Modulation Of CaV2 Research Articles

Impact of isorhamnetin for suppression of action potential aberrations associated with atrial fibrillation

Abstract Background Isorhamnetin, a natural flavonoid, has potent antioxidant, antifibrotic and L-type Cav1.2 channel modulation effects, but its antiarrhythmic effect has not been reported. Purpose The purpose of this study is to investigate the effect of isorhamnetin on angiotensin II (Ang II)-induced atrial fibrillation (AF) in mice. Methods Wild-type male mice (C57BL/6J, 8 weeks old) were randomly divided into the following three groups: 1) control group, 2) Ang II treated group, 3) Ang II+isorhamnetin treated group. Ang II (1000 ng/kg/min) was administered by an implantable osmotic pump for two consecutive weeks, and isorhamnetin (5 mg/kg) was administered intraperitoneally for three weeks, one week before the initiation of Ang II administration. HL-1 cells were also assigned three groups. Ang II (1 μM) was treated for one day, and isorhamnetin (10 μM) was pretreated one hour before the beginning of Ang II treatment. To evaluate the effect of isorhamnetin on AF, electrophysiologic study including AF induction and action potential duration (APD) measurement with atrial tissue and HL-1 cells was performed. The expression of ion channel genes and proteins, including Cav1.2, were also evaluated. Results AF induction rate was markedly increased by Ang II and significantly decreased by isorhamnetin. AF duration was remarkably prolonged by Ang II and significantly shortened by isorhamnetin. Atrial effective refractory period (AERP) shortened by Ang II and reversed by isorhamnetin. Ang II administration significantly prolonged APD; that was alleviated by isorhamnetin. In addition, Ang II increased the frequency of delayed afterdepolarization (DAD), whereas isorhamnetin reduced its frequency. Cav1.2 channel expression was enhanced by Ang II and normalized by isorhamnetin. On the other hand, Ang II and isorhamnetin had no effect on potassium channels. Conclusion The findings in this study suggest that isorhamnetin prevents APD prolongation via the modulation of Cav1.2 channel and reduced the frequency of DAD, which is one of the mechanisms to prevent AF vulnerability.Effect of isorhamnetin on APD

Modulation of CaV1.2 Channel Function by Interacting Proteins and Post-Translational Modifications: Implications in Cardiovascular Diseases and COVID-19.

CaV1.2 calcium channel is the primary conduit for Ca2+ influx into cardiac and smooth muscles that underscores its importance in the pathogenesis of hypertension, atherosclerosis, myocardial infarction, and heart failure. But, a few controversies still remain. Therefore, exploring new ways to modulate CaV1.2 channel activity will augment the arsenal of CaV1.2 channel-based therapeutics for treatment of cardiovascular diseases. Here, we will mainly introduce a couple of emerging CaV1.2 channel interacting proteins, such as Galectin-1 and Cereblon, and discuss their roles in hypertension and heart failure through fine-tuning CaV1.2 channel activity. Of current interest, we will also evaluate the implication of the role of CaV1.2 channel in SARS-CoV-2 infection and the potential treatments of COVID-19-related cardiovascular symptoms.

L-cysteine modulates visceral nociception mediated by the CaV2.3 R-type calcium channels

CaV2.3 channels are subthreshold voltage-gated calcium channels that play crucial roles in neurotransmitter release and regulation of membrane excitability, yet modulation of these channels with endogenous molecules and their role in pain processing is not well studied. Here, we hypothesized that an endogenous amino acid l-cysteine could be a modulator of these channels and may affect pain processing in mice. To test this hypothesis, we employed conventional patch-clamp technique in the whole-cell configuration using recombinant CaV2.3 subunit stably expressed in human embryonic kidney (HEK-293) cells. We found in our in vitro experiments that l-cysteine facilitated gating and increased the amplitudes of recombinant CaV2.3 currents likely by chelating trace metals that tonically inhibit the channel. In addition, we took advantage of mouse genetics in vivo using the acetic acid visceral pain model that was performed on wildtype and homozygous Cacna1e knockout male littermates. In ensuing in vivo experiments, we found that l-cysteine administered both subcutaneously and intraperitoneally evoked more prominent pain responses in the wildtype mice, while the effect was completely abolished in knockout mice. Conversely, intrathecal administration of l-cysteine lowered visceral pain response in the wildtype mice, and again the effect was completely abolished in the knockout mice. Our study strongly suggests that l-cysteine-mediated modulation of CaV2.3 channels plays an important role in visceral pain processing. Furthermore, our data are consistent with the contrasting roles of CaV2.3 channels in mediating visceral nociception in the peripheral and central pain pathways.

Serotonin-Mediated Activation of Serotonin Receptor Type 1 Oppositely Modulates Voltage-Gated Calcium Channel Currents in Rat Sensory Neurons Innervating Hindlimb Muscle.

Serotonin (5-HT) is a multifaceted neurotransmitter that has been described to play a role as a peripheral inflammatory mediator when released in ischemic or injured muscle. Dorsal root ganglia (DRG) neurons are key sensors of noxious stimuli that are released under inflammatory conditions or mechanical stress. Little information is available on the specific 5-HT receptor subtypes expressed in primary afferents that help regulate reflex pressor responses. In the present study, the whole-cell patch-clamp technique was employed to examine the modulation of voltage-gated calcium channel (CaV) 2.2 currents by 5-HT and to identify the 5-HT receptor subtype(s) mediating this response in acutely dissociated rat DRG neurons innervating triceps surae muscle. Our results indicate that exposure of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled DRG neurons to 5-HT can exert three modulatory effects on CaV currents: high inhibition, low inhibition, and enhancement. Both 5-HT-mediated inhibition responses were blocked after pretreatment with pertussis toxin (PTX), indicating that 5-HT receptors are coupled to CaV2.2 via Gα i/o protein subunits. Application of selective serotonin receptor type 1 (5-HT1) agonists revealed that modulation of CaV2.2 currents occurs primarily after 5-HT1A receptor subtype stimulation and minimally from 5-HT1D activation. Finally, the intrathecal administration of the selective 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), significantly (P < 0.05) decreased the pressor response induced by intra-arterial administration of lactic acid. This suggests that 5-HT1A receptors are expressed presynaptically on primary afferent neurons innervating triceps surae muscle. Our findings indicate that preferential stimulation of 5-HT1 receptors, expressed on thin fiber muscle afferents, serves to regulate the reflex pressor response to metabolic stimuli. SIGNIFICANCE STATEMENT: The monoamine serotonin (5-HT), released under ischemic conditions, can contribute to the development of inflammation that negatively affects the exercise pressor reflex. The 5-HT receptor subtype and signaling pathway that underlies calcium channel modulation in dorsal root ganglia afferents, innervating hindlimb muscles, are unknown. We show that 5-HT can either block (primarily via serotonin receptor type 1 (5-HT1)A subtypes) or enhance voltage-gated calcium channel (CaV2.2) currents. Our findings suggest 5-HT exhibits receptor subtype selectivity, providing a complexity of cellular responses.

The terpenes camphene and alpha-bisabolol inhibit inflammatory and neuropathic pain via Cav3.2\xa0T-type calcium channels

T-type calcium channels are known molecular targets of certain phytocannabinoids and endocannabinoids. Here we explored the modulation of Cav3.2 T-type calcium channels by terpenes derived from cannabis plants. A screen of eight commercially available terpenes revealed that camphene and alpha-bisabolol mediated partial, but significant inhibition of Cav3.2 channels expressed in tsA-201 cells, as well as native T-type channels in mouse dorsal root ganglion neurons. Both compounds inhibited peak current amplitude with IC50s in the low micromolar range, and mediated an additional small hyperpolarizing shift in half-inactivation voltage. When delivered intrathecally, both terpenes inhibited nocifensive responses in mice that had received an intraplantar injection of formalin, with alpha-bisabolol showing greater efficacy. Both terpenes reduced thermal hyperalgesia in mice injected with Complete Freund’s adjuvant. This effect was independent of sex, and absent in Cav3.2 null mice, indicating that these compounds mediate their analgesic properties by acting on Cav3.2 channels. Both compounds also inhibited mechanical hypersensitivity in a mouse model of neuropathic pain. Hence, camphene and alpha-bisabolol have a wide spectrum of analgesic action by virtue of inhibiting Cav3.2 T-type calcium channels.

Delineating an extracellular redox-sensitive module in T-type Ca2+ channels

T-type (Cav3) Ca2+ channels are important regulators of excitability and rhythmic activity of excitable cells. Among other voltage-gated Ca2+ channels, Cav3 channels are uniquely sensitive to oxidation and zinc. Using recombinant protein expression in HEK293 cells, patch clamp electrophysiology, site-directed mutagenesis, and homology modeling, we report here that modulation of Cav3.2 by redox agents and zinc is mediated by a unique extracellular module containing a high-affinity metal-binding site formed by the extracellular IS1–IS2 and IS3–IS4 loops of domain I and a cluster of extracellular cysteines in the IS1–IS2 loop. Patch clamp recording of recombinant Cav3.2 currents revealed that two cysteine-modifying agents, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) and N-ethylmaleimide, as well as a reactive oxygen species–producing neuropeptide, substance P (SP), inhibit Cav3.2 current to similar degrees and that this inhibition is reversed by a reducing agent and a zinc chelator. Pre-application of MTSES prevented further SP-mediated current inhibition. Substitution of the zinc-binding residue His191 in Cav3.2 reduced the channel's sensitivity to MTSES, and introduction of the corresponding histidine into Cav3.1 sensitized it to MTSES. Removal of extracellular cysteines from the IS1–IS2 loop of Cav3.2 reduced its sensitivity to MTSES and SP. We hypothesize that oxidative modification of IS1–IS2 loop cysteines induces allosteric changes in the zinc-binding site of Cav3.2 so that it becomes sensitive to ambient zinc.

Ghrelin Selectively Inhibits CaV3.3 Subtype of Low-Voltage-Gated Calcium Channels.

The mechanisms by which ghrelin controls electrical activity in the hypothalamus are not fully understood. One unexplored target of ghrelin is CaV3, responsible for transient calcium currents (T-currents) that control neuronal firing. We investigated the effect of ghrelin on CaV3 subtypes and how this modulation impacts on neuronal activity. We performed whole-cell patch-clamp recordings in primary mouse hypothalamic cultures to explore the effect of ghrelin on T-currents. We also recorded calcium currents from transiently transfected tsA201 cells to study the sensitivity of each CaV3 subtype to GHSR activation. Finally, we ran a computational model combining the well-known reduction of potassium current by ghrelin with the CaV3 biophysical parameter modifications induced by ghrelin to predict the impact on neuronal electrical behavior. We found that ghrelin inhibits native NiCl2 sensitive current currents in hypothalamic neurons. We determined that CaV3.3 is the only CaV3 subtype sensitive to ghrelin. The modulation of CaV3.3 by ghrelin comprises a reduction in maximum conductance, a shift to hyperpolarized voltages of the I-V and steady-state inactivation curves, and an acceleration of activation and inactivation kinetics. Our model-based prediction indicates that the inhibition of CaV3.3 would attenuate the stimulation of firing originating from the inhibition of potassium currents by ghrelin. In summary, we discovered a new target of ghrelin in neurons: the CaV3.3. This mechanism would imply a negative feed-forward regulation of the neuronal activation exerted by ghrelin. Our work expands the knowledge of the wide range of actions of GHSR, a receptor potentially targeted by therapeutics for several diseases.

Interactions of Rabconnectin-3 with Cav2 calcium channels

This study describes the interaction between Cav2 calcium channels and Rabconnectin-3, a di-subunit protein that is associated with synaptic vesicles. Immunostaining reveals that both Rabconnectin-3α (RB-3α) and Rabconnectin-3β (RB-3β) are colocalized in mouse hippocampal neurons. Co-immunoprecipitations from brain tissue is consistent with the formation of a protein complex between RB-3α and RB-3β and both Cav2.2 and the related Cav2.1 calcium channel. The coexpression of either RB-3α or RB-3β with Cav2.2 calcium channels in tsA-201 cells led to a reduction in Cav2.2 current density without any effects on the voltage-dependence of activation or inactivation. Coexpression of both Rabconnectin-3 subunits did not cause an additive effect on current densities. Finally, the presence of Rabconnectin-3 did not interfere with μ-opioid receptor mediated Gβγ modulation of Cav2.2 channels. Altogether, our findings show that Rabconnectin-3 has the propensity to regulate calcium entry mediated by Cav2.2 channels.

The ERG1a K+ Channel Increases Intracellular Calcium and Calpain Activity in C2C12 Myotubes

In skeletal muscle increased intracellular calcium concentration ([Ca2+]i; uM range) is necessary for excitation‐contraction coupling; however, smaller increases in [Ca2+]i (nM range) can modulate other physiological processes in non‐contracting muscle. Indeed, fluctuations in localized calcium concentration can serve as a second messenger. In contrast, inappropriate changes in [Ca2+]i can have detrimental effects on muscle tissue and are associated with numerous skeletal muscle pathologies, for example muscular dystrophy, amyotrophic lateral sclerosis, malignant hyperthermia, cancer cachexia, and atrophy. Therefore, [Ca2+]i must be tightly regulated in terms of time, space and amplitude for cellular processes to occur in a properly coordinated fashion. In earlier reports, we showed that the ERG1a K+ channel is upregulated in atrophying skeletal muscle and contributes to increased ubiquitin proteasome proteolysis. Here, using the ratiometric Ca2+ indicator fura‐2AM, we show that ERG1a expression in C2C12 myotubes produces an increase in basal [Ca2+]i as well as a transient increase in [Ca2+]i as a consequence of depolarization. We explored this transient increase in [Ca2+]i using pharmacological agents. The data demonstrate that the ERG1a‐induced increase is not sensitive to the L‐type calcium channel blocker nifedipine, suggesting that it does not result from modulation of Cav1.1 channels. To further support this data, immunoblots reveal that there is no change in Cav1.1 channel abundance. However, the data do demonstrate that the increase in [Ca2+]i is sensitive to the SERCA blocking agent thapsigargin, suggesting that the source of the calcium is the sarcoplasmic reticulum stores. Additionally, the data reveal that ERG1a expression also increases basal calpain activity. In summary, to date the data show that ERG1a increases [Ca2+]i levels and suggest that this increase could occur, at least in part, as a result of release of calcium from sarcoplasmic reticulum stores. Additionally, the data reveal that ERG1a expression also increases calpain activity, suggesting that the increase in intracellular calcium results in increased calpain activity and possibly contributes to the increased proteolysis that occurs in atrophic skeletal muscle.Support or Funding InformationU.S. Department of Defense, PRMRP Grant# PR170326This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Modulation of Cav2.3 channels by unconjugated bilirubin (UCB) – Candidate mechanism for UCB-induced neuromodulation and neurotoxicity

Elevated levels of unbound unconjugated bilirubin (UCB) can lead to bilirubin encephalopathy and kernicterus. In spite of a large number of studies demonstrating UCB-induced changes in central neurotransmission, it is still unclear whether these effects involve alterations in the function of specific ion channels. To assess how different UCB concentrations and UCB:albumin (U/A) molar ratios affect neuronal R-type voltage-gated Ca2+ channels, we evaluated their effects on whole-cell currents through recombinant Cav2.3 + β3 channel complexes and ex-vivo electroretinograms (ERGs) from wildtype and Cav2.3-deficient mice. Our findings show that modestly elevated levels of unbound UCB (U/A = 0.5) produce subtle but significant changes in the voltage-dependence of activation and prepulse inactivation, resulting in a stimulation of currents activated by weak depolarization and inhibition at potentials on the plateau of the activation curve. Saturation of the albumin binding capacity (U/A = 1) produced additional suppression that became significant when albumin was omitted completely and might involve a complete loss of channel function.Acutely administered UCB (U/A = 0.5) has recently been shown to affect transsynaptic signaling in the isolated vertebrate retina. The present report reveals that sustained exposure of the murine retina to UCB significantly suppresses also late responses of the inner retina (b-wave) from wildtype compared to Cav2.3-deficient mice. In addition, recovery during washout was significantly more complete and faster in retinae lacking Cav2.3 channels.Together, these findings show that UCB affects cloned and native Cav2.3 channels at clinically relevant U/A molar ratios and indicate that supersaturation of albumin is not required for modulation but associated with a loss of channel functional that could contribute to chronic neuronal dysfunction.

High doses of digoxin increase the myocardial nuclear factor-kB and CaV1.2 channels in healthy mice. A possible mechanism of digitalis toxicity.

Toxic effects of digoxin may occur with normal therapeutic serum level. However, the underlying mechanisms are not fully understood. Nuclear factor kappa-B (NF-kB) is an important transcription factor in most organ systems and is often implicated in the harmful effects of cardiac injury. NF-kB promotes inflammatory responses, mediates adverse cardiac remodeling and has a function correlation with calcium. The voltage-gated L-type calcium channel CaV1.2 mediates the influx of Ca+2 into the cell in response to membrane depolarization. Our aim was to characterize the role of NF-kB during digoxin toxicity and to assess its correlation with Cav 1.2 in healthy mice in vivo. To address these questions, digoxin was administered in doses of 0.1, 1 or 5 mg/kg orally daily for seven days to the animals. Serum digoxin, serum calcium, atrial and ventricular calcium levels were measured. We, also, looked for NF-kB and CaV1.2 channel expression in cardiac muscle of mice. Digoxin at a dose of 0.1 mg/kg did not enhance serum, atrial, and ventricular Ca+2 levels, but were increased when digoxin dose of 1 and 5 mg/kg were administered. Histologically, myocardial necrosis and cellular infiltration on day 7 were significantly more severe in the 5 mg/kg/day digoxin group. Immunohistochemical studies showed more expression of both NF-kB and CaV1.2 in 1 and 5 mg/kg/day digoxin groups. These data suggest that NF-kB may be responsible for digoxin toxicity, at least partially via modulation of CaV1.2 and intracellular calcium homeostasis in the myocardium.

Proteolytic cleavage of the hydrophobic domain in the CaVα2δ1 subunit improves assembly and activity of cardiac CaV1.2 channels

Voltage-gated L-type CaV1.2 channels in cardiomyocytes exist as heteromeric complexes with the pore-forming CaVα1, CaVβ, and CaVα2δ1 subunits. The full complement of subunits is required to reconstitute the native-like properties of L-type Ca2+ currents, but the molecular determinants responsible for the formation of the heteromeric complex are still being studied. Enzymatic treatment with phosphatidylinositol-specific phospholipase C, a phospholipase C specific for the cleavage of glycosylphosphatidylinositol (GPI)-anchored proteins, disrupted plasma membrane localization of the cardiac CaVα2δ1 prompting us to investigate deletions of its hydrophobic transmembrane domain. Patch-clamp experiments indicated that the C-terminally cleaved CaVα2δ1 proteins up-regulate CaV1.2 channels. In contrast, deleting the residues before the single hydrophobic segment (CaVα2δ1 Δ1059-1063) impaired current up-regulation. CaVα2δ1 mutants G1060I and G1061I nearly eliminated the cell-surface fluorescence of CaVα2δ1, indicated by two-color flow cytometry assays and confocal imaging, and prevented CaVα2δ1-mediated increase in peak current density and modulation of the voltage-dependent gating of CaV1.2. These impacts were specific to substitutions with isoleucine residues because functional modulation was partially preserved in CaVα2δ1 G1060A and G1061A proteins. Moreover, C-terminal fragments exhibited significantly altered mobility in denatured immunoblots of CaVα2δ1 G1060I and CaVα2δ1 G1061I, suggesting that these mutant proteins were impaired in proteolytic processing. Finally, CaVα2δ1 Δ1059-1063, but not CaVα2δ1 G1060A, failed to co-immunoprecipitate with CaV1.2. Altogether, our data support a model in which small neutral hydrophobic residues facilitate the post-translational cleavage of the CaVα2δ1 subunit at the predicted membrane interface and further suggest that preventing GPI anchoring of CaVα2δ1 averts its cell-surface expression, its interaction with CaVα1, and modulation of CaV1.2 currents.

Redox-Dependent Modulation of T-Type Ca(2+) Channels in Sensory Neurons Contributes to Acute Anti-Nociceptive Effect of Substance P.

Aims: Neuropeptide substance P (SP) is produced and released by a subset of peripheral sensory neurons that respond to tissue damage (nociceptors). SP exerts excitatory effects in the central nervous system, but peripheral SP actions are still poorly understood; therefore, here, we aimed at investigating these peripheral mechanisms. Results: SP acutely inhibited T-type voltage-gated Ca2+ channels in nociceptors. The effect was mediated by neurokinin 1 (NK1) receptor-induced stimulation of intracellular release of reactive oxygen species (ROS), as it can be prevented or reversed by the reducing agent dithiothreitol and mimicked by exogenous or endogenous ROS. This redox-mediated T-type Ca2+ channel inhibition operated through the modulation of CaV3.2 channel sensitivity to ambient zinc, as it can be prevented or reversed by zinc chelation and mimicked by exogenous zinc. Elimination of the zinc-binding site in CaV3.2 rendered the channel insensitive to SP-mediated inhibition. Importantly, peripherally applied SP significantly reduced bradykinin-induced nociception in rats in vivo; knock-down of CaV3.2 significantly reduced this anti-nociceptive effect. This atypical signaling cascade shared the initial steps with the SP-mediated augmentation of M-type K+ channels described earlier. Innovation: Our study established a mechanism underlying the peripheral anti-nociceptive effect of SP whereby this neuropeptide produces ROS-dependent inhibition of pro-algesic T-type Ca2+ current and concurrent enhancement of anti-algesic M-type K+ current. These findings will lead to a better understanding of mechanisms of endogenous analgesia. Conclusion: SP modulates T-type channel activity in nociceptors by a redox-dependent tuning of channel sensitivity to zinc; this novel modulatory pathway contributes to the peripheral anti-nociceptive effect of SP. Antioxid. Redox Signal. 25, 233–251.

Protein kinase A modulation of CaV1.4 calcium channels.

The regulation of L-type Ca2+ channels by protein kinase A (PKA) represents a crucial element within cardiac, skeletal muscle and neurological systems. Although much work has been done to understand this regulation in cardiac CaV1.2 Ca2+ channels, relatively little is known about the closely related CaV1.4 L-type Ca2+ channels, which feature prominently in the visual system. Here we find that CaV1.4 channels are indeed modulated by PKA phosphorylation within the inhibitor of Ca2+-dependent inactivation (ICDI) motif. Phosphorylation of this region promotes the occupancy of calmodulin on the channel, thus increasing channel open probability (PO) and Ca2+-dependent inactivation. Although this interaction seems specific to CaV1.4 channels, introduction of ICDI1.4 to CaV1.3 or CaV1.2 channels endows these channels with a form of PKA modulation, previously unobserved in heterologous systems. Thus, this mechanism may not only play an important role in the visual system but may be generalizable across the L-type channel family.

Cacna1g is a genetic modifier of epilepsy caused by mutation of voltage-gated sodium channel Scn2a.

More than 1,200 mutations in neuronal voltage-gated sodium channel (VGSC) genes have been identified in patients with several epilepsy syndromes. A common feature of genetic epilepsies is variable expressivity among individuals with the same mutation. The Scn2a(Q54) transgenic mouse model has a mutation in Scn2a that results in spontaneous epilepsy. Scn2a(Q54) phenotype severity varies depending on the genetic strain background, making it a useful model for identifying and characterizing epilepsy modifier genes. Scn2a(Q54) mice on the [C57BL/6JxSJL/J]F1 background exhibit earlier seizure onset, elevated spontaneous seizure frequency, and decreased survival compared to Scn2a(Q54) mice congenic on the C57BL/6J strain. Genetic mapping and RNA-Seq analysis identified Cacna1g as a candidate modifier gene at the Moe1 locus, which influences Scn2a(Q54) phenotype severity. In this study, we evaluated the modifier potential of Cacna1g, encoding the Cav3.1 voltage-gated calcium channel, by testing whether transgenic alteration of Cacna1g expression modifies severity of the Scn2a(Q54) seizure phenotype. Scn2a(Q54) mice exhibited increased spontaneous seizure frequency with elevated Cacna1g expression and decreased seizure frequency with decreased Cacna1g expression. These results provide support for Cacna1g as an epilepsy modifier gene and suggest that modulation of Cav3.1 may be an effective therapeutic strategy.

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.

Crosstalk Between Beta Subunits, Intracellular Ca2+-Signaling and SNAREs in the Modulation of Cav2.1 Channel Steady-State Inactivation

Several SNARE proteins specifically interact with CaV2.1 (P/Q-type) and CaV2.2 (N-type) channels by binding to a synaptic protein interaction site (synprint) located within the intracellular loop connecting domains II and III (LII-III) of the channels. This interaction not only allows secretory vesicles docking to the plasma membrane near the pathway for Ca2+ entry, the signal that triggers exocytotic neurotransmitter release, but also, under certain conditions, shifts the voltage dependence of steady-state inactivation towards more negative membrane potentials. Besides the important anchoring function of the synprint site in the functional interaction SNARE proteins-CaV2.1 channels, the involvement of other molecular domains has been proposed including the alpha1A first intracellular loop (LI-II). The molecular mechanism by which LI-II influences CaV2.1-SNAREs functional interaction is unknown. It has been suggested that conformational changes at LI-II produced by different regulatory beta subunits not only determine the degree of voltage-dependent inactivation but also the extent of a Ca2+-dependent inactivation component (mediated by the binding of Ca2+-calmodulin to a IQ-like motif in alpha1A C-terminal domain). We have now analyzed CaV2.1 channel modulation by syntaxin-1A in the presence of functionally different ancillary beta subunits (beta2a or beta3) and using different human alpha1A constructs containing either a LI-II partial deletion or mutations in the IQ-like region. Our results reveal that regulation by syntaxin-1A of CaV2.1 channel activity requires both the integrity of alpha1A LI-II and the lack of a Ca2+-dependent component in the channel steady-state inactivation. Supported by Spanish Ministry of Economy and Competitiveness (Grant SAF2012-31089, FEDER Funds, Fondo de Investigación Sanitaria, Instituto Carlos III, Spain (RIC RD12/0042/0014, Red HERACLES).

Modulation of CaV1.2 calcium channel by neuropeptide W regulates vascular myogenic tone via G protein-coupled receptor 7.

Neuropeptide W (NPW), an endogenous ligand for the G protein-coupled receptor 7 (GPR7), was first found to make important roles in central nerve system. In periphery, NPW was also present and regulated intracellular calcium homeostasis by L-type calcium channels. This study was designed to discover the effects of NPW-GPR7 on the function of CaV1.2 calcium channels in the vascular smooth muscle cells (VSMCs) and vasotone of arterial vessels. By whole-cell patch clamp, we studied the effects of NPW-23, the active form of NPW, on the CaV1.2 channels in the heterologously transfected human embryonic kidney 293 cells and VSMCs isolated from rat. Living system was used to explore the physiological function of NPW-23 in arterial myogenic tone. To investigate the pathological relevance, NPW mRNA level of mesenteric arteries was measured in the hypertensive and normotensive rats. NPW's receptor GPR7 was coexpressed with CaV1.2 channels in arterial smooth muscle. NPW-23 increased the ICa,L in transfected human embryonic kidney 293 cells and VSMCs via GPR7, which could be abrogated by phospholipase C (PLC)/protein kinase C (PKC) inhibitors, not protein kinase A or protein kinase G inhibitor. After NPW-23 application, the expression of pan phospho-PKC was increased; moreover, intracellular diacylglycerol level, the second messenger catalyzed by PLC, was increased 1.5-2-fold. Application with NPW-23 increased pressure-induced vasotone of the rat mesenteric arteries. Importantly, the expression of NPW was decreased in the hypertensive rats. NPW-23 regulates ICa,L via GPR7, which is mediated by PLC/PKC signaling, and such a mechanism plays a role in modulating vascular myogenic tone, which may involve in the development of vascular hypertension.

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.

Modulation of CaV2.1 channels by neuronal calcium sensor-1 induces short-term synaptic facilitation.

Facilitation and inactivation of P/Q-type Ca2+ currents mediated by Ca2+/calmodulin binding to Ca(V)2.1 channels contribute to facilitation and rapid depression of synaptic transmission, respectively. Other calcium sensor proteins displace calmodulin from its binding site and differentially modulate P/Q-type Ca2 + currents, resulting in diverse patterns of short-term synaptic plasticity. Neuronal calcium sensor-1 (NCS-1, frequenin) has been shown to enhance synaptic facilitation, but the underlying mechanism is unclear. We report here that NCS-1 directly interacts with IQ-like motif and calmodulin-binding domain in the C-terminal domain of Ca(V)2.1 channel. NCS-1 reduces Ca2 +-dependent inactivation of P/Q-type Ca2+ current through interaction with the IQ-like motif and calmodulin-binding domain without affecting peak current or activation kinetics. Expression of NCS-1 in presynaptic superior cervical ganglion neurons has no effect on synaptic transmission, eliminating effects of this calcium sensor protein on endogenous N-type Ca2+ currents and the endogenous neurotransmitter release machinery. However, in superior cervical ganglion neurons expressing wild-type Ca(V)2.1 channels, co-expression of NCS-1 induces facilitation of synaptic transmission in response to paired pulses and trains of depolarizing stimuli, and this effect is lost in Ca(V)2.1 channels with mutations in the IQ-like motif and calmodulin-binding domain. These results reveal that NCS-1 directly modulates Ca(V)2.1 channels to induce short-term synaptic facilitation and further demonstrate that CaS proteins are crucial in fine-tuning short-term synaptic plasticity.