Calcium Channel Beta Subunit Research Articles

Small-molecule CaVα1⋅CaVβ antagonist suppresses neuronal voltage-gated calcium-channel trafficking

Extracellular calcium flow through neuronal voltage-gated CaV2.2 calcium channels converts action potential-encoded information to the release of pronociceptive neurotransmitters in the dorsal horn of the spinal cord, culminating in excitation of the postsynaptic central nociceptive neurons. The CaV2.2 channel is composed of a pore-forming α1 subunit (CaVα1) that is engaged in protein-protein interactions with auxiliary α2/δ and β subunits. The high-affinity CaV2.2α1⋅CaVβ3 protein-protein interaction is essential for proper trafficking of CaV2.2 channels to the plasma membrane. Here, structure-based computational screening led to small molecules that disrupt the CaV2.2α1⋅CaVβ3 protein-protein interaction. The binding mode of these compounds reveals that three substituents closely mimic the side chains of hot-spot residues located on the α-helix of CaV2.2α1 Site-directed mutagenesis confirmed the critical nature of a salt-bridge interaction between the compounds and CaVβ3 Arg-307. In cells, compounds decreased trafficking of CaV2.2 channels to the plasma membrane and modulated the functions of the channel. In a rodent neuropathic pain model, the compounds suppressed pain responses. Small-molecule α-helical mimetics targeting ion channel protein-protein interactions may represent a strategy for developing nonopioid analgesia and for treatment of other neurological disorders associated with calcium-channel trafficking.

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.

Troponin T3 Regulates Calcium Channel Beta1a Subunit Nuclear Translocation in Skeletal Muscle

Voltage-dependent calcium channel (VDCC) beta subunits (CaVβ) are critical for CaVα subunit membrane expression and gating properties. While CaVβ interacts with a variety of other molecular partners, nonchannel functions of skeletal muscle CaVβ1a have not been reported. In a yeast two hybrid (Y2H) screening of the mouse tibialis anterior (TA) muscle cDNA library, troponin T3 (TNNT3), specific to fast skeletal muscle, was identified using full-length CaVβ1a as the bait. Further studies revealed that TNNT3, but not the slow skeletal muscle TNNT1, co-immunoprecipitated with CaVβ1a in the mouse skeletal muscle cell line C2C12 and mouse skeletal muscle. Consistently, both CaVβ1a and TNNT3 were highly expressed in mouse fast, but not slow, skeletal muscle. Surprisingly, when DsRed-tagged TNNT3 and YFP tagged CaVβ1a (CaVβ1a -YFP) were transiently co-expressed in C2C12 cells or the mouse flexor digitorum brevis (FDB) muscle, both showed a punctuate distribution pattern in the cytoplasm and nucleus. In comparison, CaVβ1a-YFP alone localized uniformly in the cytoplasm. Further truncation analysis revealed that TNNT3 C-terminus (aa 161-244) localized exclusively in FDB nuclei and enriched CaVβ1a -YFP accordingly. In contrast, the TNNT3 middle region (aa 55-160) localized only in the cytoplasm, while the N-terminus (aa 1-54) was found in both cytoplasm and nucleus. Note that the N-terminus could also recruit CaVβ1a-YFP in the nucleus. Y2H assay verified that the TNNT3 C-terminus has the strongest interaction with CaVβ1a. Immunoblotting with an antibody targeting the TNNT3 N-terminus detected an increased TNNT3 fragment in the aging mouse skeletal muscle. We conclude that TNNT3 interacts with CaVβ1a and regulates its nuclear translocation mainly through its TNNT3 C- or N-terminus. These findings strongly support a transcription-regulation function for CaVβ1a and TNNT3.

Identification of ion channel genes in the Acyrthosiphon pisum genome.

Aphids are major pests of crops, causing hundreds of millions of dollars worth of damage annually. Ion channel proteins are often the targets of modern insecticides and mutations in ion channel genes can lead to resistance to many leading classes of insecticides. The sequencing of the pea aphid, Acyrthosiphon pisum, genome has now allowed detailed in silico analysis of the aphid ion channels. The study has revealed significant differences in the composition of the ion channel families between the aphid and other insects. For example A. pisum does not appear to contain a homologue of the nACh receptor alpha 5 gene whilst the calcium channel beta subunit has been duplicated. These variations could result in differences in function or sensitivity to insecticides. The genome sequence will allow the study of aphid ion channels to be accelerated, leading to a better understanding of the function of these economically important channels. The potential for identifying novel insecticide targets within the aphid is now a step closer.

Rem2 Redistributes in Response to Neuronal Stimulation

Rem2 is a small GTP-binding protein of the RGK family. It is targeted to the cell membrane where it interacts with the beta subunit of calcium channels and abolishes or reduces endogenous or exogenous calcium currents, and also has known interactions with calmodulin and 14-3-3. Rem2 is unique in the RGK family, being found predominantly in the brain and upregulated in response to stimulation. Knockdown of Rem2 in neuronal cultures results in fewer glutamatergic synapses. We have found that fluorescent-labeled Rem2 changes its subcellular localization in neurons from a diffuse to a punctuate distribution after neuronal stimulation or after activation of NMDA receptors. This rearrangement is calcium dependent and involves the C-terminal 30 residues, suggesting the presence of a self-association domain as well as an autoinhibitory domain that keeps Rem2 diffusely distributed until stimulation. A calmodulin-binding deficient mutant shows very little rearrangement upon stimulation, supporting a role for calcium in this phenomenon.

Heart field reduction in embryos depleted for voltage-gated calcium channel beta subunit CACNB2

Heart field reduction in embryos depleted for voltage-gated calcium channel beta subunit CACNB2

Determinants of the voltage dependence of G protein modulation within calcium channel beta subunits.

CaVβ subunits of voltage-gated calcium channels contain two conserved domains, a src-homology-3 (SH3) domain and a guanylate kinase-like (GK) domain with an intervening HOOK domain. We have shown in a previous study that, although Gβγ-mediated inhibitory modulation of CaV2.2 channels did not require the interaction of a CaVβ subunit with the CaVα1 subunit, when such interaction was prevented by a mutation in the α1 subunit, G protein modulation could not be removed by a large depolarization and showed voltage-independent properties (Leroy et al., J Neurosci 25:6984–6996, 2005). In this study, we have investigated the ability of mutant and truncated CaVβ subunits to support voltage-dependent G protein modulation in order to determine the minimal domain of the CaVβ subunit that is required for this process. We have coexpressed the CaVβ subunit constructs with CaV2.2 and α2δ-2, studied modulation by the activation of the dopamine D2 receptor, and also examined basal tonic modulation. Our main finding is that the CaVβ subunit GK domains, from either β1b or β2, are sufficient to restore voltage dependence to G protein modulation. We also found that the removal of the variable HOOK region from β2a promotes tonic voltage-dependent G protein modulation. We propose that the absence of the HOOK region enhances Gβγ binding affinity, leading to greater tonic modulation by basal levels of Gβγ. This tonic modulation requires the presence of an SH3 domain, as tonic modulation is not supported by any of the CaVβ subunit GK domains alone.

Cloning, characterization and expression analysis of calcium channel β subunit from pearl oyster (Pinctada fucata)

The absorption, transport and localization of calcium underlie the basis of biomineralization, and Ca(2+) entry into epithelial cell is the primary step in shell formation. However, the related mechanism of Ca(2+) transport is poorly documented at the gene or protein level. L-type voltage-dependent calcium channels may be involved in calcium transport for biomineralization in some marine invertebrates. In this study, a full-length cDNA of a voltage-dependent calcium channel beta subunit from Pinctada fucata (PCabeta) was cloned, and its amino acid sequence was deduced. PCabeta shared 51%-67% apparently sequence identity with voltage-dependent calcium channel beta subunits from other species. However, PCabeta was much shorter than other voltage-dependent calcium channel beta subunits particularly at the carboxyl terminus, indicating that it is likely a truncated beta subunit isoform. Semi-quantitative RT-PCR analysis showed that PCabeta was expressed in all the tested tissues and that it had a higher expression level in the gill tissue and hemolymph than in other tissues, suggesting that L-type voltage-dependent calcium channels are responsible for Ca(2+) absorption in the gill and Ca(2+) entry into hemocytes. In the mantle, PCabeta mRNA was predominantly expressed in the inner and middle folds of the mantle epithelium, suggesting that L-type voltage-dependent calcium channels are involved in Ca(2+) absorption from the ambient medium in the mantle. All these results suggest that voltage-dependent calcium channels are involved in Ca(2+) uptake and transport during oyster biomineralization.

New roles for voltage gated calcium channel beta subunits in zebrafish

New roles for voltage gated calcium channel beta subunits in zebrafish

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The importance of occupancy rather than affinity of CaV(beta) subunits for the calcium channel I-II linker in relation to calcium channel function.

The Ca(V)beta subunits of voltage-gated calcium channels regulate the trafficking and biophysical properties of these channels. We have taken advantage of mutations in the tyrosine residue within the alpha interaction domain (AID) in the I-II linker of Ca(V)2.2 which reduce, but do not abolish, the binding of beta1b to the AID of Ca(V)2.2. We have found that the mutation Y388S decreased the affinity of Ca(V)beta1b binding to the Ca(V)2.2 I-II linker from 14 to 329 nm. However, the Y388S mutation had no effect on current density and cell surface expression of Ca(V)2.2/alpha2delta-2/beta1b channels expressed in human embryonic kidney tsA-201 cells, when equivalent proportions of cDNA were used. Furthermore, despite the 24-fold reduced affinity of Ca(V)beta1b for the Y388S I-II linker of Ca(V)2.2, all the key features of modulation as well as trafficking by Ca(V)beta subunits remained intact. This is in contrast to the much more marked effect of the W391A mutation, which abolished interaction with the Ca(V)2.2 I-II linker, and very markedly affected the trafficking of the channels. However, using the Xenopus oocyte expression system, where expression levels can be accurately titrated, when Ca(V)beta1b cDNA was diluted 50-fold, all evidence of interaction with Ca(V)2.2 Y388S was lost, although wild-type Ca(V)2.2 was still normally modulated by the reduced concentration of beta1b. These results indicate that high affinity interaction with the alpha1 subunit is not necessary for any of the modulatory effects of Ca(V)beta subunits, but occupancy of the interaction site is important, and this will occur, despite the reduced affinity, if the Ca(V)beta subunit is present in sufficient excess.

A single CaVβ can reconstitute both trafficking and macroscopic conductance of voltage‐dependent calcium channels

Voltage-dependent calcium-channel beta subunits (Ca(V)beta) strongly modulate pore-forming alpha(1) subunits by trafficking channel complexes to the plasma membrane and enhancing channel open probability (P(o)). Despite their central role, it is unclear whether binding of a single Ca(V)beta, or multiple Ca(V)betas, to an alpha(1) subunit governs the two distinct functions. Conventional experiments utilizing coexpression of alpha(1) and Ca(V)beta subunits have been unable to resolve the ambiguity due to difficulties in establishing their stoichiometry in functional channels. Here, we unambiguously establish a 1: 1 stoichiometry by covalently linking Ca(V)beta(2b) to the carboxyl terminus of alpha(1C) (Ca(V)1.2), creating alpha(1C).beta(2b). Recombinant L-type channels reconstituted in HEK 293 cells with alpha(1C).beta(2b) supported whole-cell currents to the same extent as channels reconstituted via coexpression of the individual subunits. Analysis of gating charge showed alpha(1C).beta(2b) fully restored channel trafficking to the plasma membrane. Co-transfecting Ca(V)beta(2a) with alpha(1C).beta(2b) had little further impact on function. To rule out the possibility that fused Ca(V)beta(2b) was interacting in trans with neighbouring alpha(1) molecules, alpha(1C).beta(2b) was cotransfected with alpha(1B) (Ca(V)2.2), and pharmacological block with nimodipine showed an absence of alpha(1B) trafficking. These results establish that association of a single Ca(V)beta with a pore-forming alpha(1) subunit captures the functional essence of HVA calcium channels, and introduce alpha(1)-Ca(V)beta fusion proteins as a powerful new tool to probe structure-function mechanisms.

Uncoupling of Calcium Channel α1 and β Subunits in Developing Neurons

Calcium channel beta subunits are key modulators of calcium channel function and membrane targeting of the pore-forming alpha1 subunit. Here we show that an invertebrate (Lymnaea stagnalis) homolog of P/Q- and N-type calcium channels (LCav2), although colocalized with beta subunits in synapses of mature neurons, is physically uncoupled from the beta subunits in the leading edge of growth cones of outgrowing neurons. Moreover, LCav2 channels that mediate transmitter release in mature synapses also participate in neuronal outgrowth in growth cones. The differential association of beta subunits with synaptic calcium channels and those expressed in emergent neuronal growth suggests that beta subunits may play a role in the transformation of Cav2 calcium channel function in immature neurons and mature synapses.

Expression, purification and crystallization of a functional core of the voltage-dependent calcium channel beta subunit.

Two versions of the functional core of the rabbit voltage-dependent calcium channel beta2a subunit were expressed in Escherichia coli. These proteins were purified to homogeneity and screened for crystallization. Crystallization conditions were refined using the hanging-drop vapour-diffusion method and two crystal forms were pursued. Crystal form I is represented by thick rods with tetragonal symmetry, unit-cell parameters a = b = 75, c = 165 A and a diffraction limit of 3.4 A which were obtained using ammonium sulfate as a precipitant. Crystal form II gives rise to plates with orthorhombic symmetry, unit-cell parameters a = 35, b = 75, c = 165 A and a diffraction limit of 2.3 A which were grown using polyethylene glycol 20K as a precipitant.

Calcium Current in Rat Cardiomyocytes Is Modulated by the Carboxyl-terminal Ahnak Domain

Ahnak, a protein of 5643 amino acids, interacts with the regulatory beta-subunit of cardiac calcium channels and with F-actin. Recently, we defined the binding sites among the protein partners in the carboxyl-terminal domain of ahnak. Here we further narrowed down the beta(2)-interaction sites to the carboxyl-terminal 188 amino acids of ahnak by the recombinant ahnak protein fragments P3 (amino acids 5456-5556) and P4 (amino acids 5556-5643). The effects of these P3 and P4 fragments on the calcium current were investigated under whole-cell patch clamp conditions on rat ventricular cardiomyocytes. P4 but not P3 increased significantly the current amplitude by 22.7 +/- 5% without affecting its voltage dependence. The slow component of calcium current inactivation was slowed down by both P3 and P4, whereas only P3 slowed significantly the fast one. The composite recombinant protein fragment P3-P4 induced similar modifications to the ones induced by each of the ahnak fragments. In the presence of carboxyl-terminal ahnak protein fragments, isoprenaline induced a similar relative increase in current amplitude and shift in current kinetics. The actin-stabilizing agents, phalloidin and jasplakinolide, did not modify the effects of these ahnak protein fragments on calcium current in control conditions nor in the presence of isoprenaline. Hence, our results suggest that the functional effects of P3, P4, and P3-P4 on calcium current are mediated by targeting the ahnak-beta(2)-subunit interaction rather than by targeting the ahnak-F-actin interaction. More specifically they suggest that binding of the beta(2)-subunit to the endogenous subsarcolemmal giant ahnak protein re-primes the alpha(1C)/beta(2)-subunit interaction and that the ahnak-derived proteins relieve the beta(2)-subunit from this inhibition.

Several Structural Domains Contribute to the Regulation of N-type Calcium Channel Inactivation by the β3 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.

Functional roles of cytoplasmic loops and pore lining transmembrane helices in the voltage-dependent inactivation of HVA calcium channels.

Voltage-dependent inactivation of calcium channels is a key mechanism for regulating intracellular calcium levels and neuronal excitability. In sodium and potassium channels, the molecular determinants that govern fast inactivation involve pore block by a cytoplasmic gating particle. As we discuss here, there is an increasing body of evidence that is consistent with a qualitatively similar inactivation mechanism in high-voltage-activated calcium channels. Work from a number of laboratories has implicated both cytoplasmic regions and the pore-lining S6 transmembrane helices in the inactivation process. Together with our recent findings, this leads us to propose a model in which the intracellular domain I-II linker region acts as a 'hinged lid' that physically occludes the pore by docking to the cytoplasmic ends of the S6 segments. We further propose that the ancillary calcium channel Beta subunits differentially modulate inactivation kinetics by binding to and thereby regulating the mobility of the putative inactivation gate. Indeed, additional evidence suggests that the carboxy terminus, amino terminus and domain III-IV linker regions of the channel modulate inactivation rates through interactions with the I-II linker per se, or indirectly via the ancillary Beta subunits. Taken together, the fast voltage-dependent inactivation of calcium channels appears reminiscent of that of sodium channels, but appears to show a more complex regulation through intramolecular interactions between the putative inactivation gate and other cytoplasmic regions.

G protein modulation of voltage-gated calcium channels.

Calcium influx into any cell requires fine tuning to guarantee the correct balance between activation of calcium-dependent processes, such as muscle contraction and neurotransmitter release, and calcium-induced cell damage. G protein-coupled receptors play a critical role in negative feedback to modulate the activity of the CaV2 subfamily of the voltage-dependent calcium channels, which are largely situated on neuronal and neuro-endocrine cells. The basis for the specificity of the relationships among membrane receptors, G proteins, and effector calcium channels will be discussed, as well as the mechanism by which G protein-mediated inhibition is thought to occur. The inhibition requires free G beta gamma dimers, and the cytoplasmic linker between domains I and II of the CaV2 alpha 1 subunits binds G beta gamma dimers, whereas the intracellular N terminus of CaV2 alpha 1 subunits provides essential determinants for G protein modulation. Evidence suggests a key role for the beta 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.

Beta subunits of voltage-gated calcium channels.

Calcium channel beta subunits have marked effects on the trafficking and on several of the biophysical properties of all high voltage activated calcium channels. In this article I shall review information on the different genes, on the structure of the beta subunits, and on their differential expression and post-translational modification. Their role in trafficking and assembly of the calcium channel heteromultimer will be described, and I will then review their effects on voltage-dependent and kinetic properties, stressing the differences between palmitoylated beta2a and the other beta subunits. Evidence for effects on calcium channel pharmacology will also be examined. I shall discuss the hypothesis that beta subunits can bind reversibly to calcium channels, and examine their role in the G protein modulation of calcium channels. Finally, I shall describe the consequences of knock-out of different beta subunit genes, and describe evidence for the involvement of beta subunits in disease.

Modified Cardiovascular L-type Channels in Mice Lacking the Voltage-dependent Ca2+ Channel β3 Subunit

The beta subunits of voltage-dependent calcium channels are known to modify calcium channel currents through pore-forming alpha1 subunits. Of the four beta subunits reported to date, the beta3 subunit is highly expressed in smooth muscle cells and is thought to consist of L-type calcium channels. To determine the role of the beta3 subunit in the voltage-dependent calcium channels of the cardiovascular system in situ, we performed a series of experiments in beta3-null mice. Western blot analysis indicated a significant reduction in expression of the alpha1 subunit in the plasma membrane of beta3-null mice. Dihydropyridine binding experiments also revealed a significant decrease in the calcium channel population in the aorta. Electrophysiological analyses indicated a 30% reduction in Ca2+ channel current density, a slower inactivation rate, and a decreased dihydropyridine-sensitive current in beta3-null mice. The reductions in the peak current density and inactivation rate were reproduced in vitro by co-expression of the calcium channel subunits in Chinese hamster ovary cells. Despite the reduced channel population, beta3-null mice showed normal blood pressure, whereas a significant reduction in dihydropyridine responsiveness was observed. A high salt diet significantly elevated blood pressure only in the beta3-null mice and resulted in hypertrophic changes in the aortic smooth muscle layer and cardiac enlargement. In conclusion, this study demonstrates the involvement and importance of the beta3 subunit of voltage-dependent calcium channels in the cardiovascular system and in regulating channel populations and channel properties in vascular smooth muscle cells.