Human Cav3 Research Articles

Large-Scale Functional RNAi Screen in C. elegans Identifies TGF-β and Notch Signaling Pathways as Modifiers of CACNA1A.

Variants in CACNA1A that encodes the pore-forming α1-subunit of human voltage-gated Cav2.1 (P/Q-type) Ca2+ channels cause several autosomal-dominant neurologic disorders, including familial hemiplegic migraine type 1, episodic ataxia type 2, and spinocerebellar ataxia type 6. To identify modifiers of incoordination in movement disorders, we performed a large-scale functional RNAi screen, using the Caenorhabditis elegans strain CB55, which carries a truncating mutation in the unc-2 gene, the worm ortholog for the human CACNA1A. The screen was carried out by the feeding method in 96-well liquid culture format, using the ORFeome v1.1 feeding library, and time-lapse imaging of worms in liquid culture was used to assess changes in thrashing behavior. We looked for genes that, when silenced, either ameliorated the slow and uncoordinated phenotype of unc-2, or interacted to produce a more severe phenotype. Of the 350 putative hits from the primary screen, 37 genes consistently showed reproducible results. At least 75% of these are specifically expressed in the C. elegans neurons. Functional network analysis and gene ontology revealed overrepresentation of genes involved in development, growth, locomotion, signal transduction, and vesicle-mediated transport. We have expanded the functional network of genes involved in neurodegeneration leading to cerebellar ataxia related to unc-2/CACNA1A, further confirming the involvement of the transforming growth factor β pathway and adding a novel signaling cascade, the Notch pathway.

Modulation of Cav3.2 T-type calcium channel permeability by asparagine-linked glycosylation

abstractLow-voltage-gated T-type calcium channels are expressed throughout the nervous system where they play an essential role in shaping neuronal excitability. Defects in T-type channel expression have been linked to various neuronal disorders including neuropathic pain and epilepsy. Currently, little is known about the cellular mechanisms controlling the expression and function of T-type channels. Asparagine-linked glycosylation has recently emerged as an essential signaling pathway by which the cellular environment can control expression of T-type channels. However, the role of N-glycans in the conducting function of T-type channels remains elusive. In the present study, we used human Cav3.2 glycosylation-deficient channels to assess the role of N-glycosylation on the gating of the channel. Patch-clamp recordings of gating currents revealed that N-glycans attached to hCav3.2 channels have a minimal effect on the functioning of the channel voltage-sensor. In contrast, N-glycosylation on specific asparagine residues may have an essential role in the conducting function of the channel by enhancing the channel permeability and / or the pore opening of the channel. Our data suggest that modulation of N-linked glycosylation of hCav3.2 channels may play an important physiological role, and could also support the alteration of T-type currents observed in disease states.

Regulation of Voltage Sensing Structures of CaV1.2 Calcium Channels by the Auxiliary β-Subunit (β3)

High Voltage-Activated channels (HVA) are composed of four non-identical repeats with distinct voltage-sensing domain (VSD). The cardiac HVA CaV1.2 is modulated by at least two auxiliary subunits, α2δ and β. The β-subunit is known to facilitate channel opening without altering gating currents or single channel conductance. By tracking the movement of individual voltage sensors in human CaV1.2 channels using voltage clamp fluorometry we learned that individual voltage-sensors contribute to channel opening and gating currents to a different degree (Pantazis et al. 2014, PNAS). Taking advantage that rabbit CaV1.2 can be expressed without auxiliary subunits, we explored the impact that β subunits exert on voltage-sensing or coupling and found that β3 regulates Voltage-sensor I (VSD I) activity. Voltage-sensors of all members of the voltage-gated ion channel family have three main conformations: resting, active and relaxed. VSD transition from the resting to the active conformation is thought to be coupled to channel opening, while the relaxed configuration is populated during prolonged depolarizations. Once in the relaxed state, strong hyperpolarizations are necessary to return to the resting state. When rabbit CaV1.2 channels are co-expressed without β subunit the voltage-dependency of VSD I indicates that near 50% of VSD I are in the relaxed state even at −90mV holding potential. In contrast, with β3 present, VSD I dwells between resting and active state. The augmented preference of VSD I for the relaxed state in the absence of β3 may explain why CaV1.2 channels lacking the β-subunit are less willing to open and together with our results with human CaV2.1 suggest that VSD I participates in inactivation rather than activation. We hypothesize that when in the relaxed state, VSD I keeps channels from opening or in an “unwilling” state.

Eminence of VSD I in the Voltage-Dependent Inactivation of the Human CaV1.2 Channel

In response to prolonged membrane depolarizations, the L-type calcium channel (CaV1.2) enters a non-conducting inactivated state that diminishes Ca2+ influx. Specifically, CaV1.2 channels undergo two types of inactivation: voltage-dependent inactivation (VDI) and a Ca2+-dependent, calmodulin-mediated inactivation (CDI). What is the structural basis for the voltage dependence of VDI? We hypothesize that at least one of the four CaV1.2 homologous but non-identical voltage-sensing domains (VSDs) should drive or regulate VDI. Using the Voltage-Clamp Fluorometry technique, we optically tracked the movement of the individual VSDs in conducting human CaV1.2 channels (α1C/α2δ-1) expressed in Xenopus oocytes under conditions that show a different extent of VDI (300ms depolarizations to 10 mV, using Ba2+ as the charge carrier to prevent CDI): β3 co-expression, VDI∼35%; β2a co-expression, VDI∼15%; Timothy-Syndrome-causing mutation G406R with β3, VDI∼8%. We found that, in all channels, upon repolarization to −90mV, VSD I deactivated with a bi-exponential decay (τ1∼1ms, τ2∼35ms). Channels with attenuated VDI (Timothy-Syndrome-causing mutation and β2a co-expression) exhibited mostly fast VSD I deactivation (Amp2∼6% and 12%, respectively), while a more pronounced VDI (β3 co-expression) correlated with an increase in the fraction of VSD I returning slowly to their resting state (Amp2∼55%). This feature is reminiscent of “charge immobilization”, whereby the inactivation of voltage-gated channels slows down VSD deactivation and produces an apparent reduction of the off gating charge. Our data support the view that VSD I undergoes “immobilization” following CaV1.2 inactivation. On the other hand, VSDs III and IV kinetics were not affected by the time-course of VDI. In conclusion, our findings reveal a strong correlation between the degree of inactivation and VSD I kinetics, suggesting the involvement of VSD I in setting the rate of VDI.

Calcium Current Properties and Leak Conductance in Mouse Muscle Fibers Overexpressing a Type 1 Hypokalemic Periodic Paralysis Mutant L-Type Calcium Channel

Missense mutations in the gene encoding the alpha1 subunit of the L-type calcium channel CaV1.1 induce type 1 Hypokalemic Periodic Paralysis (HypoPP1). These mutations mainly occur at arginine residues in the fourth transmembrane segment of voltage-sensor domains. Very few studies have investigated the acute effects of these mutations on channel function and muscle membrane electrical properties because of the difficulty to express Cav1.1 in heterologous systems. In the present study we successfully transferred by electroporation the genes encoding the turboGFP-tagged human wildtype (WT) and R1239H HypoPP1 mutant Cav1.1 into hind limb mouse muscles. The expression profile of the two channels showed a regular striated pattern indicative of the localization of the channels in the t-tubule membrane. Measurement of the L-type current using the silicone-clamp technique showed that the maximal conductance and the voltage-dependence of the Cav1.1 channel were significantly reduced and shifted towards negative potentials respectively in fibers expressing R1239H Cav1.1 as compared to fibers expressing WT Cav1.1. Applying voltage ramps from a holding potential of 0, −20, −40 or −60 mV to −120 mV in the presence of an external low-chloride, sodium-free and potassium-free solution revealed a significant higher leak conductance measured between −80 and −120 mV in fibers expressing R1239H Cav1.1. Acidification of the external solution significantly increased the leak inward current, leak conductance and the fluorescence of an internally loaded pH indicator in fibers expressing R1239H. These data suggest that an elevated leak inward current, likely carrying protons, flows at resting membrane potentials in fibers expressing R1239H Cav1.1 and that acidification of the external medium following muscle exercise could contribute to favor the onset of muscle paralysis in HypoPP1.

Block of Recombinant T-Type Calcium Channels by Gossypol, a Potential Contraceptive

Gossypol is a bioactive compound associated with some male contraceptive properties. Experimental evidences have shown that gossypol inhibits spermatogenesis as well as spermatozoa motility and viability. T-type calcium channels, or CaV3 channels, have been found in both spermatogenic and sperm mammalian cells; they play an essential role in spermatogenesis, motility, capacitation and acrosome reaction. Recent studies show that native T-type calcium currents from murine spermatogenic cells are inhibited by micromolar concentrations of gossypol in a non-reversible process. However, until now there was no data showing whether gossypol preferentially blocks any member of the CaV3 channels subfamily, neither information about the blocking mechanism. Here, we performed whole-cell patch-clamp recordings of HEK-293 cells stably expressing human CaV3 channels (CaV3.1, CaV3.2 and CaV3.3) to address these questions. We found that gossypol blocks the three CaV3 channels in a concentration-response manner with similar IC50s ranging from 6 to 11 μM. Block by gossypol is not completely irreversible although T-type currents were poorly recovered (10-30%). Voltage-dependence of activation was not modified by gossypol, but steady-state inactivation was shifted to more negative potentials (around 10 mV), and block at −30 mV was stronger when using more depolarized holding potentials. In addition, the inactivated-state of CaV3 channels was stabilized in the presence of gossypol as the recovery from inactivation was delayed and incomplete. A modest use-dependent component was observed only in CaV3.1. Current kinetics were not affected by gossypol. The results demonstrate that gossypol blocks human CaV3 channels at micromolar concentrations with preferential binding to inactivated-states, and suggest that sperm cells will be more sensitive to gossypol during the hyperpolarization that take place along the capacitation process. These results provide new insights about the potential use of gossypol as a contraceptive.Supported by CONACYT-MEXICO 167790.

Blocking of T-Type Calcium Channels by TTA-A2 Reveals a Conservative Binding Site for State-Dependent Antagonists

T-type calcium channels, or CaV3 channels, are involved in several cell functions, and they can generate low-threshold spikes that lead to burst firing and oscillatory behavior in various types of neurons. In addition, CaV3 channels are also implicated in a number of pathophysiological processes such as epilepsy, sleep disorders, hypertension, neuropathic pain and cancer. However, until recently there were no potent and selective antagonists of CaV3 channels that facilitate further investigation of T-type channel function in neurological diseases and to validate these channels as efficient drug targets. Recently, a series of potent and selective T-type calcium channel blockers (T-type antagonists or TTAs) have been reported in several studies. Here, we describe the identification of one aminoacid that is determinant for the molecular coupling of TTA-A2 ([2-(4-cyclopropylphenyl)-N-((1R)-1-{5-[(2,2,2-trifluoroethyl)oxo]pyridin-2-yl}ethyl)acetamide]) on the CaV3.1 recombinant human T-type calcium channel. By using a combination of molecular modeling, docking studies, mutagenesis and patch-clamp experiments of CaV3.1 channels expressed in HEK-293 cells, we have collected experimental evidence that supports the existence of a conservative binding site for TTA-A2 and state-dependent antagonists similar to TTA-A2, e.g., mibefradil. Blocking potency of TTA-A2 was reduced by two thirds in CaV3.1 mutants, where the identified aminoacid was replaced for alanine, proline, or arginine, among others. Sequence alignment of the three human CaV3 channels suggest that the binding site is conserved in the CaV3 subfamily, but not in the L-type CaV1.2 channel. In addition, our data also provides further evidence that TTA-Q4, a positive allosteric modulator of TTA-A2 functional inhibition, does not share the same binding site with the latter. Our results contribute to the knowledge for the rational design of T-type channel antagonists based on the structure of the TTA-A2 binding site.Supported by CONACYT-MEXICO 167790.

Simulation of L-Type Calcium Currents using Different Experimental Data Sources: from Cell Line to iPS-Derived Cardiomyocyte

Drug-induced pro-arrhythmic risk has been one of the main causes for drug candidate failure and has even led to compound withdrawn from the market. A new cardiac safety paradigm that combines in-vitro screening of multiple ion channels and in-silico tools, particularly biophysically-detailed cardiac electrophysiology models, has been encouraged by the FDA, HESI, Cardiac Safety Research Consortium & Safety Pharmacology Society.CaV1.2 channel (conducting the L-type Calcium current (ICaL)) plays an important role in cardiac electrophysiology. Blockade of ICaL can mitigate the pro-arrhythmic risk for multi-channel blockers (e.g. verapamil). For drug development, L-type calcium current data can be obtained from different experimental sources. Cell lines over-expressing the human CaV1.2 channel are normally used for HT compound screening. And recently iPS-derived cardiomyocytes have become an increasingly widespread model in pharmaceutical industry.In this study, the kinetics (steady state activation, inactivation and recovery from inactivation) of CaV1.2 CaL from over-expressed CHO cells and iPS-derived cardiomyocytes were measured using Ca2+ containing or Ba2+ containing solutions (to distinguish between Calcium-dependent and Voltage-dependent inactivations). Several in-silico models were used to emulate the experimental protocols, the simulated ICaL was extracted and compared with experimental data.Comparing simulated traces using data obtained from these two experiment sources, we can learn a lot about the biophysical and biological models and their translational value.

Evidence for redox sensing by a human cardiac calcium channel.

Ion channels are critical to life and respond rapidly to stimuli to evoke physiological responses. Calcium influx into heart muscle occurs through the ion conducting α1C subunit (Cav1.2) of the L-type Ca2+ channel. Glutathionylation of Cav1.2 results in increased calcium influx and is evident in ischemic human heart. However controversy exists as to whether direct modification of Cav1.2 is responsible for altered function. We directly assessed the function of purified human Cav1.2 in proteoliposomes. Truncation of the C terminus and mutation of cysteines in the N terminal region and cytoplasmic loop III-IV linker did not alter the effects of thiol modifying agents on open probability of the channel. However mutation of cysteines in cytoplasmic loop I-II linker altered open probability and protein folding assessed by thermal shift assay. We find that C543 confers sensitivity of Cav1.2 to oxidative stress and is sufficient to modify channel function and posttranslational folding. Our data provide direct evidence for the calcium channel as a redox sensor that facilitates rapid physiological responses.

Binding mechanisms of 1,4-dihydropyridine derivatives to L-type calcium channel Cav1.2: a molecular modeling study.

L-type Ca(2+) channels (LTCCs), the heteromultimeric proteins, are associated with electrical signaling and provide the key link between electrical signals and non-electrical processes. 1,4-Dihydropyridine (DHP) derivatives are a major class of blockers for LTCCs, and have experienced widespread use in the treatment of cardiovascular diseases. However, the precise knowledge of the binding mechanism of these ligands to LTCCs at the atomic level has remained unknown because of the unavailability of the crystal structures of LTCCs. In this study, homology modeling, molecular docking, molecular dynamics (MD) simulations, free energy calculations and decomposition were employed to explore the structural requirement of the binding of DHP derivatives to human Cav1.2, a member of LTCCs. The binding conformations of the DHPs in the active site of Cav1.2 were predicted, and the rank of the binding free energies of Cav1.2/DHPs is generally consistent with the experimental data. The structural analysis shows that most studied ligands fit into a hydrophobic pocket formed by Phe1129, Ile1173, Phe1176, Met1177 and Met1509, and form aryl-aryl interaction with Phe1129 or Tyr1508. The consistency between the predictions and experimental data suggest that the developed model is reliable and can be used as a valuable platform for the structure-based design of new potent ligands of Cav1.2.

Identification of State-Dependent Blockers for Voltage-Gated Calcium Channels Using a FLIPR-Based Assay.

The FLIPR (Fluorescent Imaging Plate Reader) system has been extensively used in the early stages of drug discovery for the identification of small molecules as a starting point for drug development, and for the pharmacological characterization of compounds. The main application of the system has been the measurement of intracellular Ca(2+) signals using fluorescent calcium indicators.This chapter describes the application of a protocol for the study and characterization of state-dependent blockers of Voltage-Gated Calcium Channels (VGCC) on the FLIPR(TETRA).The cell line suitable for the application of the protocol, and described hereafter, co-expresses the human CaV1.2 channel and the human inward rectifier K(+) channel Kir2.3. The presence of Kir2.3 allows the modulation of the plasma membrane potential and consequently of the state of the CaV1.2 channel by changing the extracellular K(+) concentration. In this way, CaV1.2 activity can be measured at different membrane voltages, corresponding to either the resting or partial inactivated state, by loading the cells with a calcium probe in extracellular low or high potassium buffer.

Voltage-Gated R-Type Calcium Channel Inhibition via Human μ-, δ-, and κ-opioid Receptors Is Voltage-Independently Mediated by Gβγ Protein Subunits.

Elucidating the mechanisms that modulate calcium channels via opioid receptor activation is fundamental to our understanding of both pain perception and how opioids modulate pain. Neuronal voltage-gated N-type calcium channels (Cav2.2) are inhibited by activation of G protein-coupled opioid receptors (ORs). However, inhibition of R-type (Cav2.3) channels by μ- or κ-ORs is poorly defined and has not been reported for δ-ORs. To investigate such interactions, we coexpressed human μ-, δ-, or κ-ORs with human Cav2.3 or Cav2.2 in human embryonic kidney 293 cells and measured depolarization-activated Ba(2+) currents (IBa). Selective agonists of μ-, δ-, and κ-ORs inhibited IBa through Cav2.3 channels by 35%. Cav2.2 channels were inhibited to a similar extent by κ-ORs, but more potently (60%) via μ- and δ-ORs. Antagonists of δ- and κ-ORs potentiated IBa amplitude mediated by Cav2.3 and Cav2.2 channels. Consistent with G protein βγ (Gβγ) interaction, modulation of Cav2.2 was primarily voltage-dependent and transiently relieved by depolarizing prepulses. In contrast, Cav2.3 modulation was voltage-independent and unaffected by depolarizing prepulses. However, Cav2.3 inhibition was sensitive to pertussis toxin and to intracellular application of guanosine 5'-[β-thio]diphosphate trilithium salt and guanosine 5'-[γ-thio]triphosphate tetralithium salt. Coexpression of Gβγ-specific scavengers-namely, the carboxyl terminus of the G protein-coupled receptor kinase 2 or membrane-targeted myristoylated-phosducin-attenuated or abolished Cav2.3 modulation. Our study reveals the diversity of OR-mediated signaling at Cav2 channels and identifies neuronal Cav2.3 channels as potential targets for opioid analgesics. Their novel modulation is dependent on pre-existing OR activity and mediated by membrane-delimited Gβγ subunits in a voltage-independent manner.

Phosphorylation of the Cav3.2 T-type calcium channel directly regulates its gating properties

Phosphorylation is a major mechanism regulating the activity of ion channels that remains poorly understood with respect to T-type calcium channels (Cav3). These channels are low voltage-activated calcium channels that play a key role in cellular excitability and various physiological functions. Their dysfunction has been linked to several neurological disorders, including absence epilepsy and neuropathic pain. Recent studies have revealed that T-type channels are modulated by a variety of serine/threonine protein kinase pathways, which indicates the need for a systematic analysis of T-type channel phosphorylation. Here, we immunopurified Cav3.2 channels from rat brain, and we used high-resolution MS to construct the first, to our knowledge, in vivo phosphorylation map of a voltage-gated calcium channel in a mammalian brain. We identified as many as 34 phosphorylation sites, and we show that the vast majority of these sites are also phosphorylated on the human Cav3.2 expressed in HEK293T cells. In patch-clamp studies, treatment of the channel with alkaline phosphatase as well as analysis of dephosphomimetic mutants revealed that phosphorylation regulates important functional properties of Cav3.2 channels, including voltage-dependent activation and inactivation and kinetics. We also identified that the phosphorylation of a locus situated in the loop I-II S442/S445/T446 is crucial for this regulation. Our data show that Cav3.2 channels are highly phosphorylated in the mammalian brain and establish phosphorylation as an important mechanism involved in the dynamic regulation of Cav3.2 channel gating properties.

Contrasting the roles of the I-II loop gating brake in CaV3.1 and CaV3.3 calcium channels.

Low-voltage-activated CaV3 channels are distinguished among other voltage-activated calcium channels by the most negative voltage activation threshold. The voltage dependence of current activation is virtually identical in all three CaV3 channels while the current kinetics of the CaV3.3 current is one order slower than that of the CaV3.1 and CaV3.2 channels. We have analyzed the voltage dependence and kinetics of charge (Q) movement in human recombinant CaV3.3 and CaV3.1 channels. The voltage dependence of voltage sensor activation (Qon-V) of the CaV3.3 channel was significantly shifted with respect to that of the CaV3.1 channel by +18.6mV and the kinetic of Qon activation in the CaV3.3 channel was significantly slower than that of the CaV3.1 channel. Removal of the gating brake in the intracellular loop connecting repeats I and II in the CaV3.3 channel in the ID12 mutant channel shifted the Qon-V relation to a value even more negative than that for the CaV3.1 channel. The kinetic of Qon activation was not significantly different between ID12 and CaV3.1 channels. Deletion of the gating brake in the CaV3.1 channel resulted in a GD12 channel with the voltage dependence of the gating current activation significantly shifted toward more negative potentials. The Qon kinetic was not significantly altered. ID12 and GD12 mutants did not differ significantly in voltage dependence nor in the kinetic of voltage sensor activation. In conclusion, the putative gating brake in the intracellular loop connecting repeats I and II controls the gating current of the CaV3 channels. We suggest that activation of the voltage sensor in domain I is limiting both the voltage dependence and the kinetics of CaV3 channel activation.

Less is More: Design of a Highly Stable Disulfide-Deleted Mutant of Analgesic Cyclic α-Conotoxin Vc1.1.

Cyclic α-conotoxin Vc1.1 (cVc1.1) is an orally active peptide with analgesic activity in rat models of neuropathic pain. It has two disulfide bonds, which can have three different connectivities, one of which is the native and active form. In this study we used computational modeling and nuclear magnetic resonance to design a disulfide-deleted mutant of cVc1.1, [C2H,C8F]cVc1.1, which has a larger hydrophobic core than cVc1.1 and, potentially, additional surface salt bridge interactions. The new variant, hcVc1.1, has similar structure and serum stability to cVc1.1 and is highly stable at a wide range of pH and temperatures. Remarkably, hcVc1.1 also has similar selectivity to cVc1.1, as it inhibited recombinant human α9α10 nicotinic acetylcholine receptor-mediated currents with an IC50 of 13 μM and rat N-type (Cav2.2) and recombinant human Cav2.3 calcium channels via GABAB receptor activation, with an IC50 of ~900 pM. Compared to cVc1.1, the potency of hcVc1.1 is reduced three-fold at both analgesic targets, whereas previous attempts to replace Vc1.1 disulfide bonds by non-reducible dicarba linkages resulted in at least 30-fold decreased activity. Because it has only one disulfide bond, hcVc1.1 is not subject to disulfide bond shuffling and does not form multiple isomers during peptide synthesis.

Abnormal Excitability and Episodic Low-Frequency Oscillations in the Cerebral Cortex of thetotteringMouse

The Ca(2+) channelopathies caused by mutations of the CACNA1A gene that encodes the pore-forming subunit of the human Cav2.1 (P/Q-type) voltage-gated Ca(2+) channel include episodic ataxia type 2 (EA2). Although, in EA2 the emphasis has been on cerebellar dysfunction, patients also exhibit episodic, nonmotoric abnormalities involving the cerebral cortex. This study demonstrates episodic, low-frequency oscillations (LFOs) throughout the cerebral cortex of tottering (tg/tg) mice, a widely used model of EA2. Ranging between 0.035 and 0.11 Hz, the LFOs in tg/tg mice can spontaneously develop very high power, referred to as a high-power state. The LFOs in tg/tg mice are mediated in part by neuronal activity as tetrodotoxin decreases the oscillations and cortical neuron discharge contain the same low frequencies. The high-power state involves compensatory mechanisms because acutely decreasing P/Q-type Ca(2+) channel function in either wild-type (WT) or tg/tg mice does not induce the high-power state. In contrast, blocking l-type Ca(2+) channels, known to be upregulated in tg/tg mice, reduces the high-power state. Intriguingly, basal excitatory glutamatergic neurotransmission constrains the high-power state because blocking ionotropic or metabotropic glutamate receptors results in high-power LFOs in tg/tg but not WT mice. The high-power LFOs are decreased markedly by acetazolamide and 4-aminopyridine, the primary treatments for EA2, suggesting disease relevance. Together, these results demonstrate that the high-power LFOs in the tg/tg cerebral cortex represent a highly abnormal excitability state that may underlie noncerebellar symptoms that characterize CACNA1A mutations.

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.

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.

Inhibition of Human CaV2.3 Channels via μ-, δ- and κ-Opioid Receptor Activation

Neuronal voltage-gated Cav2.3 channels are widely expressed in the central and peripheral nervous system where they contribute to neurotransmission and pain sensation. However, modulation of the Cav2.3 channel through G protein-coupled (GPC) μ- and δ-opioid receptors is poorly defined and has not previously been reported for κ-opioid receptors. We hypothesized that activation of human μ-, δ- or κ-opioid receptors modulates Cav2.3 channels via G protein signaling. Whole-cell Ba2+ currents were recorded in HEK293T cells co-expressing human Cav2.2 or Cav2.3 channels and μ-, δ- or κ-opioid receptors. Selective opioid receptor agonists and antagonists were used to study receptor modulation. The involvement of intracellular signaling pathways was investigated using specific inhibitors of GPC receptor-G protein coupling. Activation of μ-, δ- or κ-opioid receptors inhibited Cav2.3 and Cav2.2 channel current amplitude by ∼45% and ∼60%, respectively. Inhibition of Cav2.3 was not dependent on the type of subunit co-expressed. Inhibition of the Cav2.3 channel was primarily voltage independent, as depolarizing prepulses could not relieve the inhibited current. This was in marked contrast with the primarily voltage-dependent modulation of Cav2.2 channels that showed nearly complete recovery of the inhibited current with depolarizing prepulses. For all three types of opioid receptors, the pathway leading to Cav2.3 channel inhibition was sensitive to pertussis toxin and intracellular application of GDP-β-S. Similarly, the overexpression of a G protein subunit scavenger, myristoylated-phosducin, significantly reduced the magnitude of Cav2.3 channel inhibition. Here we demonstrate that Cav2.3 channels are efficiently inhibited by activation of μ-, δ- or κ-opioid receptors. Inhibition occurs via voltage-independent G protein signaling mechanisms. These results suggest opioid receptor controls specific members of the Cav2 channel family via differential signaling pathways. Neuronal Cav2.3 channels are therefore potential targets for opioid analgesics.