting the CaV1.2-Gal-1 interaction, we administered Tat-e9c, a peptide that competed for binding of Gal-1 by a miniosmotic pump, and this specific disruption of CaV1.2-Gal-1 coupling increased smooth muscle CaV1.2 currents, induced larger arterial contraction, and caused hypertension in rats. In contrasting experiments, overexpression of Gal-1 in smooth muscle by a single bolus of AAV5-Gal-1 significantly reduced blood pressure in spontaneously hypertensive rats. We have defined molecularly that Gal-1 promotes CaV1.2 degradation by replacing CaVβ and thereby exposing specific lysines for polyubiquitination and by masking I-II loop endoplasmic reticulum export signals. This mechanistic understanding provided the basis for targeting CaV1.2-Gal-1 interaction to demonstrate clearly the modulatory role that Gal-1 plays in regulating blood pressure, and offering a potential approach for therapeutic management of hypertension.

Constitutive activity of the Ghrelin receptor reduces surface expression of voltage-gated Ca2+ channels in a CaVβ-dependent manner.

Voltage-gated Ca2+ (CaV) channels couple membrane depolarization to Ca2+ influx, triggering a range of Ca2+-dependent cellular processes. CaV channels are, therefore, crucial in shaping neuronal activity and function, depending on their individual temporal and spatial properties. Furthermore, many neurotransmitters and drugs that act through G protein coupled receptors (GPCRs), modulate neuronal activity by altering the expression, trafficking, or function of CaV channels. GPCR-dependent mechanisms that downregulate CaV channel expression levels are observed in many neurons but are, by comparison, less studied. Here we show that the growth hormone secretagogue receptor type 1a (GHSR), a GPCR, can inhibit the forwarding trafficking of several CaV subtypes, even in the absence of agonist. This constitutive form of GPCR inhibition of CaV channels depends on the presence of a CaVβ subunit. CaVβ subunits displace CaVα1 subunits from the endoplasmic reticulum. The actions of GHSR on CaV channels trafficking suggest a role for this signaling pathway in brain areas that control food intake, reward, and learning and memory.

The CaVβ Subunit Protects the I-II Loop of the Voltage-gated Calcium Channel CaV2.2 from Proteasomal Degradation but Not Oligoubiquitination

CaVβ subunits interact with the voltage-gated calcium channel CaV2.2 on a site in the intracellular loop between domains I and II (the I-II loop). This interaction influences the biophysical properties of the channel and leads to an increase in its trafficking to the plasma membrane. We have shown previously that a mutant CaV2.2 channel that is unable to bind CaVβ subunits (CaV2.2 W391A) was rapidly degraded (Waithe, D., Ferron, L., Page, K. M., Chaggar, K., and Dolphin, A. C. (2011) J. Biol. Chem. 286, 9598–9611). Here we show that, in the absence of CaVβ subunits, a construct consisting of the I-II loop of CaV2.2 was directly ubiquitinated and degraded by the proteasome system. Ubiquitination could be prevented by mutation of all 12 lysine residues in the I-II loop to arginines. Including a palmitoylation motif at the N terminus of CaV2.2 I-II loop was insufficient to target it to the plasma membrane in the absence of CaVβ subunits even when proteasomal degradation was inhibited with MG132 or ubiquitination was prevented by the lysine-to-arginine mutations. In the presence of CaVβ subunit, the palmitoylated CaV2.2 I-II loop was protected from degradation, although oligoubiquitination could still occur, and was efficiently trafficked to the plasma membrane. We propose that targeting to the plasma membrane requires a conformational change in the I-II loop that is induced by binding of the CaVβ subunit.

Phosphorylation sites in the Hook domain of CaVβ subunits differentially modulate CaV1.2 channel function

Regulation of L-type calcium current is critical for the development, function, and regulation of many cell types. CaV1.2 channels that conduct L-type calcium currents are regulated by many protein kinases, but the sites of action of these kinases remain unknown in most cases. We combined mass spectrometry (LC–MS/MS) and whole-cell patch clamp techniques in order to identify sites of phosphorylation of CaVβ subunits in vivo and test the impact of mutations of those sites on CaV1.2 channel function in vitro. Using the CaV1.1 channel purified from rabbit skeletal muscle as a substrate for phosphoproteomic analysis, we found that Ser193 and Thr205 in the HOOK domain of CaVβ1a subunits were both phosphorylated in vivo. Ser193 is located in a potential consensus sequence for casein kinase II, but it was not phosphorylated in vitro by that kinase. In contrast, Thr205 is located in a consensus sequence for cAMP-dependent phosphorylation, and it was robustly phosphorylated in vitro by PKA. These two sites are conserved in multiple CaVβ subunit isoforms, including the principal CaVβ subunit of cardiac CaV1.2 channels, CaVβ2b. In order to assess potential modulatory effects of phosphorylation at these sites separately from the effects of phosphorylation of the α11.2 subunit, we inserted phosphomimetic or phosphoinhibitory mutations in CaVβ2b and analyzed their effects on CaV1.2 channel function in transfected nonmuscle cells. The phosphomimetic mutation CaVβ2bS152E decreased peak channel currents and shifted the voltage dependence of both activation and inactivation to more positive membrane potentials. The phosphoinhibitory mutation CaVβ2bS152A had opposite effects. There were no differences in peak CaV1.2 currents or voltage dependence between the phosphomimetic mutation CaVβ2bT164D and the phosphoinhibitory mutation CaVβ2bT164A. However, calcium-dependent inactivation was significantly increased for the phosphomimetic mutation CaVβ2bT164D. This effect was subunit-specific, as the corresponding mutation in the palmitoylated isoform, CaVβ2a, had no effect. Overall, our data identify two conserved sites of phosphorylation of the Hook domain of CaVβ subunits in vivo and reveal differential modulatory effects of phosphomimetic mutations in these sites. These results reveal a new dimension of regulation of CaV1.2 channels through phosphorylation of the Hook domains of their β subunits.

Regulation of neuronal high-voltage activated CaV2 Ca2+ channels by the small GTPase RhoA

High-Voltage-Activated (HVA) Ca2+ channels are known regulators of synapse formation and transmission and play fundamental roles in neuronal pathophysiology. Small GTPases of Rho and RGK families, via their action on both cytoskeleton and Ca2+ channels are key molecules for these processes. While the effects of RGK GTPases on neuronal HVA Ca2+ channels have been widely studied, the effects of RhoA on the HVA channels remains however elusive. Using heterologous expression in Xenopus laevis oocytes, we show that RhoA activity reduces Ba2+ currents through CaV2.1, CaV2.2 and CaV2.3 Ca2+ channels independently of CaVβ subunit. This inhibition occurs independently of RGKs activity and without modification of biophysical properties and global level of expression of the channel subunit. Instead, we observed a marked decrease in the number of active channels at the plasma membrane. Pharmacological and expression studies suggest that channel expression at the plasma membrane is impaired via a ROCK-sensitive pathway. Expression of constitutively active RhoA in primary culture of spinal motoneurons also drastically reduced HVA Ca2+ current amplitude. Altogether our data revealed that HVA Ca2+ channels regulation by RhoA might govern synaptic transmission during development and potentially contribute to pathophysiological processes when axon regeneration and growth cone kinetics are impaired.

Molecular characterization and functional expression of the Apis mellifera voltage-dependent Ca2+ channels

Voltage-gated Ca2+ channels allow the influx of Ca2+ ions from the extracellular space upon membrane depolarization and thus serve as a transducer between membrane potential and cellular events initiated by Ca2+ transients. Most insects are predicted to possess three genes encoding Cavα, the main subunit of Ca2+ channels, and several genes encoding the two auxiliary subunits, Cavβ and Cavα2δ; however very few of these genes have been cloned so far. Here, we cloned three full-length cDNAs encoding the three Cavα subunits (AmelCav1a, AmelCav2a and AmelCav3a), a cDNA encoding a novel variant of the Cavβ subunit (AmelCavβc), and three full-length cDNAs encoding three Cavα2δ subunits (AmelCavα2δ1 to 3) of the honeybee Apis mellifera. We identified several alternative or mutually exclusive exons in the sequence of the AmelCav2 and AmelCav3 genes. Moreover, we detected a stretch of glutamine residues in the C-terminus of the AmelCav1 subunit that is reminiscent of the motif found in the human Cav2.1 subunit of patients with Spinocerebellar Ataxia type 6. All these subunits contain structural domains that have been identified as functionally important in their mammalian homologues. For the first time, we could express three insect Cavα subunits in Xenopus oocytes and we show that AmelCav1a, 2a and 3a form Ca2+ channels with distinctive properties. Notably, the co-expression of AmelCav1a or AmelCav2a with AmelCavβc and AmCavα2δ1 produces High Voltage-Activated Ca2+ channels. On the other hand, expression of AmelCav3a alone leads to Low Voltage-Activated Ca2+ channels.

β2A and β3 Differentially Modulate Time- and Voltage-Dependent Properties of Individual Voltage Sensors in the Human Cav1.2 Channel

The human L-type calcium channel (CaV1.2) is a multi-protein complex consisting of the pore-forming α1c subunit and at least two auxiliary subunits, β and α2δ, which assemble in a 1:1:1 stoichiometry. β subunits regulate multiple aspects of CaV1.2 channel physiology, including surface expression, degradation and gating. Investigating the effect of β2a and β3 subunits on human CaV1.2 channels (α1c+α2δ) expressed in Xenopus oocytes, we found that the voltage dependences of total charge displacement (QV) and conductance (GV) were ∼10mV more negative in the presence of β2a. We tested the hypothesis that β2a and β3 subunits differently modulate the activation of the four voltage-sensor domains (VSDs) in the α1c subunit. Using the Voltage-Clamp Fluorometry technique, we fluorescently tracked the movement of individual VSD in conducting human CaV1.2 channels. We found that, in the presence of β2a, the voltage-dependent activation of VSDs II and III was left-shifted as compared to β3-associated CaV1.2 (VSD II with β2a: Vhalf= −31.1±1.0 mV, z= 4.5±0.8 e0; VSD II with β3: Vhalf= −26.5±1.8 mV, z= 2.4±0.1 e0; VSD III with β2a: Vhalf= −36.6±3.9 mV, z= 1.8±0.1 e0; VSD III with β3: Vhalf= −16.9±1.4 mV, z= 1.2±0.1 e0). Moreover, activation and deactivation kinetics of VSD II in channels associated with β2a were dramatically slower than in the presence of β3, while voltage- and time-dependent properties of VSD IV remained the same in the presence of either Cavβ subunit. These results suggest that, in spite of their highly-conserved sequence homology, different β subunit isoforms distinctly modulate the biophysical properties of CaV1.2 voltage sensors to mediate their effect on Ca conductance.

Ancient origins of RGK protein function: modulation of voltage-gated calcium channels preceded the protostome and deuterostome split.

RGK proteins, Gem, Rad, Rem1, and Rem2, are members of the Ras superfamily of small GTP-binding proteins that interact with Ca2+ channel β subunits to modify voltage-gated Ca2+ channel function. In addition, RGK proteins affect several cellular processes such as cytoskeletal rearrangement, neuronal dendritic complexity, and synapse formation. To probe the phylogenetic origins of RGK protein–Ca2+ channel interactions, we identified potential RGK-like protein homologs in genomes for genetically diverse organisms from both the deuterostome and protostome animal superphyla. RGK-like protein homologs cloned from Danio rerio (zebrafish) and Drosophila melanogaster (fruit flies) expressed in mammalian sympathetic neurons decreased Ca2+ current density as reported for expression of mammalian RGK proteins. Sequence alignments from evolutionarily diverse organisms spanning the protostome/deuterostome divide revealed conservation of residues within the RGK G-domain involved in RGK protein – Cavβ subunit interaction. In addition, the C-terminal eleven residues were highly conserved and constituted a signature sequence unique to RGK proteins but of unknown function. Taken together, these data suggest that RGK proteins, and the ability to modify Ca2+ channel function, arose from an ancestor predating the protostomes split from deuterostomes approximately 550 million years ago.

BARP suppresses voltage-gated calcium channel activity and Ca2+-evoked exocytosis.

Voltage-gated calcium channels (VGCCs) are key regulators of cell signaling and Ca(2+)-dependent release of neurotransmitters and hormones. Understanding the mechanisms that inactivate VGCCs to prevent intracellular Ca(2+) overload and govern their specific subcellular localization is of critical importance. We report the identification and functional characterization of VGCC β-anchoring and -regulatory protein (BARP), a previously uncharacterized integral membrane glycoprotein expressed in neuroendocrine cells and neurons. BARP interacts via two cytosolic domains (I and II) with all Cavβ subunit isoforms, affecting their subcellular localization and suppressing VGCC activity. Domain I interacts at the α1 interaction domain-binding pocket in Cavβ and interferes with the association between Cavβ and Cavα1. In the absence of domain I binding, BARP can form a ternary complex with Cavα1 and Cavβ via domain II. BARP does not affect cell surface expression of Cavα1 but inhibits Ca(2+) channel activity at the plasma membrane, resulting in the inhibition of Ca(2+)-evoked exocytosis. Thus, BARP can modulate the localization of Cavβ and its association with the Cavα1 subunit to negatively regulate VGCC activity.

Gene splicing of an invertebrate beta subunit (LCavβ) in the N-terminal and HOOK domains and its regulation of LCav1 and LCav2 calcium channels.

The accessory beta subunit (Cavβ) of calcium channels first appear in the same genome as Cav1 L-type calcium channels in single-celled coanoflagellates. The complexity of this relationship expanded in vertebrates to include four different possible Cavβ subunits (β1, β2, β3, β4) which associate with four Cav1 channel isoforms (Cav1.1 to Cav1.4) and three Cav2 channel isoforms (Cav2.1 to Cav2.3). Here we assess the fundamentally-shared features of the Cavβ subunit in an invertebrate model (pond snail Lymnaea stagnalis) that bears only three homologous genes: (LCav1, LCav2, and LCavβ). Invertebrate Cavβ subunits (in flatworms, snails, squid and honeybees) slow the inactivation kinetics of Cav2 channels, and they do so with variable N-termini and lacking the canonical palmitoylation residues of the vertebrate β2a subunit. Alternative splicing of exon 7 of the HOOK domain is a primary determinant of a slow inactivation kinetics imparted by the invertebrate LCavβ subunit. LCavβ will also slow the inactivation kinetics of LCav3 T-type channels, but this is likely not physiologically relevant in vivo. Variable N-termini have little influence on the voltage-dependent inactivation kinetics of differing invertebrate Cavβ subunits, but the expression pattern of N-terminal splice isoforms appears to be highly tissue specific. Molluscan LCavβ subunits have an N-terminal “A” isoform (coded by exons: 1a and 1b) that structurally resembles the muscle specific variant of vertebrate β1a subunit, and has a broad mRNA expression profile in brain, heart, muscle and glands. A more variable “B” N-terminus (exon 2) in the exon position of mammalian β3 and has a more brain-centric mRNA expression pattern. Lastly, we suggest that the facilitation of closed-state inactivation (e.g. observed in Cav2.2 and Cavβ3 subunit combinations) is a specialization in vertebrates, because neither snail subunit (LCav2 nor LCavβ) appears to be compatible with this observed property.

Structural Flexibility of CaV1.2 and CaV2.2 I-II Proximal Linker Fragments in Solution

Voltage-dependent calcium channels (CaV) enable the inward flow of calcium currents for a wide range of cells. CaV1 and CaV2 subtype α1 subunits form the conducting pore using four repeated membrane domains connected by intracellular linkers. The domain I-II linker connects to the membrane gate (IS6), forming an α-helix, and is bound to the CaVβ subunit. Previous studies indicated that this region may or may not form a continuous helix depending on the CaV subtype, thereby modulating channel activation and inactivation properties. Here, we used small-angle x-ray scattering and ensemble modeling analysis to investigate the solution structure of these linkers, extending from the membrane domain and including the CaVβ-binding site, called the proximal linker (PL). The results demonstrate that the CaV1.2 PL is more flexible than the CaV2.2 PL, the flexibility is intrinsic and not dependent on CaVβ binding, and the flexibility can be most easily explained by the presence of conserved glycines. Our analysis also provides a robust example of investigating protein domains in which flexibility plays an essential role.

Characterization of the first honeybee Ca2+ channel subunit reveals two novel species- and splicing-specific modes of regulation of channel inactivation

The honeybee is a model system to study learning and memory, and Ca(2+) signals play a key role in these processes. We have cloned, expressed, and characterized the first honeybee Ca(2+) channel subunit. We identified two splice variants of the Apis CaVβ Ca(2+) channel subunit (Am-CaVβ) and demonstrated expression in muscle and neurons. Although AmCaVβ shares with vertebrate CaVβ subunits the SH3 and GK domains, it beholds a unique N terminus that is alternatively spliced in the first exon to produce a long (a) and short (b) variant. When expressed with the CaV2 channels both, AmCaVβa and AmCaVβb, increase current amplitude, shift the voltage-sensitivity of the channel, and slow channel inactivation as the vertebrate CaVβ2a subunit does. However, as opposed to CaVβ2a, slow inactivation induced by Am-CaVβa was insensitive to palmitoylation but displayed a unique PI3K sensitivity. Inactivation produced by the b variant was PI3K-insensitive but staurosporine/H89-sensitive. Deletion of the first exon suppressed the sensitivity to PI3K inhibitors, staurosporine, or H89. Recording of Ba(2+) currents in Apis neurons or muscle cells evidenced a sensitivity to PI3K inhibitors and H89, suggesting that both AmCaVβ variants may be important to couple cell signaling to Ca(2+) entry in vivo. Functional interactions with phospho-inositide and identification of phosphorylation sites in AmCaVβa and AmCaVβb N termini, respectively, suggest that AmCaVβ splicing promoted two novel and alternative modes of regulation of channel activity with specific signaling pathways. This is the first description of a splicing-dependent kinase switch in the regulation of Ca(2+) channel activity by CaVβ subunit.