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Calmodulin (CaM) was reported to interact with PreIQ and IQ of CaV1.2 channels, but to date, no explicit binding sites of CaM were illustrated. Therefore, in the present study, we firstly used MOE (Molecular Operating Environment) for protein-protein docking and we found that the most likely residues of CaM that play an important role in the interface are concentrated in central linker region. Next we examined the binding properties of CaM and its mutants to PreIQ and IQ by GST pull-down assays. Here we confirmed that CaM binds to PreIQ and IQ in a concentration-dependent and [Ca2+]-dependent manner. However, silencing the effect of N-lobe and C-lobe by mutating two Ca2+ binding sites of each lobe abolished [Ca2+]-dependence of CaM binding, but could not influence the combination. And the mutant in central linker reduced the binding of CaM/PreIQ and CaM/IQ especially at low [Ca2+]. We confirmed that N-lobe and C-lobe play vital role in sensing the change of Ca2+, and found that the central linker of CaM is involved in the binding of CaM to CaV1.2 channels in particular at low [Ca2+], not only participates in the combination with PreIQ, but also with IQ.

Sick sinus syndrome (SSS) is primarily a disease of the elderly, and age-dependent decrease in Cav1.2 and Cav1.3 Ca2+ channels within the sinus node has been shown to play an important role in sinoatrial node (SAN) degeneration; however, posttranscriptional mechanisms regulating decrease in Cav1.2 and Cav1.3 Ca2+ channels remain unclear. Some studies have reported that microRNAs (miRNAs) are involved in age-related cardiovascular diseases. Nevertheless, little is known about the roles of miRNAs in age-related SSS. This study investigated whether miR-1976 was involved in the regulation of SAN degeneration by targeting Cav1.2 and Cav1.3 Ca2+ channels. First, using microarray-based miRNA expression profiling and qRT-PCR, we confirmed that miR-1976 was upregulated in the plasma of patients with age-related SSS relative to healthy controls. By employing target gene prediction software, luciferase assay and western blotting, we further confirmed Cav1.2 and Cav1.3 as direct targets of miR-1976. Furthermore, miR-1976 levels in rabbit SAN tissues were negatively correlated with Cav1.2 and Cav1.3 expression and intrinsic heart rates but positively correlated with corrected sinus node recovery time (CSNRT). Additionally, miR-1976 transgenic mice displayed attenuated Cav1.2 and Cav1.3 protein expression, which led to sinus node dysfunction. These results suggest that miR-1976 plays an important role in the SAN aging process by targeting Cav1.2 and Cav1.3. Thus, miR-1976 could have great potential as a noninvasive diagnostic tool and therapeutic target for SSS. These findings may reveal important insights into the pathogenesis of SSS.

The first clinically used antiarrhythmic, antianginal and anti-hypertensive phenylalkylamine, verapamil's cardiovascular activity is inextricably linked to its ability to antagonize Ca2+ overload via blocking CaV1.2, a cardiac L-type Ca2+ channel of undisputed physiological and pharmacological importance in cardiovascular disorders such as myocardial ischemia-reperfusion injury. From a structural point of view, however, the action mechanism of verapamil is still elusive. Therefore, incorporating previous findings for verapamil and CaV1.2, this review article puts forward two experimental data-derived and -supported 3D structure models for CaV1.2's α1 subunit and its verapamil-bound form. Furthermore, this article suggests three biophysical mechanisms, namely competitive binding, steric hindrance and electrostatic repulsion, towards an atomic level understanding of how verapamil blocks the L-type Ca2+ current mediated by CaV1.2 in reality, which can be useful for the design and development of next-generation Ca2+ antagonists to provide safer and more effective treatment of cardiovascular diseases.

Background 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. Methods 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. Results 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. Conclusions 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.

Manganese-enhanced magnetic resonance imaging (MEMRI) exploits the biophysical similarity of Ca2+ and Mn2+ to map the brain's activity in vivo. However, to what extent different Ca2+ channels contribute to the enhanced signal that MEMRI provides and how Mn2+ dynamics influence Mn2+ brain accumulation after systemic administration of MnCl2 are not yet fully understood. Here, we demonstrate that mice lacking the L-type Ca2+ channel 1.2 (Cav1.2) in the CNS show approximately 50% less increase in MEMRI contrast after repeated systemic MnCl2 injections, as compared to control mice. In contrast, genetic deletion of L-type Ca2+ channel 1.3 (Cav1.3) did not reduce signal. Brain structure- or cell type-specific deletion of Cav1.2 in combination with voxel-wise MEMRI analysis revealed a preferential accumulation of Mn2+ in projection terminals, which was confirmed by local MnCl2 administration to defined brain areas. Taken together, we provide unequivocal evidence that Cav1.2 represents an important channel for neuronal Mn2+ influx after systemic injections. We also show that after neuronal uptake, Mn2+ preferentially accumulates in projection terminals.

The L-type calcium channel (LTCC) is an important determinant of cardiac contractility. Therefore, changes in LTCC activity or protein levels could be expected to affect cardiac function. Several studies describing LTCC regulation are available, but only a few examine LTCC protein stability. Polycystin-1 (PC1) is a mechanosensor that regulates heart contractility and is involved in mechanical stretch-induced cardiac hypertrophy. PC1 was originally described as an unconventional Gi/o protein-coupled receptor in renal cells. We recently reported that PC1 regulates LTCC stability in cardiomyocytes under stress; however, the mechanism underlying this effect remains unknown. Here, we use cultured neonatal rat ventricular myocytes and hypo-osmotic stress (HS) to model mechanical stretch. The model shows that the Cavβ2 subunit is necessary for LTCC stabilization in cardiomyocytes during mechanical stretch, acting through an AKT-dependent mechanism. Our data also shows that AKT activation depends on the G protein-coupled receptor activity of PC1, specifically its G protein-binding domain, and the associated Gβγ subunit of a heterotrimeric Gi/o protein. In fact, over-expression of the human PC1 C-terminal mutant lacking the G protein-binding domain blunted the AKT activation-induced increase in Cav1.2 protein in cardiomyocytes. These findings provide novel evidence that PC1 is involved in the regulation of cardiac LTCCs through a Giβγ-AKT-Cavβ2 pathway, suggesting a new mechanism for regulation of cardiac function.

Voltage-gated L-type CaV1.2 channels in cardiomyocytes exist as heteromeric complexes. Co-expression of CaVα2δ1 with CaVβ/CaVα1 proteins reconstitutes the functional properties of native L-type currents, but the interacting domains at the CaV1.2/CaVα2δ1 interface are unknown. Here, a homology-based model of CaV1.2 identified protein interfaces between the extracellular domain of CaVα2δ1 and the extracellular loops of the CaVα1 protein in repeats I (IS1S2 and IS5S6), II (IIS5S6), and III (IIIS5S6). Insertion of a 9-residue hemagglutinin epitope in IS1S2, but not in IS5S6 or in IIS5S6, prevented the co-immunoprecipitation of CaV1.2 with CaVα2δ1. IS1S2 contains a cluster of three conserved negatively charged residues Glu-179, Asp-180, and Asp-181 that could contribute to non-bonded interactions with CaVα2δ1. Substitutions of CaV1.2 Asp-181 impaired the co-immunoprecipitation of CaVβ/CaV1.2 with CaVα2δ1 and the CaVα2δ1-dependent shift in voltage-dependent activation gating. In contrast, single substitutions in CaV1.2 in neighboring positions in the same loop (179, 180, and 182-184) did not significantly alter the functional up-regulation of CaV1.2 whole-cell currents. However, a negatively charged residue at position 180 was necessary to convey the CaVα2δ1-mediated shift in the activation gating. We also found a more modest contribution from the positively charged Arg-1119 in the extracellular pore region in repeat III of CaV1.2. We conclude that CaV1.2 Asp-181 anchors the physical interaction that facilitates the CaVα2δ1-mediated functional modulation of CaV1.2 currents. By stabilizing the first extracellular loop of CaV1.2, CaVα2δ1 may up-regulate currents by promoting conformations of the voltage sensor that are associated with the channel's open state.

This study investigated the effects of fluoride exposure on the mRNA expression of Cav1.2 calcium signaling pathway and apoptosis regulatory molecules in PC12 cells. The viability of PC12 cell receiving high fluoride (5.0 mM) and low fluoride (0.5 mM) alone or fluoride combined with L-type calcium channel (LTCC) agonist/inhibitor (5 umol/L FPL6417/2 u mol/L nifedipine) was detected using cell counting kit-8 at different time points (2, 4, 6, 8, 12, 10, and 24 h). Changes in the cell configuration were observed after exposing the cells to fluoride for 24 h. The expression levels of molecules related to the LTCC were examined, particularly, Cav1.2, c-fos, CAMK II, Bax, and Bcl-2. Fluoride poisoning induced severe cell injuries, such as decreased PC12 cell activity, enhanced cell apoptosis, high c-fos, CAMKII, and Bax mRNA expression levels. Bcl-2 expression level was also reduced. Meanwhile, high fluoride, high fluoride with FPL64176, and low fluoride with FPL64176 enhanced the Cav1.2 expression level. In contrast, low fluoride, high fluoride with nifedipine, and low fluoride with nifedipine reduced the Cav1.2 expression level. Thus, Cav1.2 may be an important molecular target for the fluorosis treatment, and the LTCC inhibitor nifedipine may be an effective drug for fluorosis.

To characterize the role of cAMP-dependent protein kinase (PKA) in regulating vascular Ca2+ current through Cav1.2 channels [ICa1.2], we have documented a marked capacity of the isoquinoline H-89, widely used as a PKA inhibitor, to reduce current amplitude. We hypothesized that the ICa1.2 inhibitory activity of H-89 was mediated by mechanisms unrelated to PKA inhibition. To support this, an in-depth analysis of H-89 vascular effects on both ICa1.2 and contractility was undertaken by performing whole-cell patch-clamp recordings and functional experiments in rat tail main artery single myocytes and rings, respectively. H-89 inhibited ICa1.2 with a pIC50 (M) value of about 5.5, even under conditions where PKA activity was either abolished by both the PKA antagonists KT5720 and protein kinase inhibitor fragment 6-22 amide or enhanced by the PKA stimulators 6-Bnz-cAMP and 8-Br-cAMP. Inhibition of ICa1.2 by H-89 appeared almost irreversible upon washout, was charge carrier- and voltage-dependent, and antagonised by the Cav1.2 channel agonist (S)-(-)-Bay K 8644. H-89 did not alter both potency and efficacy of verapamil, did not affect current kinetics or voltage-dependent activation, while shifting to the left the 50% voltage of inactivation in a concentration-dependent manner. H-89 docked at the α1C subunit in a pocket region close to that of (S)-(-)-Bay K 8644 docking, forming a hydrogen bond with the same, key amino acid residue Tyr-1489. Finally, both high K+- and (S)-(-)-Bay K 8644-induced contractions of rings were fully reverted by H-89. In conclusion, these results indicate that H-89 inhibited vascular ICa1.2 and, consequently, the contractile function through a PKA-independent mechanism. Therefore, caution is recommended when interpreting experiments where H-89 is used to inhibit vascular smooth muscle PKA.

Nicotine use is one of the most common forms of drug addiction. Although L-type calcium channels (LTCCs) are involved in nicotine addiction, the contribution of the two primary LTCC subtypes (Cav1.2 and 1.3) is unknown. This study aims to determine the contribution of these two LTCC subtypes to nicotine-induced conditioned place preference (CPP) responses by using transgenic mouse models that do not express Cav1.3 (Cav1.3-/-) or contain a mutation in the dihydropyridine (DHP) site of the Cav1.2 (Cav1.2DHP-/-). We found a hyperbolic dose dependent nicotine (0.1-1mg/kg; 0.5mg/kg optimum) effect on place preference in wild type (WT) mice, that could be prevented by the DHP LTCC blocker nifedipine pretreatment. Similarly, Cav1.3-/- mice showed nicotine-induced place preference which was antagonized by nifedipine. In contrast, nifedipine pretreatment of Cav1.2DHP-/- mice had no effect on nicotine-induced CPP responses, suggesting an involvement of Cav1.2 subtype in the nicotine-induced CPP response. Nifedipine alone failed to produce either conditioned place aversion or CPP in WT mice. These results collectively indicate Cav1.2, but not Cav1.3 LTCC subtype regulates, at least in part, the reinforcing effects of nicotine use.

Objective Voltage-gated inward Ca 2+ currents (ICa) are triggered by cell depolarization and commonly produce transient increases in the cytoplasmic free Ca 2+ concentration. The Ca V 1.2 distal C-terminus is susceptible to proteolytic cleavage, which yields a truncated Ca V 1.2 subunit and a cleaved C-terminal fragment (CCt or DCT). Stem cells from the apical papilla (SCAPs) has a capacity for differentiation into the odontoblastic-like cells in vitro and dentin forming in vivo, which makes SCAPs advantages in tissue engineering and regenerative endodontic. The aim of this study was to investigate the effect of Ca V 1.2 and its distal C-terminal fragment in the odontoblastic differentiation of rat SCAPs (stem cells from the apical papilla). Design In this study, we generated stable Ca V 1.2 knockdown and DCT over-expressed rSCAPs using short hairpin RNA and DCT gene containing Lentivirus vectors, respectively. The transfected apical papilla cells were induced to differentiate into the odontoblast-like cells, and the expression of markers for odontoblastic differentiation were analyzed by alizarin red staining, Real-time Polymerase chain reaction (RT-PCR), and Western blot analysis. Results The knockdown of Ca V 1.2 and excess expression of DCT both suppressed the expression of DSPP, ALP in mRNA level and the formation of calcium nodules. Conclusions Our results suggest that Ca V 1.2 and DCT play important roles in the differentiation of rSCAPs, DCT might act as a transcription factor and regulate the differentiation of rSCAPs.

Aims Azelnidipine, a third-generation dihydropyridine calcium channel blocker (DHP CCB), has a characteristic hypotensive effect that persists even after it has disappeared from the plasma, which is thought to be due to its high hydrophobicity. However, because azelnidipine is unique, it might have other unknown effects on L-type Cav1.2 channels that result in the long-lasting decrease of blood pressure. The aim of this study was to investigate the potential quantitative modification of Cav1.2 by azelnidipine. Main methods HEK293 cells were used to express Cav1.2 channels. Immunocytochemical analysis was performed to detect changes in the surface expression of the pore-forming subunit of the Cav1.2 channel, Cav1.2α1c. Western blotting analysis was performed to evaluate changes in expression levels of total Cav1.2α1c and Cavβ2c. Key findings The surface expression of Cav1.2α1c was markedly reduced by treatment with azelnidipine, but not with other DHP CCBs (amlodipine and nicardipine). Results obtained with a dynamin inhibitor and an early endosome marker suggested that the reduction of surface Cav1.2α1c was not likely caused by internalization. Azelnidipine reduced the total amount of Cav1.2α1c protein in HEK293 cells and rat pulmonary artery smooth muscle cells. The reduction of Cav1.2α1c was rescued by inhibiting proteasome activity. In contrast, azelnidipine did not affect the amount of auxiliary Cavβ2c subunits that function as a chaperone of Cav1.2. Significance This study is the first to demonstrate that azelnidipine reduces the expression of Cav1.2α1c, which might partly explain its long-lasting hypotensive effect.

CaV1.2 channels play a fundamental role in the regulation of vascular smooth muscle tone. The aim of the present study was to synthesize morin derivatives bearing the nitrophenyl moiety of dihydropyridine Ca2+ antagonists to increase the flavonoid vasorelaxant activity. The effects of morin and its derivatives were assessed on CaV1.2 and KCa1.1 channels, both in vitro and in silico, as well as on the contractile responses of rat aorta rings. All compounds were effective CaV1.2 channel blockers, positioning in the α1C subunit region where standard blockers bind. Among the four newly synthesized morin derivatives, the penta-acetylated morin-1 was the most efficacious Ca2+ antagonist, presenting a vasorelaxant profile superior to that of the parent compound and, contrary to morin, antagonized also the release of Ca2+ from the sarcoplasmic reticulum; surprisingly, it also stimulated KCa1.1 channel current. Computational analysis demonstrated that morin-1 bound close to the KCa1.1 channel S6 segment. In conclusion, these findings open a new avenue for the synthesis of valuable multi-functional, vasorelaxant morin derivatives capable to target several pathways underpinning the pathogenesis of hypertension.

Careful analysis of previously published reports and some new insights into the structure activity studies revealed an important role of Threonine 1143 in drug binding. Substituting T1143 by alanine and other residues significantly reduced channel inhibition by qDil and Dil. Mutation T1143A did not affect channel activation or inactivation while almost completely diminishing channel block by Dil or qDil. These findings support the view that T1143 serves as drug binding determinant. Other mutations in this position than T1143A (T1143L/Y/S/N/C/V/E) diminished channel inhibition by qDil but additionally affected channel activation and inactivation and may therefore affect channel block allosterically. Collectively, our data suggest that T1143 is an essential diltiazem binding determinant.

Ca 2+ -activated Cl − current (I Cl(Ca) ) mediated by TMEM16A and/or Bestrophin-3 may contribute to cardiac arrhythmias. The true profile of I Cl(Ca) during an actual ventricular action potential (AP), however, is poorly understood. We aimed to study the profile of I Cl(Ca) systematically under physiological conditions (normal Ca 2+ cycling and AP voltage-clamp) as well as in conditions designed to change [Ca 2+ ] i . The expression of TMEM16A and/or Bestrophin-3 in canine and human left ventricular myocytes was examined. The possible spatial distribution of these proteins and their co-localization with Ca v 1.2 was also studied. The profile of I Cl(Ca), identified as a 9-anthracene carboxylic acid-sensitive current under AP voltage-clamp conditions, contained an early fast outward and a late inward component, overlapping early and terminal repolarizations, respectively. Both components were moderately reduced by ryanodine, while fully abolished by BAPTA, but not EGTA. [Ca 2+ ] i was monitored using Fura-2-AM. Setting [Ca 2+ ] i to the systolic level measured in the bulk cytoplasm (1.1 μM) decreased I Cl(Ca), while application of Bay K8644, isoproterenol, and faster stimulation rates increased the amplitude of I Cl(Ca) . Ca 2+ -entry through L-type Ca 2+ channels was essential for activation of I Cl(Ca) . TMEM16A and Bestrophin-3 showed strong co-localization with one another and also with Ca v 1.2 channels, when assessed using immunolabeling and confocal microscopy in both canine myocytes and human ventricular myocardium. Activation of I Cl(Ca) in canine ventricular cells requires Ca 2+ -entry through neighboring L-type Ca 2+ channels and is only augmented by SR Ca 2+ -release. Substantial activation of I Cl(Ca) requires high Ca 2+ concentration in the dyadic clefts which can be effectively buffered by BAPTA, but not EGTA.

Seventeen compounds, rather selective, direct or indirect inhibitors and activators of PKA, PKG, and PKC, were analysed for effects on vascular CaV1.2 channel current (ICa1.2) by using the patch-clamp technique in single rat tail artery myocytes. The aim was to investigate how PKs regulate ICa1.2 and disclose any unexpected modulation of CaV1.2 channel function by these agents. The cAMP analogues 8-Br-cAMP and 6-Bnz-cAMP partially reduced ICa1.2 in dialysed cells, while weakly increasing it under the perforated configuration. The β-adrenoceptor agonist isoproterenol and the adenylate cyclase activator forskolin concentration-dependently increased ICa1.2; this effect was reversed by PKA inhibitors H-89 and KT5720, but not by PKI 6-22. The cGMP analogue 8-Br-cGMP, similarly to the NO-donor SNP, moderately reduced ICa1.2, this effect being reversed to a slight stimulation under the perforated configuration. Among PKG inhibitors, Rp-8-Br-PET-cGMPS decreased current amplitude in a concentration-dependent manner while Rp-8-Br-cGMPS was ineffective. The non-specific phosphodiesterase inhibitor IBMX increased ICa1.2, while H-89, KT5720, and PKI 6-22 antagonized this effect. The PKC activator PMA, but not the diacylglycerol analogue OAG, stimulated ICa1.2 in a concentration-dependent manner; conversely, the PKCα inhibitor Go6976 markedly reduced basal ICa1.2 and, similarly to the PKCδ (rottlerin) and PKCe translocation inhibitors antagonised PMA-induced current stimulation. The ensemble of findings indicates that the stimulation of cAMP/PKA, in spite of the paradoxical effect of both 8-Br-cAMP and 6-Bnz-cAMP, or PKC pathways enhanced, while that of cGMP/PKG weakly inhibited ICa1.2 in rat tail artery myocytes. Since Rp-8-Br-PET-cGMPS and Go6976 appeared to block directly CaV1.2 channel, their docking to the channel protein was investigated. Both compounds appeared to bind the α1C subunit in a region involved in CaV1.2 channel inactivation, forming an interaction network comparable to that of CaV1.2 channel blockers. Therefore, caution should accompany the use of these agents as pharmacological tools to elucidate the mechanism of action of drugs on vascular preparations.

CaV1.2 and transient receptor potential canonical channel 3 (TRPC3) are two proteins known to have important roles in pathological cardiac hypertrophy; however, such roles still remain unclear. A better understanding of these roles is important for furthering the clinical understanding of heart failure. We previously reported that Trpc3-knockout (KO) mice are resistant to pathologic hypertrophy and that their CaV1.2 protein expression is reduced. In this study, we aimed to examine the relationship between these two proteins and characterize their role in neonatal cardiomyocytes. We measured CaV1.2 expression in the hearts of wild-type (WT) and Trpc3-/- mice, and examined the effects of Trpc3 knockdown and overexpression in the rat cell line H9c2. We also compared the hypertrophic responses of neonatal cardiomyocytes cultured from Trpc3-/- mice to a representative hypertrophy-causing drug, isoproterenol (ISO), and measured the activity of nuclear factor of activated T cells 3 (NFAT3) in neonatal cardiomyocytes (NCMCs). We inhibited the L-type current with nifedipine, and measured the intracellular calcium concentration using Fura-2 with 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced Ba2+ influx. When using the Trpc3-mediated Ca2+ influx, both intracellular calcium concentration and calcium influx were reduced in Trpc3-KO myocytes. Not only was the expression of CaV1.2 greatly reduced in Trpc3-KO cardiac lysate, but the size of the CaV1.2 currents in NCMCs was also greatly reduced. When NCMCs were treated with Trpc3 siRNA, it was confirmed that the expression of CaV1.2 and the intracellular nuclear transfer activity of NFAT decreased. In H9c2 cells, the ISO activated- and verapamil inhibited- Ca2+ influxes were dramatically attenuated by Trpc3 siRNA treatment. In addition, it was confirmed that both the expression of CaV1.2 and the size of H9c2 cells were regulated according to the expression and activation level of TRPC3. We found that after stimulation with ISO, cell hypertrophy occurred in WT myocytes, while the increase in size of Trpc3-KO myocytes was greatly reduced. These results suggest that not only the cell hypertrophy process in neonatal cardiac myocytes and H9c2 cells were regulated according to the expression level of CaV1.2, but also that the expression level of CaV1.2 was regulated by TRPC3 through the activation of NFAT.

Introduction We investigated whether abnormal activity of the L-type Ca2+ channels (LTCCs), Cav1.2 and Cav1.3, under extracellular hypercalcemia, impaired heart pacemaker activity. Membrane currents and intracellular Ca2+ ([Ca2 +]i) dynamics of the sino-atrial node (SAN) generate the heart pacemaker activity. Impairments of SAN activity, also called SAN dysfunction (SND), harms heart automaticity. Recent studies have associated abnormal [Ca2 +]i dynamics with SNDs. Cav1.2 and Cav1.3 carry the main Ca2+ influx of SAN cells to sustain their [Ca2 + ]i dynamics. Modified extracellular Ca2+ ([Ca2 +]o) could alter Ca2+ influx through these channels. For example, cancer and hyperparathyroidism can raise [Ca2 + ]o, causing an extracellular hypercalcemia that could alter [Ca2 + ]i dynamics and impair SAN activity and heart automaticity. Methods and results To test this hypothesis, we measured contractions, [Ca2 +]i release and L-type Ca2+ current (ICa,L) in spontaneous cells of the murine SAN. Then, we recorded rate and propagation of the spontaneous action potentials (APs) generated by the SAN tissue ex-vivo. In spontaneous SAN cells, we observed that the modification of [Ca2 +]o modulates [Ca2 +]i and cells contraction through changes of ICa,L. In particular, the increase of [Ca2 +]o dysregulated pacemaker activity, likely through excessive Ca2+ influx mediated by Cav1.2. [Ca2 +]o increase to hypercalcemic levels induced arrhythmia also in the intact SAN tissues, activating ectopic leading regions of pacemaking and impairing conduction towards the atria. We recovered these pacemaker dysfunctions by blocking Ca2 + -activated small-conductance K+ (SK) channels. Conclusions Hypercalcemia causes excessive Cav1.2-mediated Ca2+ influx, which alters [Ca2 +]i and could hyperactivate SK channels, leading to pacemaker impairment. Cav1.2 or SK modulation may reduce pacemaker dysfunctions, preventing SND progression.

Certain voltage-activated Ca2+ channels have been reported to act as potential zinc entry routes. However, it remains to be determined whether zinc can permeate individual Ca2+ channel isoforms. We expressed recombinant Ca2+ channel isoforms in Xenopus oocytes and attempted to record zinc currents from them using a two-electrode voltage clamp method. We found that, in an extracellular zinc solution, inward currents arising from zinc permeation could be recorded from Xenopus oocytes expressing L-type Cav1.2 or Cav1.3 isoforms, but not from oocytes expressing Cav2.2, Cav2.3, Cav3.1, or Cav3.2. Zinc currents through Cav1.2 and Cav1.3 were blocked by nimodipine, but enhanced by (±)Bay K8644, supporting the finding that zinc can permeate both L-type Cav1.2 and Cav1.3 channel isoforms. We also examined the blocking effects of low concentrations of zinc on Ca2+ currents through the L-type channel isoforms. Low micro-molar zinc potently blocked Ca2+ currents through Cav1.2 and Cav1.3 with different sensitivities (IC50 for Cav1.2 and Cav1.3=18.4 and 34.1μM) and de-accelerated the activation and inactivation kinetics in a concentration-dependent manner. Notably, mild acidifications of the external zinc solution increased zinc currents through Cav1.2 and Cav1.3, with the increment level for Cav1.3 being greater than that for Cav1.2. In overall, we provide evidence that Cav1.2 and Cav1.3 isoforms are capable of potentially functioning as zinc permeation routes, through which zinc entry can be differentially augmented by mild acidifications.

Cav1.2 and Cav1.3 are the major L-type voltage-gated Ca(2+) channels in the CNS. Yet, their individual in vivo functions are largely unknown. Both channel subunits are expressed in the auditory brainstem, where Cav1.3 is essential for proper maturation. Here, we investigated the role of Cav1.2 by targeted deletion in the mouse embryonic auditory brainstem. Similar to Cav1.3, loss of Cav1.2 resulted in a significant decrease in the volume and cell number of auditory nuclei. Contrary to the deletion of Cav1.3, the action potentials of lateral superior olive (LSO) neurons were narrower compared with controls, whereas the firing behavior and neurotransmission appeared unchanged. Furthermore, auditory brainstem responses were nearly normal in mice lacking Cav1.2. Perineuronal nets were also unaffected. The medial nucleus of the trapezoid body underwent a rapid cell loss between postnatal days P0 and P4, shortly after circuit formation. Phosphorylated cAMP response element-binding protein (CREB), nuclear NFATc4, and the expression levels of p75NTR, Fas, and FasL did not correlate with cell death. These data demonstrate for the first time that both Cav1.2 and Cav1.3 are necessary for neuronal survival but are differentially required for the biophysical properties of neurons. Thus, they perform common as well as distinct functions in the same tissue.

The activity of Cav1.2 Ca(2+) channels is maintained in the presence of calmodulin and ATP, even in cell-free patches, and thus a channel ATP-binding site has been suggested. In this study, we examined whether other nucleotides, such as GTP, UTP, CTP, ADP and AMP, could be substituted for ATP in guinea-pig ventricular myocytes. We found that all the nucleotides tested could re-prime the Ca(2+) channels in the presence of 1 μM calmodulin in the inside-out mode. The order of efficacy was ATP > GTP > UTP > ADP > CTP ≈ AMP. Thus, the presumed nucleotide-binding site in the channel seemed to favor a purine rather than pyrimidine base and a triphosphate rather than a di- or mono-phosphate group. Furthermore, a high concentration (10 mM) of GTP, UTP, CTP, ADP and AMP had inhibitory effects on the channel activity. These results provide information on the putative nucleotide-binding site(s) in Cav1.2 Ca(2+) channels.

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.

Gain-of-function mutations in CACNA1C, encoding the L-type Ca(2+) channel Cav1.2, cause Timothy syndrome (TS), a multi-systemic disorder with dysmorphic features, long-QT syndrome (LQTS) and autism spectrum disorders. TS patients have heterozygous mutations (G402S and G406R) located in the alternatively spliced exon 8, causing a gain-of-function by reduced voltage-dependence of inactivation. Screening 540 unrelated patients with non-syndromic forms of LQTS, we identified six functional relevant CACNA1C mutations in different regions of the channel. All these mutations caused a gain-of-function combining different mechanisms, including changes in current amplitude, rate of inactivation and voltage-dependence of activation or inactivation, similar as in TS. Computer simulations support the theory that the novel CACNA1C mutations prolong action potential duration. We conclude that genotype-negative LQTS patients should be investigated for mutations in CACNA1C, as a gain-of-function in Cav1.2 is likely to cause LQTS and only specific and rare mutations, i.e. in exon 8, cause the multi-systemic TS.

Voltage-gated Ca(2+) channels play a key role in initiating muscle excitation-contraction coupling, neurotransmitter release, gene expression, and hormone secretion. The association of CaV1.2 with a supramolecular complex impacts trafficking, localization, turnover, and, most importantly, multifaceted regulation of its function in the heart. Several studies hint at an important role for the C terminus of the α1C subunit as a hub for multidimensional regulation of CaV1.2 channel trafficking and function. Recent studies have demonstrated an important role for the four-residue PDZ binding motif at the C terminus of α1C in interacting with scaffold proteins containing PDZ domains, in the subcellular localization of CaV1.2 in neurons, and in the efficient signaling to cAMP-response element-binding protein in neurons. However, the role of the α1C PDZ ligand domain in the heart is not known. To determine whether the α1C PDZ motif is critical for CaV1.2 trafficking and function in cardiomyocytes, we generated transgenic mice with inducible expression of an N-terminal FLAG epitope-tagged dihydropyridine-resistant α1C with the PDZ motif deleted (ΔPDZ). These mice were crossed with α-myosin heavy chain reverse transcriptional transactivator transgenic mice, and the double-transgenic mice were fed doxycycline. The ΔPDZ channels expressed, trafficked to the membrane, and supported robust excitation-contraction coupling in the presence of nisoldipine, a dihydropyridine Ca(2+) channel blocker, providing functional evidence that they appropriately target to dyads. The ΔPDZ Ca(2+) channels were appropriately regulated by isoproterenol and forskolin. These data indicate that the α1C PDZ motif is not required for surface trafficking, localization to the dyad, or adrenergic stimulation of CaV1.2 in adult cardiomyocytes.

We have recently shown that freshly isolated astrocytes from the mouse brain express mRNA for the L-channel gene Cav1.3 to at least the same degree (per mg mRNA) as corresponding neurons. The amount of extracellular Ca(2+) actually entering cultured astrocytes by its opening is modest, but due to secondary Ca(2+)-mediated stimulation of the ryanodine receptor (RyR) the increase in free cytosolic Ca(2+) [Ca(2+)]i is substantial. The other Cav1 subtype expressed in brain is Cav1.2, which is even expressed in higher density. Although the different primers used for the two genes preclude exact quantitative comparison, the present study suggests that this is also the case in the freshly isolated astrocytes and neurons, which express equal Cav1.2 densities. Again, most of the increase in [Ca(2+)]i occurred by RyR activity. In contrast to Cav1.3 the expression of Cav1.2 was greatly increased (doubled) after two weeks of treatment with fluoxetine hydrochloride (10mg/kg). Accordingly [Ca(2+)]i in cultured astrocytes exposed to the addition of 10-60mM KCl increased substantially in cultured astrocytes treated chronically with fluoxetine with the lag time until the effect was observed depending upon the fluoxetine concentration. This effect was inhibited by nifedipine or siRNA against Cav1.2. The increase in K(+)-induced rise in [Ca(2+)]i after fluoxetine treatment is directly opposite to a decrease in [Ca(2+)]i after treatment with any of the anti-bipolar drugs lithium, carbamazepine or valproic acid, due to reduced capacitative Ca(2+) influx. We have previously shown a similar effect after fluoxetine treatment, but it becomes overridden by the Cav1.2 up-regulation.

Aims Studies suggest that cardiovascular function in offspring can be epigenetically programmed by environmental changes during pregnancy. CaV1.2 channel plays a major role in the regulation of the vascular tone. This study investigated the effects and underlying mechanisms of exercise during pregnancy on CaV1.2 channel functional remodeling in hypertensive offspring. Main methods Exercise groups were subjected to swimming at the first day of pregnancy and on a regular schedule thereafter for 3 weeks. Their offspring (6-month-old, male) were tested for baseline blood pressure, cardiovascular response, and vascular tone of the mesenteric artery. Mesenteric artery smooth muscle cells were taken to study the whole-cell current of the CaV1.2 channel. Western blotting, RT-PCR and DNA bisulfite sequencing PCR were performed to study the protein, mRNA expression and DNA methylation of the CaV1.2 channel α1C subunit. Key findings Exercise during pregnancy reduced the pressor response to norepinephrine and Bay K8644, and the depressor response to nifedipine in offspring of hypertensive rats. The level of the CaV1.2 channel in norepinephrine-induced vasoconstrictions decreased, and the whole-cell current of the CaV1.2 channel declined in the SHR-EX group. Further studies found that exercise during pregnancy reduced the protein and mRNA expression of the CaV1.2 channel α1C subunit and upregulated DNA methylation of the Cacna1c gene promoter region in the hypertensive offspring. Significance These data suggest that exercise during pregnancy improves vascular functional remodeling in offspring of hypertensive rats, downregulating the CaV1.2 channel function and protein expression, a change that is most likely caused by DNA methylation.

Ca(2+) flux through l-type CaV1.2 channels shapes the waveform of the ventricular action potential (AP) and is essential for excitation-contraction (EC) coupling. Timothy syndrome (TS) is a disease caused by a gain-of-function mutation in the CaV1.2 channel (CaV1.2-TS) that decreases inactivation of the channel, which increases Ca(2+) influx, prolongs APs, and causes lethal arrhythmias. Although many details of the CaV1.2-TS channels are known, the cellular mechanisms by which they induce arrhythmogenic changes in intracellular Ca(2+) remain unclear. We found that expression of CaV1.2-TS channels increased sarcolemmal Ca(2+) "leak" in resting TS ventricular myocytes. This resulted in higher diastolic [Ca(2+)]i in TS ventricular myocytes compared to WT. Accordingly, TS myocytes had higher sarcoplasmic reticulum (SR) Ca(2+) load and Ca(2+) spark activity, larger amplitude [Ca(2+)]i transients, and augmented frequency of Ca(2+) waves. The large SR Ca(2+) release in TS myocytes had a profound effect on the kinetics of CaV1.2 current in these cells, increasing the rate of inactivation to a high, persistent level. This limited the amount of influx during EC coupling in TS myocytes. The relationship between the level of expression of CaV1.2-TS channels and the probability of Ca(2+) wave occurrence was non-linear, suggesting that even low levels of these channels were sufficient to induce maximal changes in [Ca(2+)]i. Depolarization of WT cardiomyocytes with a TS AP waveform increased, but did not equalize [Ca(2+)]i, compared to depolarization of TS myocytes with the same waveform. We propose that CaV1.2-TS channels increase [Ca(2+)] in the cytosol and the SR, creating a Ca(2+)overloaded state that increases the probability of arrhythmogenic spontaneous SR Ca(2+) release.

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

The modulation and regulation of voltage-gated Ca2+ channels is affected by the pore-forming segments, the cytosolic parts of the channel, and interacting intracellular proteins. In this study we demonstrate a direct physical interaction between the N terminus (NT) and C terminus (CT) of the main subunit of the L-type Ca2+ channel CaV1.2, α1C, and explore the importance of this interaction for the regulation of the channel. We used biochemistry to measure the strength of the interaction and to map the location of the interaction sites, and electrophysiology to investigate the functional impact of the interaction. We show that the full-length NT (amino acids 1-154) and the proximal (close to the plasma membrane) part of the CT, pCT (amino acids 1508-1669) interact with sub-micromolar to low-micromolar affinity. Calmodulin (CaM) is not essential for the binding. The results further suggest that the NT-CT interaction regulates the channel's inactivation, and that Ca2+, presumably through binding to calmodulin (CaM), reduces the strength of NT-CT interaction. We propose a molecular mechanism in which NT and CT of the channel serve as levers whose movements regulate inactivation by promoting changes in the transmembrane core of the channel via S1 (NT) or S6 (pCT) segments of domains I and IV, accordingly, and not as a kind of pore blocker. We hypothesize that Ca2+-CaM-induced changes in NT-CT interaction may, in part, underlie the acceleration of CaV1.2 inactivation induced by Ca2+ entry into the cell.

Dihydropyridine Ca2+ channel antagonists (DHPs) block Ca(V)1.2 L-type Ca2+ channels (LTCCs) by stabilizing their voltage-dependent inactivation (VDI); however, it is still not clear how DHPs allosterically interact with the kinetically distinct (fast and slow) VDI. Thus, we analyzed the effect of a prototypical DHP, nifedipine on LTCCs with or without the Timothy syndrome mutation that resides in the I-II linker (LI-II) of Ca(V)1.2 subunits and impairs VDI. Whole-cell Ba2+ currents mediated by rabbit Ca(V)1.2 with or without the Timothy mutation (G436R) (analogous to the human G406R mutation) were analyzed in the presence and absence of nifedipine. In the absence of nifedipine, the mutation significantly impaired fast closed-and open-state VDI (CSI and OSI) at -40 and 0 mV, respectively, but did not affect channels' kinetics at -100 mV. Nifedipine equipotently blocked these channels at -80 mV. In wild-type LTCCs, nifedipine promoted fast CSI and OSI at -40 and 0 mV and promoted or stabilized slow CSI at -40 and -100 mV, respectively. In LTCCs with the mutation, nifedipine resumed the impaired fast CSI and OSI at -40 and 0 mV, respectively, and had the same effect on slow CSI as in wild-type LTCCs. Therefore, nifedipine has two mechanistically distinct effects on LTCCs: the promotion of fast CSI/OSI caused by LI-II at potentials positive to the sub-threshold potential and the promotion or stabilization of slow CSI caused by different mechanisms at potentials negative to the subthreshold potential., Article, EUROPEAN JOURNAL OF PHARMACOLOGY. 685(1-3):15-23 (2012)

An important regulatory mechanism of excitation-evoked Ca2+ influx is voltage-dependent, or pre-pulse, facilitation (VDF): a memory property of some CaV channels, whereby channel activation is enhanced following a pre-conditioning pulse of sufficient duration and amplitude. While VDF can greatly increase Ca2+ influx and confer memory of excitation, the molecular determinants for its voltage- and time-dependent properties are unclear. We posited that VDF emerges from the activity of one, or more, of the four homologous, but non-identical, voltage-sensing domains (VSDs) of the pore-forming α1 subunit. Using voltage clamp fluorometry, we optically tracked the activation of each VSD in human CaV1.2 (α1C) channels expressed in Xenopus oocytes in association with β3 subunits, voltage-clamped by the cut-open oocyte method, in the absence or presence of facilitating pre-pulses (80 mV, 200 ms). Ba2+ was used as the charge carrier to exclude Ca2+-dependent processes. Pre-pulses increased the activation of VSD-I and -II at 20 mV by ∼60% and accelerated VSD-I activation kinetics by ∼10-fold. VSD-III activation was shifted to more hyperpolarized potentials by ∼20mV and doubled in effective valence. In non-facilitated α1C/β3 channels, VSD-II and -III are weakly-coupled to channel opening (Savalli et al., 2016 J Gen Physiol); pre-pulse-induced facilitation of their activation suggests that VDF engages these VSDs with the channel gate. This premise is supported by the finding that association of the α2δ-1 subunit (which mimics facilitation; Platano et al., 2000 Biophys J) strongly couples VSD-II and -III to the channel gate (Pantazis et al., 2014 PNAS). Since VSD-I (i) is also potentiated by pre-pulses, but (ii) is weakly-coupled to channel opening in both non-facilitated α1C/β3 and “constitutively facilitated” α1C/β3/α2δ channels, our work highlights it as a plausible candidate for the structural determinant of VDF.

Within the voltage-gated calcium channels (Cav channels) family, there are four genes coding for the L-type Cav channels (Cav1). The Cav1 channels underly many important physiological functions like excitation–contraction coupling, hormone secretion, neuronal excitability and gene transcription. Mutations found in the genes encoding the Cav channels define a wide variety of diseases called calcium channelopathies and all four genes coding the Cav1 channels are carrying such mutations. L-type calcium channelopathies include muscular, neurological, cardiac and vision syndromes. Among them, the Timothy syndrome (TS) is linked to missense mutations in CACNA1C, the gene that encodes the Ca v 1.2 subunit. Here we review the important features of the Cav1 channelopathies. We also report on the specific properties of TS–Ca v 1.2 channels, which display non-inactivating calcium current as well as higher plasma membrane expression. Overall, we conclude that both electrophysiological and surface expression properties must be investigated to better account for the functional consequences of mutations linked to calcium channelopathies.

The binding site within the L-type Ca(2+) channel Ca(v)1.2 for neutral dihydropyridines is well characterized. However, the contributions of the alkylamino side chains of charged dihydropyridines such as amlodipine and nicardipine to channel block are not clear. We tested the hypothesis that the distinct locations of the charged side chains on amlodipine and nicardipine would confer distinct properties of channel block by these two drugs. Using whole-cell voltage clamp, we investigated block of wild type Ca(v) 2.1, wild type Ca(v)1.2, and Ca(v)1.2/Dihydropyridine insensitive, a mutant channel insensitive to neutral DHPs, by amlodipine and nicardipine. The potency of nicardipine and amlodipine for block of closed (stimulation frequency of 0.05 Hz) Ca(v)1.2 channels was not different (IC(50) values of 60 nM and 57 nM, respectively), but only nicardipine block was enhanced by increasing the stimulation frequency to 1 Hz. The frequency-dependent block of Ca(v)1.2 by nicardipine is the result of a strong interaction of nicardipine with the inactivated state of Ca(v)1.2. However, nicardipine block of Ca(v)1.2/Dihydropyridine insensitive was much more potent than block by amlodipine (IC(50) values of 2.0 μM and 26 μM, respectively). A mutant Ca(v)2.1 channel containing the neutral DHP binding site (Ca(v)2.1/Dihydropyridine sensitive) was more potently blocked by amlodipine (IC(50)=41 nM) and nicardipine (IC(50)=175 nM) than the parent Ca(v)2.1 channel. These data suggest that the alkylamino group of nicardipine and amlodipine project into distinct regions of Ca(v)1.2 such that the side chain of nicardipine, but not amlodipine, contributes to the potency of closed-channel block, and confers frequency-dependent block.

Cav1.2 Ca(2+) channel activity diminishes in inside-out patches (run-down). Previously, we have found that with ATP, calpastatin domain L (CSL) and calmodulin (CaM) recover channel activity from the run-down in guinea pig cardiac myocytes. Because the potency of the CSL repriming effect was smaller than that of CaM, we hypothesized that CSL might act as a partial agonist of CaM in the channel-repriming effect. To examine this hypothesis, we investigated the effect of the competitions between CSL and CaM on channel activity and on binding in the channel. We found that CSL suppressed the channel-activating effect of CaM in a reversible and concentration-dependent manner. The channel-inactivating effect of CaM seen at high concentrations of CaM, however, did not seem to be affected by CSL. In the GST pull-down assay, CSL suppressed binding of CaM to GST fusion peptides derived from C-terminal regions in a competitive manner. The inhibition of CaM binding by CSL was observed with the IQ peptide but not the PreIQ peptide, which is the CaM-binding domain in the C terminus. The results are consistent with the hypothesis that CSL competes with CaM as a partial agonist for the site in the IQ domain in the C-terminal region of the Cav1.2 channel, which may be involved in activation of the channel.

The Timothy syndrome mutations G402S and G406R abolish inactivation of Ca(V)1.2 and cause multiorgan dysfunction and lethal arrhythmias. To gain insights into the consequences of the G402S mutation on structure and function of the channel, we systematically mutated the corresponding Gly-432 of the rabbit channel and applied homology modeling. All mutations of Gly-432 (G432A/M/N/V/W) diminished channel inactivation. Homology modeling revealed that Gly-432 forms part of a highly conserved structure motif (G/A/G/A) of small residues in homologous positions of all four domains (Gly-432 (IS6), Ala-780 (IIS6), Gly-1193 (IIIS6), Ala-1503 (IVS6)). Corresponding mutations in domains II, III, and IV induced, in contrast, parallel shifts of activation and inactivation curves indicating a preserved coupling between both processes. Disruption between coupling of activation and inactivation was specific for mutations of Gly-432 in domain I. Mutations of Gly-432 removed inactivation irrespective of the changes in activation. In all four domains residues G/A/G/A are in close contact with larger bulky amino acids from neighboring S6 helices. These interactions apparently provide adhesion points, thereby tightly sealing the activation gate of Ca(V)1.2 in the closed state. Such a structural hypothesis is supported by changes in activation gating induced by mutations of the G/A/G/A residues. The structural implications for Ca(V)1.2 activation and inactivation gating are discussed.

In the present study, we investigated the role of Vps34, a kinase classified into PI 3-kinase class III, in the brain of mouse after repeated in vivo treatment with morphine. The intracerebroventricular (i.c.v.) administration of nifedipine caused a dose-dependent inhibition of morphine-induced place preference. The protein levels of Vps34 in the frontal cortex including the cingulate cortex and the limbic forebrain including the nucleus accumbens significantly increased in morphine-induced place preference. I.c.v. administration of PI 3-kinase inhibitors such as wortmannin and LY294002 inhibited morphine-induced place preference in a dose-dependent manner. Ca(v)1.2 protein level of L-type voltage-dependent calcium channels significantly increased in the frontal cortex and the limbic forebrain of the morphine psychologically dependent mice, which was significantly inhibited by PI 3-kinase inhibitors. Similar changes in the expression of Ca(v)1.2 and Vps34 proteins are found in the primary culture of cerebral cortical neurons during short-term exposure to morphine. Under such conditions, facilitated translocation of Ca(v)1.2 to the plasma membrane remarkably increased by the treatment of the cultured neurons with morphine when measuring protein level of Ca(v)1.2, and such changes in Ca(v)1.2 were also suppressed by PI 3-kinase inhibitors. These findings indicate a critical role of Vps34 in the up-regulation of Ca(v)1.2 in the neuronal plasma membrane and the development of morphine-induced place preference.

We analyzed the distribution of ryanodine receptor (RyR) and Cav1.2 clusters in adult rat ventricular myocytes using three-dimensional object-based colocalization metrics. We found that ∼75% of the Cav1.2 clusters and 65% of the RyR clusters were within couplons, and both were roughly two and a half times larger than their extradyadic counterparts. Within a couplon, Cav1.2 was concentrated near the center of the underlying RyR cluster and accounted for ∼67% of its size. These data, together with previous findings from binding studies, enable us to estimate that a couplon contains 74 RyR tetramers and 10 copies of the α-subunit of Cav1.2. Extradyadic clusters of RyR contained ∼30 tetramers, whereas the extradyadic Cav1.2 clusters contained, on average, only four channels. Between 80% and 85% of both RyR and Cav1.2 molecules are within couplons. RyR clusters were in the closest proximity, with a median nearest-neighbor distance of 552 nm; comparable values for Cav1.2 clusters and couplons were 619 nm and 735 nm, respectively. Extradyadic RyR clusters were significantly closer together (624 nm) and closer to the couplons (674 nm) than the couplons were to each other. In contrast, the extradyadic clusters of Cav1.2 showed no preferential localization and were broadly distributed. These results provide a wealth of morphometric data that are essential for understanding intracellular Ca2+ regulation and modeling Ca2+ dynamics.

Ca(V)beta subunits modulate cell surface expression and voltage-dependent gating of high voltage-activated (HVA) Ca(V)1 and Ca(V)2 alpha1 subunits. High affinity Ca(V)beta binding onto the so-called alpha interaction domain of the I-II linker of the Ca(V)alpha1 subunit is required for Ca(V)beta modulation of HVA channel gating. It has been suggested, however, that Ca(V)beta-mediated plasma membrane targeting could be uncoupled from Ca(V)beta-mediated modulation of channel gating. In addition to Ca(V)beta, Ca(V)alpha2delta and calmodulin have been proposed to play important roles in HVA channel targeting. Indeed we show that co-expression of Ca(V)alpha2delta caused a 5-fold stimulation of the whole cell currents measured with Ca(V)1.2 and Ca(V)beta3. To gauge the synergetic role of auxiliary subunits in the steady-state plasma membrane expression of Ca(V)1.2, extracellularly tagged Ca(V)1.2 proteins were quantified using fluorescence-activated cell sorting analysis. Co-expression of Ca(V)1.2 with either Ca(V)alpha2delta, calmodulin wild type, or apocalmodulin (alone or in combination) failed to promote the detection of fluorescently labeled Ca(V)1.2 subunits. In contrast, co-expression with Ca(V)beta3 stimulated plasma membrane expression of Ca(V)1.2 by a 10-fold factor. Mutations within the alpha interaction domain of Ca(V)1.2 or within the nucleotide kinase domain of Ca(V)beta3 disrupted the Ca(V)beta3-induced plasma membrane targeting of Ca(V)1.2. Altogether, these data support a model where high affinity binding of Ca(V)beta to the I-II linker of Ca(V)alpha1 largely accounts for Ca(V)beta-induced plasma membrane targeting of Ca(V)1.2.

L-type Ca(2+) channels in mammalian brain neurons have either a Ca(V)1.2 or Ca(V)1.3 pore-forming subunit. Recently, it was shown that Ca(V)1.3 Ca(2+) channels underlie autonomous pacemaking in adult dopaminergic neurons in the substantia nigra pars compacta, and this reliance renders them sensitive to toxins used to create animal models of Parkinson's disease. Antagonism of these channels with the dihydropyridine antihypertensive drug isradipine diminishes the reliance on Ca(2+) and the sensitivity of these neurons to toxins, pointing to a potential neuroprotective strategy. However, for neuroprotection without an antihypertensive side effect, selective Ca(V)1.3 channel antagonists are required. In an attempt to identify potent and selective antagonists of Ca(V)1.3 channels, 124 dihydropyridines (4-substituted-1,4-dihydropyridine-3,5-dicarboxylic diesters) were synthesized. The antagonism of heterologously expressed Ca(V)1.2 and Ca(V)1.3 channels was then tested using electrophysiological approaches and the FLIPR Calcium 4 assay. Despite the large diversity in substitution on the dihydropyridine scaffold, the most Ca(V)1.3 selectivity was only about twofold. These results support a highly similar dihydropyridine binding site at both Ca(V)1.2 and Ca(V)1.3 channels and suggests that other classes of compounds need to be identified for Ca(V)1.3 selectivity.

The role of the L-type calcium channel (Cav1.2) as a molecular switch that triggers secretion prior to Ca(2+) transport has previously been demonstrated in bovine chromaffin cells and rat pancreatic beta cells. Here, we examined the effect of specific Cav1.2 allosteric modulators, BayK 8644 (BayK) and FPL64176 (FPL), on the kinetics of catecholamine release, as monitored by amperometry in single bovine chromaffin cells. We show that 2 microm BayK or 0.5 microm FPL accelerates the rate of catecholamine secretion to a similar extent in the presence either of the permeable Ca(2+) and Ba(2+) or the impermeable charge carrier La(3+). These results suggest that structural rearrangements generated through the binding of BayK or FPL, by altering the channel activity, could affect depolarization-evoked secretion prior to cation transport. FPL also accelerated the rate of secretion mediated by a Ca(2+)-impermeable channel made by replacing the wild type alpha(1)1.2 subunit was replaced with the mutant alpha(1)1.2/L775P. Furthermore, BayK and FPL modified the kinetic parameters of the fusion pore formation, which represent the initial contact between the vesicle lumen and the extracellular medium. A direct link between the channel activity and evoked secretion lends additional support to the view that the voltage-gated Ca(2+) channels act as a signaling molecular switch, triggering secretion upstream to ion transport into the cell.

Background: The neurohypophyseal hormones oxytocin (OT) and [Arg 8 ]-vasopressin (AVP) have recently been implicated in cardiac function. This study was designed to investigate actions of OT and AVP on regulation of the voltage-gated Ca 2+ channel in cardiomyocytes. Methods and Results: Cultured neonatal rat cardiomyocytes were stimulated with OT or AVP (10 -8 M to 10 -6 M) for 24-72 h, followed by real-time PCR and patch clamp studies. Although OT did not exert any modulatory effect on L-type Ca 2+ channel current, as a shortterm effect, stimulation of cardiomyocytes with OT but not AVP decreased the expression of Ca v 1.2 mRNA with a reduced L-type Ca 2+ channel current. A transcription factor CREB mRNA expression was down-regulated by OT treatment, although T-type Ca 2+ channels (Ca v 3.1, Ca v 3.2) were unaffected by OT or AVP. Conclusion: OT but not AVP down-regulates L-type Ca 2+ channel expression through the inhibition of CREB transcriptional, suggesting a novel mechanism of OT action in the cardiac electrophysiology.