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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.

β-adrenergic stimulation enhances Ca(2+) currents via L-type, voltage-gated CaV 1.2 channels, strengthening cardiac contraction. The signaling via β-adrenergic receptors (β-ARs) involves elevation of cyclic AMP (cAMP) levels and activation of protein kinase A (PKA). However, how PKA affects the channel remains controversial. Recent studies in heterologous systems and genetically engineered mice stress the importance of the posttranslational proteolytic truncation of distal C-terminus (dCT) of the main (α1C ) subunit. Here, we successfully reconstituted the cAMP/PKA regulation of the dCT-truncated CaV 1.2 in Xenopus oocytes, which previously failed with the non-truncated α1C . cAMP and purified catalytic subunit of PKA, PKA-CS, injected into intact oocytes, enhanced CaV 1.2 currents by 40%(rabbit α1C)to 130% (mouse α1C). PKA blockers were used to confirm specificity and the need for dissociation of the PKA holoenzyme. The regulation persisted in the absence of the clipped dCT (as a separate protein), the A kinase anchoring protein AKAP15, and the phosphorylation sites S1700 and T1704, previously proposed as essential for PKA effect. CaV β2b subunit was not involved, as suggested by extensive mutagenesis. Using deletion/chimeric mutagenesis, we have identified the initial segment of the cardiac long-N-terminal isoform of α1C as a previously unrecognized essential element involved in PKA regulation. We propose that the observed regulation, that exclusively involves the α1C subunit, is one of several mechanisms underlying the overall PKA action on CaV 1.2 in the heart. We hypothesize that PKA is acting on CaV 1.2, in part, by affecting a structural "scaffold" comprising the interacting cytosolic N- and C-termini of α1C .

© 2017 Taylor & FrancisEnhanced L-type Ca2+ channel (LTCC) activity in arterial myocytes contributes to vascular dysfunction during diabetes. Modulation of LTCC activity under hyperglycemic conditions could result from membrane potential-dependent and independent mechanisms. We have demonstrated that elevations in extracellular glucose (HG), similar to hyperglycemic conditions during diabetes, stimulate LTCC activity through phosphorylation of CaV1.2 at serine 1928. Prior studies have also shown that HG can suppress the activity of K+ channels in arterial myocytes, which may contribute to vasoconstriction via membrane depolarization. Here, we used a mathematical model of membrane and Ca2+ dynamics in arterial myocytes to predict the relative roles of LTCC and K+ channel activity in modulating global Ca2+ in response to HG. Our data revealed that abolishing LTCC potentiation normalizes [Ca2+]i, despite the concomitant reduction in K+ currents in response to HG. These results suggest that LTCC stimulation may be the primary mechanism underlying vasoconstriction during hyperglycemia.

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.

Astrocytes are electrically non excitable cells but calcium signaling is widely considered a form of their excitability. Astroglial calcium signaling can be regulated by multiple pathway and the L-type Cav1.2 calcium channel is one of the most important mediator of calcium influx into astrocytes in central nervous system (CNS). A recent study demonstrated that 17β-Estradiol (E2) upregulated Cav1.2 protein mainly through estrogen receptor α (ERα) in rat-cultured cortical astrocytes. Given the larger and more complex architecture of human astrocytes than murine, here, we assessed the E2-mediated effect of Cav1.2 protein in human astrocytoma cell line U87-MG to examine whether there is species difference between rat and human by using Western blots. We found that in cultured U87 cells, E2 increased Cav1.2 protein though ERα like in primary cultures of rat cortical astrocytes. This provides further evidence about the effect of E2 on Cav1.2 protein in astrocytes.

Cav1.2 L-type calcium channels play key roles in long-term synaptic plasticity, sensory transduction, muscle contraction, and hormone release. De novo mutations in the gene encoding Cav1.2 (CACNA1C) causes two forms of Timothy syndrome (TS1, TS2), characterized by a multisystem disorder inclusive of cardiac arrhythmias, long QT, autism, and adrenal gland dysfunction. In both TS1 and TS2, the missense mutation G406R is on the alternatively spliced exon 8 and 8A coding for the IS6-helix of Cav1.2 and is responsible for the penetrant form of autism in most TS individuals. The mutation causes specific gain-of-function changes to Cav1.2 channel gating: a "leftward shift" of voltage-dependent activation, reduced voltage-dependent inactivation, and a "leftward shift" of steady-state inactivation. How this occurs and how Cav1.2 gating changes alter neuronal firing and synaptic plasticity is still largely unexplained. Trying to better understanding the molecular basis of Cav1.2 gating dysfunctions leading to autism, here, we will present and discuss the properties of recently reported typical and atypical TS phenotypes and the effective gating changes exhibited by missense mutations associated with long QTs without extracardiac symptoms, unrelated to TS. We will also discuss new emerging views achieved from using iPSCs-derived neurons and the newly available autistic TS2-neo mouse model, both appearing promising for understanding neuronal mistuning in autistic TS patients. We will also analyze and describe recent proposals of molecular pathways that might explain mistuned Ca2+-mediated and Ca2+-independent excitation-transcription signals to the nucleus. Briefly, we will also discuss possible pharmacological approaches to treat autism associated with L-type channelopathies.

Residue Thr1604 in the CaV1.2 channel is a Ca2+/calmodulin dependent protein kinase II (CaMKII) phosphorylation site, and its phosphorylation status maintains the basic activity of the channel. However, the role of CaV1.2 phosphorylation at Thr1604 in myocardial hypertrophy is incompletely understood. Isoproterenol (ISO) was used to induce cardiomyocyte hypertrophy, and autocamtide-2-related inhibitory peptide (AIP) was added as a treatment. Rats in a myocardial hypertrophy development model were subcutaneously injected with ISO for two or three weeks. The heart and left ventricle weights, each of which were normalized to the body weight and cross-sectional area of the myocardial cells, were used to describe the degree of hypertrophy. Protein expression levels were detected by western blotting. CaMKII-induced CaV1.2 (Thr1604) phosphorylation (p-CaV1.2) was assayed by coimmunoprecipitation. The results showed that CaMKII, HDAC, MEF2 C, and atrial natriuretic peptide (ANP) expression was increased in the ISO group and downregulated by AIP treatment in vitro. There was no difference in the expression of these proteins between the ISO 2-week group and the ISO 3-week group in vivo. CaV1.2 channel expression did not change, but p-CaV1.2 expression was increased after ISO stimulation and decreased by AIP. In the rat model, p-CaV1.2 levels and CaMKII activity were much higher in the ISO 3-week group than in the ISO 2-week group. CaMKII-induced CaV1.2 channel phosphorylation at residue Thr1604 may be one of the key features of myocardial hypertrophy and disease development.Abbreviations: CaMKII: Ca2+/calmodulin dependent protein kinase II; p-CaMKII: autophosphorylated Ca2+/calmodulin dependent protein kinase II; CaM: calmodulin; AIP: autocamtide-2-related inhibitory peptide; ECC: excitation-contraction coupling; ISO: isoproterenol; BW: body weight; HW: heart weight; LVW: left ventricle weight; HDAC: histone deacetylase; p-HDAC: phosphorylated histone deacetylase; MEF2C: myocyte-specific enhancer factor 2C; ANP: atrial natriuretic peptide; PKC: protein kinase C

Dalbergia species heartwood, widely used in traditional medicine to treat various cardiovascular diseases, might represent a rich source of vasoactive agents. In Vietnam, Dalbergia tonkinensis is an endemic tree. Therefore, the aim of the present work was to investigate the vascular activity of R-(−)-3′-hydroxy-2,4,5-trimethoxydalbergiquinol isolated from the heartwood of D. tonkinensis and to provide circular dichroism features of its R absolute configuration. The vascular effects of R-(−)-3′-hydroxy-2,4,5-trimethoxydalbergiquinol were assessed on the in vitro mechanical activity of rat aorta rings, under isometric conditions, and on whole-cell Ba2+ currents through CaV1.2 channels (IBa1.2) recorded in single, rat tail main artery myocytes by means of the patch-clamp technique. R-(−)-3′-Hydroxy-2,4,5-trimethoxydalbergiquinol showed concentration-dependent, vasorelaxant activity on both endothelium-deprived and endothelium intact rings precontracted with the α 1 receptor agonist phenylephrine. Neither the NO (nitric oxide) synthase inhibitor Nω-nitro-L-arginine methyl ester nor the cyclooxygenase inhibitor indomethacin affected its spasmolytic activity. R-(−)-3′-Hydroxy-2,4,5-trimethoxydalbergiquinol-induced vasorelaxation was antagonized by (S)-(−)-Bay K 8644 and unaffected by tetraethylammonium plus glibenclamide. In patch-clamp experiments, R-(−)-3′-hydroxy-2,4,5-trimethoxydalbergiquinol inhibited IBa1.2 in a concentration-dependent manner and significantly decreased the time constant of current inactivation. R-(−)-3′-Hydroxy-2,4,5-trimethoxydalbergiquinol likely stabilized the channel in its closed state, as suggested by molecular modelling and docking simulation to the CaV1.2 channel α 1c subunit. In conclusion, D. tonkinensis species may represent a source of agents potentially useful for the development of novel antihypertensive drugs.

L-type calcium channel (LTCC) gene Cav1.2 on astrocytes play an essential role in the alteration of Ca2+ homeostasis and a line of calcium-related glial-cell functions and neuro-glial dialogue. A wealth of experiment revealed that oxidative stress and reactive astrogliosis account for most neurological diseases. Existing studies shows oxidative stress induces influx of Ca2+ into the cell through LTCC. In this study, we directly investigated the effect of oxidative stress inducing by hydrogen peroxide (H2O2) on LTCC Cav1.2 α1-subunit. We found that H2O2 upregulated LTCC Cav1.2 α1-subunit protein but not mRNA, suggesting H2O2-induced changes in Cav1.2 α1-subunits most likely through posttranscriptional mechanism.

Timothy syndrome (TS) is a neurodevelopmental disorder caused by mutations in the pore-forming subunit α11.2 of the L-type voltage-gated Ca2+-channel Cav1.2, at positions G406R or G402S. Although both mutations cause cardiac arrhythmias, only Cav1.2G406R is associated with the autism-spectrum-disorder (ASD). We show that transcriptional activation by Cav1.2G406R and Cav1.2G402S is driven by membrane depolarization through the Ras/ERK/CREB pathway in a process called excitation-transcription (ET) coupling, as previously shown for wt Cav1.2. This process requires the presence of the intracellular β-subunit of the channel. We found that only the autism-associated mutant Cav1.2G406R, as opposed to the non-autistic mutated channel Cav1.2G402S, exhibits a depolarization-independent CREB phosphorylation, and spontaneous transcription of cFos and MeCP2. A leftward voltage-shift typical of Cav1.2G406R activation, increases channel opening at subthreshold potentials, resulting in an enhanced channel activity, as opposed to a rightward shift in Cav1.2G402S. We suggest that the enhanced spontaneous Cav1.2G406R activity accounts for the increase in basal transcriptional activation. This uncontroled transcriptional activation may result in the manifestation of long-term dysregulations such as autism. Thus, gating changes provide a mechanistic framework for understanding the molecular events underlying the autistic phenomena caused by the G406R Timothy mutation. They might clarify whether a constitutive transcriptional activation accompanies other VGCC that exhibit a leftward voltage-shift of activation and are also associated with long-term cognitive disorders.

TAR DNA-binding protein 43 kDa (TDP-43), encoded by TARDBP, is an RNA-binding protein, the nuclear depletion of which is the histopathological hallmark of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder affecting both upper and lower motor neurons. Besides motor symptoms, patients with ALS often develop nonneuronal signs including glucose intolerance, but the underlying pathomechanism is still controversial, i.e., whether it is impaired insulin secretion and/or insulin resistance. Here, we showed that ALS subjects reduced early-phase insulin secretion and that the nuclear localization of TDP-43 was lost in the islets of autopsied ALS pancreas. Loss of TDP-43 inhibited exocytosis by downregulating CaV1.2 calcium channels, thereby reducing early-phase insulin secretion in a cultured β cell line (MIN6) and β cell–specific Tardbp–knockout mice. Overexpression of CaV1.2 restored early-phase insulin secretion in Tardbp–knocked-down MIN6 cells. Our findings suggest that TDP-43 regulates cellular exocytosis mediated by L-type voltage–dependent calcium channels and, thus, plays an important role in the early phase of insulin secretion by pancreatic islets. Thus, nuclear loss of TDP-43 is implicated in not only the selective loss of motor neurons, but also in glucose intolerance due to impaired insulin secretion at an early stage of ALS.

The L-type Ca2+channel CaV1.2 governs gene expression, cardiac contraction, and neuronal activity. Binding of α-actinin to the IQ motif of CaV1.2 supports its surface localization and postsynaptic targeting in neurons. We report a bi-functional mechanism that restricts CaV1.2 activity to its target sites. We solved separate NMR structures of the IQ motif (residues 1646-1664) bound to α-actinin-1 and to apo-calmodulin (apoCaM). The CaV1.2 K1647A and Y1649A mutations, which impair α-actinin-1 but not apoCaM binding, but not the F1658A and K1662E mutations, which impair apoCaM but not α-actinin-1 binding, decreased single channel open probability, gating charge movement, and its coupling to channel opening. Thus, α-actinin recruits CaV1.2 to defined surface regions and simultaneously boosts its open probability so that CaV1.2 is mostly active when appropriately localized.

Bipolar disorder (BPD) is a common mental illness with significant morbidity and mortality.1 Although evidence have suggested changes in oxidative stress, dopamine and inflammation in BPD, it is hard to define the aetiological mechanism of BPD clearly. Recently, some but not all candidate gene association studies, family-based association studies, linkage studies, genome-wide association studies (GWASs) and meta-analyses showed that mutation of L-type voltage-gated calcium channels (LTCCs) gene CACNA1C is implicated in the mechanism of BPD.2–8 These findings support the possibility that BPD might have calcium channelopathy.9 LTCCs, which consist of a complex of alpha-1, alpha-2/delta and beta subunits in a 1:1:1 ratio, mediate the influx of calcium ions (Ca2+) into the cell on membrane polarization. The alpha-1 subunit consists of 24 transmembrane segments and forms the pore through which ions pass into the cell. Cav1.2 is crucial in modulating kinetics of the LTCCs.10 Cav1.2 is widely expressed in the heart11 12 and brain.13 So far, it is well known for its function in the heart.11 12 LTCCs are located at both presynaptic nerve terminals and postsynaptic dendrites and dendritic spines. It initiates many physiological responses, including secretion, muscle contraction and gene transcription. LTCCs have prominent roles in learning and memory processes, which might be related with the pathology of BPD.14 LTCCs might control gene expression through …

Cav1.2 is the pore‐forming subunit of L‐type voltage‐gated calcium channel (LTCC) that plays an important role in calcium overload and cell death in Alzheimer's disease. LTCC activity can be regulated by estrogen, a sex steroid hormone that is neuroprotective. Here, we investigated the potential mechanisms in estrogen‐mediated regulation of Cav1.2 protein. We found that in cultured primary neurons, 17β‐estradiol (E2) reduced Cav1.2 protein through estrogen receptor α (ERα). This effect was offset by a proteasomal inhibitor MG132, indicating that ubiquitin–proteasome system was involved. Consistently, the ubiquitin (UB) mutant at lysine 29 (K29R) or the K29‐deubiquitinating enzyme TRAF‐binding protein domain (TRABID) attenuated the effect of ERα on Cav1.2. We further identified that the E3 ligase Mdm2 (double minute 2 protein) and the PEST sequence in Cav1.2 protein played a role, as Mdm2 overexpression and the membrane‐permeable PEST peptides prevented ERα‐mediated Cav1.2 reduction, and Mdm2 overexpression led to the reduced Cav1.2 protein and the increased colocalization of Cav1.2 with ubiquitin in cortical neurons in vivo. In ovariectomized (OVX) APP/PS1 mice, administration of ERα agonist PPT reduced cerebral Cav1.2 protein, increased Cav1.2 ubiquitination, and improved cognitive performances. Taken together, ERα‐induced Cav1.2 degradation involved K29‐linked UB chains and the E3 ligase Mdm2, which might play a role in cognitive improvement in OVX APP/PS1 mice.

Summary: The excitability of CA1 hippocampal pyramidal cells is mediated by a slow AHP (sAHP) that responds to calcium increases by Cav1 calcium channels and ryanodine receptors (RyR). We used super-resolution and FRET microscopy to investigate the proximity and functional coupling among Cav1.3/Cav1.2, RyR2, and KCa3.1 potassium channels that contribute to the sAHP. dSTORM and FRET imaging shows that Cav1.3, RyR2, and KCa3.1 are organized as a triprotein complex that colocalizes with junctophilin (JPH) 3 and 4 proteins that tether the plasma membrane to the endoplasmic reticulum. JPH3 and JPH4 shRNAs dissociated a Cav1.3-RyR2-KCa3.1 complex and reduced the IsAHP. Infusing JPH3 and JPH4 antibodies into CA1 cells reduced IsAHP and spike accommodation. These data indicate that JPH3 and JPH4 proteins maintain a Cav1-RyR2-KCa3.1 complex that allows two calcium sources to act in tandem to define the activation properties of KCa3.1 channels and the IsAHP. : Sahu et al. establish that junctophilin proteins maintain a triprotein complex that assembles a voltage-gated plasma membrane calcium channel and endoplasmic reticular ryanodine receptors with a calcium-gated potassium channel to generate the long-duration slow afterhyperpolarization that regulates spike output in hippocampal pyramidal cells. Keywords: CaV1.3, Cav1.2, KCa3.1, ryanodine receptor, RyR2, junctophilin, sAHP, hippocampus, dSTORM, FRET

Aims Pathogenic gain-of-function variants in CACAN1C cause type-8 long QT syndrome (LQT8). We sought to describe the electrocardiographic features in LQT8 and utilize molecular modelling to gain mechanistic insights into its genetic culprits. Methods and results Rare variants in CACNA1C were identified from genetic testing laboratories. Treating physicians provided clinical information. Variant pathogenicity was independently assessed according to recent guidelines. Pathogenic (P) and likely pathogenic (LP) variants were mapped onto a 3D modelled structure of the Cav1.2 protein. Nine P/LP variants, identified in 23 patients from 19 families with non-syndromic LQTS were identified. Six variants, found in 79% of families, clustered to a 4-residue section in the cytosolic II–III loop region which forms a region capable of binding STAC SH3 domains. Therefore, variants may affect binding of SH3-domain containing proteins. Arrhythmic events occurred in similar proportions of patients with II–III loop variants and with other P/LP variants (53% vs. 48%, P = 0.41) despite shorter QTc intervals (477 ± 31 ms vs. 515 ± 37 ms, P = 0.03). A history of sudden death was reported only in families with II–III loop variants (60% vs. 0%, P = 0.03). The predominant T-wave morphology was a late peaking T wave with a steep descending limb. Exercise testing demonstrated QTc prolongation on standing and at 4 min recovery after exercise. Conclusion The majority of P/LP variants in patients with CACNA1C-mediated LQT8 cluster in an SH3-binding domain of the cytosolic II–III loop. This represents a ‘mutation hotspot’ in LQT8. A late-peaking T wave with a steep descending limb and QT prolongation on exercise are commonly seen.

Calcium-dependent inactivation (CDI) is a fundamental autoregulatory mechanism in Ca(V)1 and Ca(V)2 voltage-gated calcium channels. Although CDI initiates with the cytoplasmic calcium sensor, how this event causes CDI has been elusive. Here, we show that a conserved selectivity filter (SF) domain II (DII) aspartate is essential for CDI. Mutation of this residue essentially eliminates CDI and leaves key channel biophysical characteristics untouched. DII mutants regain CDI by placing an aspartate at the analogous SF site in DIII or DIV, but not DI, indicating that Ca(V) SF asymmetry is key to CDI. Together, our data establish that the Ca(V) SF is the CDI endpoint. Discovery of this SF CDI gate recasts the Ca(V) inactivation paradigm, placing it squarely in the framework of voltage-gated ion channel (VGIC) superfamily members in which SF-based gating is important. This commonality suggests that SF inactivation is an ancient process arising from the shared VGIC pore architecture.

Timothy syndrome (TS) is a very rare multisystem disorder almost exclusively associated with mutations G402S and G406R in helix IS6 of Cav1.2. Recently, mutations R518C/H in helix IIS0 of the voltage sensing domain II (VSD-II) were described as a cause of cardiac-only Timothy syndrome. The three mutations are known to decelerate voltage-dependent inactivation (VDI). Here we report a case of cardiac-only Timothy syndrome caused by mutation R518C. To explore possible impact of the three mutations on interdomain contacts, we modeled channel Cav1.2 using as templates Class Ia and Class II cryo-EM structures of presumably inactivated channel Cav1.1. In both models, R518 and several other residues in VSD-II donated H-bonds to the IS6-linked α1-interaction domain (AID). We further employed steered Monte Carlo energy minimizations to move helices S4-S5, S5 and S6 from the inactivated-state positions to those seen in the X-ray structures of the open and closed NavAb channel. In the open-state models, positions of AID and VSD-II were similar to those in Cav1.1. In the closed-state models, AID moved along the β subunit (Cavβ) towards the pore axis and shifted AID-bound VSD-II. In all the models R518 retained strong contacts with AID. Our calculations suggest that conformational changes in VSD-II upon its deactivation would shift AID along Cavβ towards the pore axis. The AID-linked IS6 would bend at flexible G402 and G406, facilitating the activation gate closure. Mutations R518C/H weakened the IIS0-AID contacts and would retard the AID shift. Mutations G406R and G402S stabilized the open state and would resist the pore closure. Several Cav1.2 mutations associated with long QT syndromes are consistent with this proposition. Our results provide a mechanistic rationale for the VDI deceleration caused by TS-associated mutations and suggest targets for further studies of calcium channelopathies.

Oxytocin (OT) and its receptor (OTR) are expressed in the heart and are involved in the physiological cardiovascular functional system. Although it is known that OT/OTR signaling is cardioprotective by reducing the inflammatory response and improving cardiovascular function, the role of OT in the cardiac electrical excitation modulation has not been clarified. This study investigates the molecular mechanism of the action of OT on cardiomyocyte membrane excitation focusing on the L-type Ca2+ channel. Our methodology uses molecular biological methods and a patch-clamp technique on rat cardiomyocytes with OT, combined with several signal inhibitors and/or activators. Our results show that long-term treatment of OT significantly decreases the expression of Cav1.2 mRNA, and reduces the L-type Ca2+ channel current (ICa.L) in cardiomyocytes. OT downregulates the phosphorylated component of a transcription factor adenosine-3′,5′-cyclic monophosphate (cAMP) response element binding protein (CREB), whose action is blocked by OTR antagonist and pertussis toxin, a specific inhibitor of the inhibitory GTP-binding regulators of adenylate cyclase, Gi. On the other hand, the upregulation of Cav1.2 mRNA expression by isoproterenol is halted by OT. Furthermore, inhibition of phospholipase C (PLC) was without effect on the OT action to downregulate Cav1.2 mRNA—which suggests a signal pathway of Gi/protein kinase A (PKA)/CREB mediated by OT/OTR. These findings indicate novel signaling pathways of OT contributing to a downregulation of the Cav1.2-L-type Ca2+ channel in cardiomyocytes.

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.

In order to specify the role of individual S4 segments in CaV1.2 gating, charged residues of segments IS4-IVS4 were replaced by glutamine and the corresponding effects on activation/deactivation of calcium channel currents were analysed. Almost all replacements of charges in IS4 and IIIS4 decreased the slope of the Boltzmann curve of channel activation (activation curve) while charge neutralisations in IIS4 and IVS4 did not significantly affect the slope. S4 mutations caused either left or rightward shifts of the activation curve, and in wild-type channels, these S4 mutations hardly affected current kinetics. In slowly gating pore (S6) mutants (G432W, A780T, G1193T or A1503G), neutralisations in S4 segments significantly accelerated current kinetics. Likewise in wild type, charge replacements in IS4 and IIIS4 of pore mutants reduced the slope of the activation curves while substitutions of charges in IIS4 and IVS4 had less or no impact. We propose a gating model where the structurally different S4 segments leave their resting positions not simultaneously. Upward movement of segments IS4 and (to a lesser extend) IIIS4 appear to be a rate-limiting stage for releasing the pore gates. These segments carry most of the effective charge for channel activation. Our study suggests that S4 segments of CaV1.2 control the closed state in domain specific manner while stabilizing the open state in a non-specific manner.

L-type calcium channel (LTCC) gene Cav1.2 is believed to play an important role in the alteration of Ca(2+) homeostasis in brain astrocytes. Increasing evidence shows that alteration of intracellular Ca(2+) concentration is related to the effect of 17β-estradiol (E2) in a variety of neurophysiological and neuropathological conditions. In this study, we measured immunoreactivity of Cav1.2 protein expression in rat primary cortical astrocytes by using Western blots. We demonstrated that E2 upregulated Cav1.2 expression in a dose- and time-dependent manner and the effect of E2 on Cav1.2 expression were blocked by an estrogen receptor (ER) antagonist, ICI-182,780. The ER subtype-selective ERα agonists propylpyrazole triole (PPT) and ERβ agonist diarylpropionitrile (DPN) both increase the expression of Cav1.2 in a dose-dependent manner. Also, the PPT most closely mimicked the upregulation of Cav1.2 protein expression by E2. Similar experiments of 10 nM E2-treated ERα- or ERβ-knockdown astrocytes have also shown that the E2 regulation of Cav1.2 protein expression is mediated through an ERα-dependent pathway. Furthermore, we established that E2 did not change the level of Cav1.2 mRNA. The induction of E2-mediated Cav1.2 expression was inhibited by cycloheximide (CHX) but not by actinomycin D (Act-D), suggesting that E2 regulation of Cav1.2 expression occurred at a posttranscriptional level. We also found that E2 may increase Cav1.2 levels by decreasing its ubiquitination and degradation rate. These findings provide new information about the effect of E2 on Cav1.2 in astrocytes, particularly necessary for the treatment of neurological disease.

Clevidipine, a dihydropyridine (DHP) analogue, lowers blood pressure (BP) by inhibiting l-type calcium channels (CaV1.2; gene CACNA1C) predominantly located in vascular smooth muscle (VSM). However, clinical observations suggest that clevidipine acts by a more complex mechanism. Clevidipine more potently reduces pulmonary vascular resistance (PVR) than systemic vascular resistance and its spectrum of effects on PVR are not shared by other DHPs. Clevidipine has potent spasmolytic effects in peripheral arteries at doses that are sub-clinical for BP lowering and, in hypertensive acute heart failure, clevidipine, but not other DHPs, provides dyspnea relief, partially independent of BP reduction. These observations suggest that a molecular variation in CaV1.2 may exist which confers unique pharmacology to different DHPs. We sequenced CACNA1C transcripts from human lungs and measured their affinity for clevidipine. Human lung tissue contains CACNA1C mRNA with many different splice variations. CaV1.2 channels with a specific combination of variable exons showed higher affinity for clevidipine, well below the concentration associated with BP reduction. Co-expression with pannexin 1 further increased the clevidipine affinity for this CaV1.2 splice variant. A high-affinity splice variant of CaV1.2 in combination with pannexin 1 could underlie the selective effects of clevidipine on pulmonary arterial pressure and on dyspnea. Research in Context Clevidipine lowers blood pressure by inhibiting calcium channels in vascular smooth muscle. In patients with acute heart failure, clevidipine was shown to relieve breathing problems. This was only partially related to the blood pressure lowering actions of clevidipine and not conferred by another calcium channel inhibitor. We here found calcium channel variants in human lung that are more selectively inhibited by clevidipine, especially when associated with pannexin channels. This study gives a possible mechanism for clevidipine's relief of breathing problems and supports future clinical trials testing the role of clevidipine in the treatment of acute heart failure., Highlights • CaV1.2 splice variants are found in human lung that have increased affinity for clevidipine. • Co-expression of CaV1.2 splice variant with Pannexin 1 further increases affinity for clevidipine but not for nicardipine. • Study supports future clinical trials testing the role of clevidipine in the treatment of acute hypertensive heart failure.

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.

Key points L-type calcium channels in the CNS exist as two subunit forming channels, Cav1.2 and Cav1.3, which are involved in short- and long-term plasticity. We demonstrate that Cav1.3 but not Cav1.2 is essential for wind-up. These results identify Cav1.3 as a key conductance responsible for short-term sensitization in physiological pain transmission. We confirm the role of Cav1.2 in a model of long-term plasticity associated with neuropathic pain. Up-regulation of Cav1.2 and down-regultation of Cav1.3 in neuropathic pain underlies the switch from physiology to pathology. Finally, the results of the present study reveal that therapeutic targeting molecular pathways involved in wind-up may be not relevant in the treatment of neuropathy. Abstract Short-term central sensitization to pain temporarily increases the responsiveness of nociceptive pathways after peripheral injury. In dorsal horn neurons (DHNs), short-term sensitization can be monitored through the study of wind-up. Wind-up, a progressive increase in DHNs response following repetitive peripheral stimulations, depends on the post-synaptic L-type calcium channels. In the dorsal horn of the spinal cord, two L-type calcium channels are present, Cav1.2 and Cav1.3, each displaying specific kinetics and spatial distribution. In the present study, we used a mathematical model of DHNs in which we integrated the specific patterns of expression of each Cav subunits. This mathematical approach reveals that Cav1.3 is necessary for the onset of wind-up, whereas Cav1.2 is not and that synaptically triggered wind-up requires NMDA receptor activation. We then switched to a biological preparation in which we knocked down Cav subunits and confirmed the prominent role of Cav1.3 in both naive and spinal nerve ligation model of neuropathy (SNL). Interestingly, although a clear mechanical allodynia dependent on Cav1.2 expression was observed after SNL, the amplitude of wind-up was decreased. These results were confirmed with our model when adapting Cav1.3 conductance to the changes observed after SNL. Finally, our mathematical approach predicts that, although wind-up amplitude is decreased in SNL, plateau potentials are not altered, suggesting that plateau and wind-up are not fully equivalent. Wind-up and long-term hyperexcitability of DHNs are differentially controlled by Cav1.2 and Cav1.3, therefore confirming that short- and long-term sensitization are two different phenomena triggered by distinct mechanisms.