CaVβ Subunits Research Articles

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.

Alternative Splicing Generates a Novel Truncated Cav1.2 Channel in Neonatal Rat Heart

L-type Cav1.2 Ca(2+) channel undergoes extensive alternative splicing, generating functionally different channels. Alternatively spliced Cav1.2 Ca(2+) channels have been found to be expressed in a tissue-specific manner or under pathological conditions. To provide a more comprehensive understanding of alternative splicing in Cav1.2 channel, we systematically investigated the splicing patterns in the neonatal and adult rat hearts. The neonatal heart expresses a novel 104-bp exon 33L at the IVS3-4 linker that is generated by the use of an alternative acceptor site. Inclusion of exon 33L causes frameshift and C-terminal truncation. Whole-cell electrophysiological recordings of Cav1.233L channels expressed in HEK 293 cells did not detect any current. However, when co-expressed with wild type Cav1.2 channels, Cav1.233L channels reduced the current density and altered the electrophysiological properties of the wild type Cav1.2 channels. Interestingly, the truncated 3.5-domain Cav1.233L channels also yielded a dominant negative effect on Cav1.3 channels, but not on Cav3.2 channels, suggesting that Cavβ subunits is required for Cav1.233L regulation. A biochemical study provided evidence that Cav1.233L channels enhanced protein degradation of wild type channels via the ubiquitin-proteasome system. Although the physiological significance of the Cav1.233L channels in neonatal heart is still unknown, our report demonstrates the ability of this novel truncated channel to modulate the activity of the functional Cav1.2 channels. Moreover, the human Cav1.2 channel also contains exon 33L that is developmentally regulated in heart. Unexpectedly, human exon 33L has a one-nucleotide insertion that allowed in-frame translation of a full Cav1.2 channel. An electrophysiological study showed that human Cav1.233L channel is a functional channel but conducts Ca(2+) ions at a much lower level.

Solution NMR and calorimetric analysis of Rem2 binding to the Ca2+ channel β4 subunit: a low affinity interaction is required for inhibition of Cav2.1 Ca2+ currents.

Rem, Rad, Kir/Gem (RGK) proteins, including Rem2, mediate profound inhibition of high-voltage activated Ca(2+) channels containing intracellular regulatory β subunits. All RGK proteins bind to voltage-gated Ca(2+) channel β subunit (Cavβ) subunits in vitro, but the necessity of the interaction for current inhibition remains controversial. This study applies NMR and calorimetric techniques to map the binding site for Rem2 on human Cavβ4a and measure its binding affinity. Our experiments revealed 2 binding surfaces on the β4 guanylate kinase domain contributing to a 156 ± 18 µM Kd interaction: a hydrophobic pocket lined by 4 critical residues (L173, N261, H262, and V303), mutation of any of which completely disrupted binding, and a nearby surface containing 3 residues (D206, L209, and D258) that when individually mutated decreased affinity. Voltage-gated Ca(2+) channel α1A subunit (Cav2.1) Ca(2+) currents were completely inhibited by Rem2 when co-expressed with wild-type Cavβ4a, but were unaffected by Rem2 when coexpressed with a Cavβ4a site 1 (L173A/V303A) or site 2 (D258A) mutant. These results provide direct evidence for a low-affinity Rem2/Cavβ4 interaction and show definitively that the interaction is required for Cav2.1 inhibition.

L-Type Ca2+ Channel Cavb Subunits Associate with and Differentially Regulate the Cardiac Cav3.2 T-Type Ca2+ Channel Currents

Low voltage activated T-type calcium channels (TTCC) play a pivotal role in the developing heart. Although the TTCC isoforms, Cav3.1 and Cav3.2, underlie cardiac TTCC current (ICa,T) and are expressed in atrial and ventricular myocytes during development, their expression and roles recede in the adult heart. However, previous studies have demonstrated the re-expression of ICa,T in pathological cardiac hypertrophy, suggesting that TTCCs contribute to the altered Ca2+ cycling and signaling in these pathological conditions. In addition, the reported altered expression of some Cavβ subunits (specifically β1 and β2 subunits) of the high voltage activated L-type calcium channels (LTCC) has been reported in hypertrophy and heart failure. We hypothesize that the altered expression of the Cavβ subunits in cardiac hypertrophy and heart failure results in the altered coupling and regulation of ICa,T. We found an increase in the Cavβ1 and Cavβ3 mRNA and Cavβ2 and Cavβ4 subunit isoform at the protein level in mouse ventricles in a transthoracic aortic constriction (TAC) induced pathological cardiac hypertrophy model. Whole-cell patch clamp electrophysiology using transiently transfected HEK293 cells revealed that co-expression of Cavβ1 or Cavβ2 with Cav3.2 channel isoform resulted in a significant increase in peak ICav3.2 and a rightward shift in the V1/2 of activation and significantly slower inactivation of ICav3.2. On the other hand, co-expression of Cavβ3 or Cavβ4 significantly reduced the peak ICav3.2. In contrast, co-expression of Cavβ isoforms did not alter ICav3.1. Furthermore, co-immunoprecipitation studies in transiently transfected HEK293 cells also demonstrated that the Cav3.2 channel separately co-immunoprecitated with anti-Cavβ1 or anti-Cavβ2 antibody. In conclusion, our data suggest that the Cavβ1 and Cavβ2 subunits of the LTCC may regulate ICav3.2 in cardiomyocytes during pathological cardiac hypertrophy.

Potentiation of high voltage-activated calcium channels by 4-aminopyridine depends on subunit composition.

4-Aminopyridine (4-AP, fampridine) is used clinically to improve neuromuscular function in patients with multiple sclerosis, spinal cord injury, and myasthenia gravis. 4-AP can increase neuromuscular and synaptic transmission by directly stimulating high voltage-activated (HVA) Ca(2+) channels independent of its blocking effect on voltage-activated K(+) channels. Here we provide new evidence that the potentiating effect of 4-AP on HVA Ca(2+) channels depends on the specific combination of voltage-activated calcium channel α1 (Cavα1) and voltage-activated calcium channel β (Cavβ) subunits. Among the four Cavβ subunits examined, Cavβ3 was the most significant subunit involved in the 4-AP-induced potentiation of both L-type and N-type currents. Of particular note, 4-AP at micromolar concentrations selectively potentiated L-type currents reconstituted with Cav1.2, α2δ1, and Cavβ3. In contrast, 4-AP potentiated N-type currents only at much higher concentrations and had little effect on P/Q-type currents. In a phrenic nerve-diaphragm preparation, blocking L-type Ca(2+) channels eliminated the potentiating effect of low concentrations of 4-AP on end-plate potentials. Furthermore, 4-AP enhanced the physical interaction of Cav1.2 and Cav2.2 subunits to Cavβ3 and also increased their trafficking to the plasma membrane. Site-directed mutagenesis identified specific regions in the guanylate kinase, HOOK, and C-terminus domains of the Cavβ3 subunit crucial to the ability of 4-AP to potentiate L-type and N-type currents. Our findings indicate that 4-AP potentiates HVA Ca(2+) channels by enhancing reciprocal Cav1.2-Cavβ3 and Cav2.2-Cavβ3 interactions. The therapeutic effect of 4-AP on neuromuscular function is probably mediated by its actions on Cavβ3-containing L-type Ca(2+) channels.

Inhibition of protein kinase C (PKC) response of voltage-gated calcium (Cav)2.2 channels expressed in Xenopus oocytes by Cavβ subunits

Cav2.2 channels are a substrate for phosphorylation by protein kinase C (PKC) isozymes. The contribution of Cavβ, an auxiliary subunit of these channels, in the PKC modulation was studied. Cav2.2 channels were expressed in Xenopus oocytes in various subunit combinations with or without Cavβ subunits. Currents were recorded using a two-electrode voltage clamp with barium as the charge carrier (IBa). Acetyl-β-methylcholine (MCh), an activator of PKCα, potentiated Cav2.2 currents expressed with Cav2.2α1 alone or Cav2.2α1α2/δ. Similarly PKC isozymes α, βII or ɛ potentiated IBa through Cav2.2α1 subunit channels. In contrast, MCh failed to potentiate currents expressed with Cav2.2α1 and Cavβ1b, β2a, β3 or β4 subunits. Similarly, in the presence of Cavβ1b subunits, PKC isozymes failed to potentiate these currents; contrarily, PKCs α or βII decreased the IBa. MCh failed to potentiate Cav2.2α1 subunit currents in the serine/threonine (Ser/Thr)→alanine mutants, T422A, S1757A or S2132A of Cav2.2α1 subunits. Hence Thr-422, Ser-1757 and Ser-2132 may be PKCα isozyme target sites. The action of PKC on these sites was further substantiated by the increased basal IBa along with the loss of MCh potentiation when Ser/Thr was mutated to aspartate. The observation that MCh or PKC isozymes failed to affect Cav2.2 currents in the presence of Cavβ subunits suggests that these subunits may have interfered with the interaction between PKC and Ser/Thr sites of Cav2.2α1 subunits. In addition to affecting channel expression and current kinetics, Cavβ subunits may also modulate the response of these channels to neurochemicals.

The Cavβ1a subunit regulates gene expression and suppresses myogenin in muscle progenitor cells.

Voltage-gated calcium channel (Cav) β subunits are auxiliary subunits to Cavs. Recent reports show Cavβ subunits may enter the nucleus and suggest a role in transcriptional regulation, but the physiological relevance of this localization remains unclear. We sought to define the nuclear function of Cavβ in muscle progenitor cells (MPCs). We found that Cavβ1a is expressed in proliferating MPCs, before expression of the calcium conducting subunit Cav1.1, and enters the nucleus. Loss of Cavβ1a expression impaired MPC expansion in vitro and in vivo and caused widespread changes in global gene expression, including up-regulation of myogenin. Additionally, we found that Cavβ1a localizes to the promoter region of a number of genes, preferentially at noncanonical (NC) E-box sites. Cavβ1a binds to a region of the Myog promoter containing an NC E-box, suggesting a mechanism for inhibition of myogenin gene expression. This work indicates that Cavβ1a acts as a Cav-independent regulator of gene expression in MPCs, and is required for their normal expansion during myogenic development.

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.

Contribution of protein kinase Cα in the stimulation of insulin by the down-regulation of Cavβ subunits

Voltage-gated calcium (Cav) channels and protein kinase C (PKC) isozymes are involved in insulin secretion. In addition, Cavβ, one of the auxiliary subunits of Cav channels, also regulates the secretion of insulin as knockout of Cavβ3 (β3(-/-)) subunits in mice led to efficient glucose homeostasis and increased insulin levels. We examined whether other types of Cavβ subunits also have similar properties. In this regard, we used small interfering RNA (siRNA) of these subunits (20 μg each) to down-regulate them and examined blood glucose, serum insulin and PKC translocation in isolated pancreatic β cells of mice. While the down-regulation of Cavβ2 and β3 subunits increased serum insulin levels and caused efficient glucose homeostasis, the down-regulation of Cavβ1 and β4 subunits failed to affect both these parameters. Examination of PKC isozymes in the pancreatic β-cells of Cavβ2- or β3 siRNA-injected mice showed that three PKC isozymes, viz., PKC α, βII and θ, translocated to the membrane. This suggests that when present, Cavβ2 and β3 subunits inhibited PKC activation. Among these three isozymes, only PKCα siRNA inhibited insulin and increased glucose concentrations. It is possible that the activation of PKCs βII and θ is not sufficient for the release of insulin and PKCα is the mediator of insulin secretion under the control of Cavβ subunits. Since Cavβ subunits are present intracellularly, it is possible that they (1) inhibited the translocation of PKC isozymes to the membrane and (2) decreased the interaction between Cav channels and PKC isozymes and thus the secretion of insulin.

Three Splice Variants of the Calcium Channel Beta4 Subunit Display Differential Targeting and Gene Regulation in Neurons

The β subunits of voltage-gated calcium channels regulate surface expression and gating properties of CaV1 and CaV2 α1 subunits. All four CaVβ genes are expressed in the brain, but only mutation/lack of β4 causes a neurological phenotype (epilepsy, ataxia) in humans and mice. The β4 isoform is also targeted into the nucleus. There it directly interacts with the epigenetic machinery, suggesting a calcium channel-independent role of β4 in transcriptional regulation. CaVβ subunits are subject to abundant alternative splicing. However, little is known about the specific functions of individual β splice variants in excitable cells. Here we identified a new alternatively spliced β4 transcript, β4e. mRNA and protein of this splice are highly expressed in mouse cerebellum and cultured cerebellar granule cells (CGC). Overexpression of β4e modulates P/Q-type calcium currents in tsA cells and promotes surface expression of native synaptic CaV2.1 channels in hippocampal neurons. Compared to the other two known full-length β4 variants (β4a, β4b) β4e is most abundantly expressed in the distal axon. Consistent with the described role of N-terminal sequences in nuclear import, β4e, which lacks these sequences, does not show nuclear targeting. The importance of nuclear targeting for the putative role of β4 in transcriptional regulation was examined by whole genome expression profiling of CGCs from β4-null mice individually reconstituted with β4a, β4b, or β4e. Strikingly, the capacity of β4 splice variants to regulate neuronal genes depended on their nuclear targeting with a rank order β4b>β4a>β4e. Together these findings indicate that in neurons the three β4 splice variants serve distinct functions. Whereas β4b plays a dual role in channel modulation and gene regulation, the newly detected β4e variant functions primarily as calcium channel subunit. Support: FWF P23479, P24079, W1101, F4406.

Differential Stability of CaVβ2a and CaVβ3 in a CaV1.2 Calcium Channel Complex

In voltage-gated calcium channels, auxiliary CaVβ subunits function in membrane targeting of the channel and modulation of its gating properties. Structural studies have revealed that the high affinity CaVα1/β interaction relies on a conserved CaVβ-binding sequence in the I-II loop of the CaVα1 subunit, called the α-interaction domain (AID). We have previously shown that in skeletal muscle triads the β1a subunit forms stable complexes with both CaV1.1 and CaV1.2, while β2a forms dynamic complexes (Campiglio et al., JCS 2013). To further test the reversibility of the CaV1.2/β2a interaction, here we injected an AID peptide into Xenopus oocytes expressing CaV1.2/β2a/α2δ-1 complexes and examined the effect of the competing peptide on current inactivation. Unexpectedly, the AID peptide had no effect on the currents of wildtype CaV1.2/β2a/α2δ-1 channel complexes. However, when a CaV1.2 AID residue known to decrease the interaction with β2a by 1000-fold was mutated (Y437A), injection of AID peptide changed the inactivation properties of the channels similar to properties of channels expressed without a β subunit. These results suggest that under our experimental conditions binding of the wild-type CaV1.2/β2a pair is too strong for peptide competition. In contrast, in wild-type CaV1.2 channel complexes containing the β3 subunit injection of the AID peptide caused a strong effect on inactivation properties, indicating efficient competition. Consistent with a lower stability of CaV1.2/β3 complexes compared to CaV1.2/β2a, β3 also showed a significantly reduced extent of co-clustering with CaV1.2 in the triads of dysgenic myotubes. Ongoing FRAP experiments will reveal whether diminished co-clustering of β3 correlates with higher mobility and dynamic β subunit exchange in a native calcium channel signaling complex. Funding: FWF P23479 and W1101 (BEF), NIH R01 HL080050 (DLM).

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.

Auxiliary Cavβ subunits of the high voltage L‐type Ca2+ channels modulate the low voltage activated cardiac Cav3.2 T‐type Ca2+ channel currents

Ca2+ signaling and contraction are initiated by the activation of high voltage L‐type Ca2+ channels (LTCC) in cardiomyocytes. However, pathological hypertrophic Ca2+ signaling results from the re‐expression of low voltage T‐type Ca2+ channel (TTCC) current. But the precise regulation of the cardiac TTCC isoforms, Cav3.1 and Cav3.2, in cardiac hypertrophy is unclear. While the Cavβ subunits (isoforms β1, β2, β3 and β4) of the LTCC are required for membrane trafficking and functional regulation of the Cav1.2 subunit of LTCC, auxiliary subunits for the TTCCs have not been identified. We investigated if the β subunits of the LTCC regulate the function of low voltage activated cardiac TTCCs. Co‐immunoprecipitation performed in transiently expressing HEK293 cells or hypertrophic mouse cardiomyocytes demonstrated that the β1, β2 and β3 but not the β4 subunits co‐IP with Cav3.2 channel protein. Whole cell patch clamp analysis in transiently expressed HEK293 cells demonstrate that β1 and β2 subunits caused a significant increase in ICav3.2 density but not ICav3.1. In contrast the β3 and β4 did not alter the ICav3.2 or ICav3.1 density. In addition, the co‐expression of β1 caused a rightward shift in the V1/2 of ICav3.2 activation. We also found that the expression of the β1 and β2 subunits was increased in ventricular myocytes in mouse model of pressure overload induced cardiac hypertrophy. We conclude that an increased expression of the β1 and β2 subunits may regulate the ICav3.2 and contribute to the altered Ca2+ signaling in pathological cardiac hypertrophy. Research support NHLBI R01HL105713 to RCB.

Electrostatic Association of Beta-Subunits to Membrane Reduces the PIP2 Sensitivity of Ca2+ Channels

High voltage-gated Ca2+ (CaV) channels are protein complexes mostly containing α1, β and α2δ subunits. G protein-coupled receptors modulate these channels through diverse pathways. Recently, membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) was identified as a key regulator of these channels and sensitivity to PIP2 exclusively dependent on CaV β-subunits. using mouse β2a (mβ2a) subunit, we report that simple binding of this subunit to the membrane surface is sufficient for reducing PIP2 sensitivity. Compared to rat β2a (rb2a) subunits which are distributed on the plasma membrane following post-translational palmitoylation, mβ2a displayed the same distribution without any lipid modification, merely through its own N-terminus. Multi-alignment analysis between rβ2a and mβ2a revealed 98% sequence homology with main differences in N-terminus. To characterize the contribution of the N-terminus to mβ2a membrane targeting, the 23 amino acids of this region was sequentially deleted and membrane targeting and current properties of these mutants were measured. The results demonstrate the last basic amino acid, K2 residue to be critical for membrane distribution of β subunits and slow inactivation of Cav current. We also found W5 to be important for the membrane targeting of mβ2a subunit. Finally, binding of mβ2a subunit was diminished upon receptor stimulation, suggesting the involvement of electrostatic interaction during binding of mβ2a subunit to lipids. Consequently, we propose that the N-terminus of mβ2a, without lipidation, still plays a key role in membrane targeting and this interaction determines the low sensitivity of CaV channels to PIP2 depletion. Taken together, the results validate that lipidation of β subunit is not a critical factor for the reduction of CaV regulation by PIP2.

The β3 Subunit Contributes to Vascular Calcium Channel Upregulation and Hypertension in Angiotensin II–Infused C57BL/6 Mice

Voltage-gated L-type Ca(2+) (Ca(v)1.2) channels in vascular smooth muscle cells are a predominant Ca(2+) influx pathway that mediates arterial tone. Channel biogenesis is accomplished when the pore-forming α(1C) subunit coassembles with regulatory Ca(v)β subunits intracellularly, and the multiprotein Ca(v)1.2 channel complex translocates to the plasma membrane to form functional Ca(2+) channels. We hypothesized that the main Ca(v)β isoform in vascular smooth muscle cells, Ca(v)β3, is required for the upregulation of arterial Ca(v)1.2 channels during the development of hypertension, an event associated with abnormal Ca(2+)-dependent tone. Ca(v)1.2 channel expression and function were compared between second-order mesenteric arteries of C57BL/6 wild-type (WT) and Ca(v)β3(-/-) mice infused with saline (control) or angiotensin II (Ang II) for 2 weeks to induce hypertension. The mesenteric arteries of Ang II-infused WT mice showed increased Ca(v)1.2 channel expression and accentuated Ca(2+)-mediated contractions compared with saline-infused WT mice. In contrast, Ca(v)1.2 channels failed to upregulate in mesenteric arteries of Ang II-infused Ca(v)β3(-/-) mice, and Ca(2+)-dependent reactivity was normal in these arteries. Basal systolic blood pressure was not significantly different between WT and Ca(v)β3(-/-) mice (98 ± 2 and 102 ± 3 mm Hg, respectively), but the Ca(v)β3(-/-) mice showed a blunted pressor response to Ang II infusion. Two weeks after the start of Ang II administration, the systolic blood pressure of Ca(v)β3(-/-) mice averaged 149 ± 4 mm Hg compared with 180 ± 5 mm Hg in WT mice. Thus, the Ca(v)β3 subunit is a critical regulatory protein required to upregulate arterial Ca(v)1.2 channels and fully develop Ang II-dependent hypertension in C57BL/6 mice.

Up-regulation of Cavβ3 Subunit in Primary Sensory Neurons Increases Voltage-activated Ca2+ Channel Activity and Nociceptive Input in Neuropathic Pain

High voltage-activated calcium channels (HVACCs) are essential for synaptic and nociceptive transmission. Although blocking HVACCs can effectively reduce pain, this treatment strategy is associated with intolerable adverse effects. Neuronal HVACCs are typically composed of α(1), β (Cavβ), and α(2)δ subunits. The Cavβ subunit plays a crucial role in the membrane expression and gating properties of the pore-forming α(1) subunit. However, little is known about how nerve injury affects the expression and function of Cavβ subunits in primary sensory neurons. In this study, we found that Cavβ(3) and Cavβ(4) are the most prominent subtypes expressed in the rat dorsal root ganglion (DRG) and dorsal spinal cord. Spinal nerve ligation (SNL) in rats significantly increased mRNA and protein levels of the Cavβ(3), but not Cavβ(4), subunit in the DRG. SNL also significantly increased HVACC currents in small DRG neurons and monosynaptic excitatory postsynaptic currents of spinal dorsal horn neurons evoked from the dorsal root. Intrathecal injection of Cavβ(3)-specific siRNA significantly reduced HVACC currents in small DRG neurons and the amplitude of monosynaptic excitatory postsynaptic currents of dorsal horn neurons in SNL rats. Furthermore, intrathecal treatment with Cavβ(3)-specific siRNA normalized mechanical hyperalgesia and tactile allodynia caused by SNL but had no significant effect on the normal nociceptive threshold. Our findings provide novel evidence that increased expression of the Cavβ(3) subunit augments HVACC activity in primary sensory neurons and nociceptive input to dorsal horn neurons in neuropathic pain. Targeting the Cavβ(3) subunit at the spinal level represents an effective strategy for treating neuropathic pain.

The Cavβ1 Subunit Regulates Gene Expression in Muscle Progenitor Cells

CaVβ subunits are traditionally considered constituents of CaV complexes (CaV1or2, CaVβ, and CaV α2/δ), where they localize at the plasma membrane and serve to regulate channel expression and gating properties. Recent publications also show CaVβ subunit localization in the nucleus. This phenomenon has been observed under a variety of conditions (different cell types, β subunit isoforms, co-expressed proteins, etc). However, the mechanisms responsible for CaVβ subunit nuclear shuttling, as well as a physiological role for this nuclear localization, remain major questions. Ongoing work in our laboratory has shown that muscle progenitor cells (myoblasts) express CaVβ1 protein (but not CaV1 subunits) in both the cytoplasm and nucleus and that the loss of CaVβ1 expression impairs proliferation in these cells. To better understand the mechanisms that link CaVβ1 nuclear localization with control of proliferation, we have conducted large-scale screening experiments designed to identify which genes are directly regulated by CaVβ1, as well as its protein binding partners. To test if CaVβ1 may regulate gene expression, we conducted microarray experiments on RNA extracted from wild type, heterozygous, and CaVβ1 -null mouse primary myoblasts. A number of gene transcripts were found to be differentially regulated based on the relative amount of CaVβ1 expression. To identify specific CaVβ1 target genes, we performed chromatin immunoprecipitation -on-a-chip experiments to locate which promoter regions CaVβ1 bound to across the entire mouse genome. Nuclear binding partners of CaVβ1 were screened using affinity purification of CaVβ1a-YFP from myoblast nuclear fractions coupled with mass spectrometry. Finally, the importance of CaVβ1 in embryonic myogenesis was explored in CaVβ1 -null mice. Our results support the idea of CaVβ subunits acting as transcription factors and regulating gene expression independently from CaV's, and suggest these functions may be particularly important to progenitor cell growth.

L-type calcium channel β subunit modulates angiotensin II responses in cardiomyocytes

Angiotensin II regulation of L-type calcium currents in cardiac muscle is controversial and the underlying signaling events are not completely understood. Moreover, the possible role of auxiliary subunit composition of the channels in Angiotensin II modulation of L-type calcium channels has not yet been explored. In this work we study the role of Cavβ subunits and the intracellular signaling responsible for L-type calcium current modulation by Angiotensin II. In cardiomyocytes, Angiotensin II exposure induces rapid inhibition of L-type current with a magnitude that is correlated with the rate of current inactivation. Semi-quantitative PCR of cardiomyocytes at different days of culture reveals changes in the Cavβ subunits expression pattern that are correlated with the rate of current inactivation and with Angiotensin II effect. Over-expression of individual b subunits in heterologous systems reveals that the magnitude of Angiotensin II inhibition is dependent on the Cavβ subunit isoform, with Cavβ1bcontaining channels being more strongly regulated. Cavβ2acontaining channels were insensitive to modulation and this effect was partially due to the N-terminal palmitoylation sites of this subunit. Moreover, PLC or diacylglycerol lipase inhibition prevents the Angiotensin II effect on L-type calcium channels, while PKC inhibition with chelerythrine does not, suggesting a role of arachidonic acid in this process. Finally, we show that in intact cardiomyocytes the magnitude of calcium transients on spontaneous beating cells is modulated by Angiotensin II in a Cavβ subunit-dependent manner. These data demonstrate that Cavβ subunits alter the magnitude of inhibition of L-type current by Angiotensin II.

Ca Vβ 1a Nuclear Translocation: Potential Mechanisms and Functions

CaVβ1a Nuclear Translocation: Potential Mechanisms and Functions CaVβ subunits are traditionally considered constituents of CaV complexes (CaV1or2, CaVβ, CaV α2/δ), where they localize at the plasma membrane and serve to regulate channel expression and gating properties. Several recent publications also show CaVβ subunit localization in the nucleus. This phenomenon has been observed under a variety of conditions (different cell types, β subunit isoforms, co-expressed proteins, etc). However, the exact mechanisms responsible for CaVβ subunit nuclear shuttling, as well as a physiological role for this nuclear localization, remain major questions. In the present work, we use primary mouse myoblasts as a model to study CaVβ subunit nuclear localization. These mitotic cells express CaVβ1a protein, despite the absence of any CaV1 subunits, and thus provide an ideal model to study non-CaV- related function (i.e. nuclear) of CaVβ subunits. Using both immunofluorescence and subcellular fractionation, we show that endogenous and exogenous CaVβ1a proteins are rapidly translocated into the nucleus of myoblasts. We also use shRNA knockdown and transgenic CaVβ1-null myoblasts to examine the importance of CaVβ1 in gene expression and cell proliferation. A mouse model of partial CaVβ1 knockdown is used to determine the role of CaVβ1a in skeletal muscle regeneration following injury in vivo. Finally, we attempt to identify nuclear binding partners of CaVβ1a-YFP using affinity purification from nuclear fractions coupled with mass spectrometry. Our results support the idea of CaVβ subunits having alternate functions separate from CaV's, within the nucleus, and suggest these functions may be specific to progenitor cells.

Regulation of Voltage-Gated Calcium Channel Activity by Palmitoylation: A Fatty Acid Tale

The phospholipid phosphatidylinositol 4,5-bisphospate (PIP2) interacts with voltage-gated Ca2+ channels to facilitate their opening. Conversely, inhibition of channel activity following activation of Gq-protein coupled receptors is associated with PIP2 breakdown. PIP2's freed fatty acid tails appear to remain associated with channels, stabilizing closed conformations. My lab is testing a model where an accessory β-subunit CaVβ2a acts as a phospholipid mimic resistant to metabolism; its two palmitoyl groups bind to the channel at a site normally occupied by PIP2's two fatty acid tails. To test this model, we have concentrated on examining N-type Ca2+ channel modulation in a recombinant system where CaV2.2 is coexpressed with α2δ-1 and one of four CaVβ subunits. M1 muscarinic or neurokinin-1 receptor stimulation inhibits N-current from CaVβ1b-, CaVβ3-, or CaVβ4-containing channels, but enhances N-current from CaVβ2a-containing channels. Exogenously applied arachidonic acid produces the same pattern of modulation. Further studies with mutated, chimeric CaVβ subunits and free palmitic acid revealed palmitoylation of CaVβ2a is essential for loss of inhibition. Loss of inhibition appears independent of kinetic changes that occur with different channel complexes. In contrast, channel mutations that reorient CaVβ2a's relative position to CaV2.2 rescue inhibition suggesting that in these experiments, the palmitoyl groups become sufficiently displaced that endogenously released arachidonic acid can once again bind to the inhibitory site. These findings suggest a new dynamic function for palmitoylation and predict that other doubly palmitoylated proteins reach up into the membrane with their lipid fingers to interact with and change the functioning of transmembrane proteins.(Funded by the NIH, AHA and the UMass Medical School).