Ca Channel Currents Research Articles

Bitter tastants induce relaxation of rat thoracic aorta precontracted with high K+

It has been reported that bitter tastants decrease blood pressure and relax precontracted vascular smooth muscle. However, the underlying mechanisms remain unclear. The aim of the present study was to determine the mechanism underlying the vasorelaxant effect of the bitter tastants. Thoracic aortic rings were isolated from Wistar rats and contractions were measured using an isometric myograph. Intracellular Ca(2+) ([Ca(2+)]i) in single rat thoracic aortic smooth muscle cells was recorded by calcium imaging. Calcium currents in single cells were recorded using patch-clamp techniques. High K(+) (140 mmol/L) induced contractions in rat thoracic aortic rings that were inhibited by 3 mmol/L chloroquine, 3 mmol/L denatonium and 10 μmol/L nifedipine. In single rat thoracic aortic smooth muscle cells, high K(+) increased [Ca(2+)]i and this effect was also blocked by 3 mmol/L chloroquine and 10 μmol/L nifedipine. Under Ca(2+) -free conditions, high K(+) failed to induce contractions in rat thoracic aortic rings. On its own, chloroquine had no effect on the muscle tension of rat aortic rings and [Ca(2+) ]i. The vasorelaxant effects of chloroquine on precontracted rat thoracic aortic rings were not altered by either 1 μg/mL pertussis toxin (PTX), an inhibitor of Gαo/i-protein, or 1 mmol/L gallein, an inhibitor of Gβγ-protein. The results of patch-clamp analysis in single cells indicate that 1 mmol/L chloroquine blocks voltage-dependent L-type Ca(2+) channel (VDLCC) currents from both extracellular and intracellular sides. Together, the results indicate that chloroquine can block VDLCC, independent of PTX- and gallein-sensitive G-proteins, resulting in relaxation of high K(+)-precontracted thoracic aortic smooth muscle.

FGF12 is a candidate Brugada syndrome locus

Less than 30% of the cases of Brugada syndrome (BrS) have an identified genetic cause. Of the known BrS-susceptibility genes, loss-of-function mutations in SCN5A or CACNA1C and their auxiliary subunits are most common. On the basis of the recent demonstration that fibroblast growth factor (FGF) homologous factors (FHFs; FGF11-FGF14) regulate cardiac Na(+) and Ca(2+) channel currents, we hypothesized that FHFs are candidate BrS loci. The goal of this study was to test whether FGF12 is a candidate BrS locus. We used quantitative polymerase chain reaction to identify the major FHF expressed in the human ventricle and then queried a phenotype-positive, genotype-negative BrS biorepository for FHF mutations associated with BrS. We queried the effects of an identified mutant with biochemical analyses combined with electrophysiological assessment. We designed a novel rat ventricular cardiomyocyte system in which we swapped the endogenous FHF with the identified mutant and defined its effects on multiple ionic currents in their native milieu and on the cardiac action potential. We identified FGF12 as the major FHF expressed in the human ventricle. In 102 individuals in the biorepository, we identified a single missense mutation in FGF12-B (Q7R-FGF12). The mutant reduced binding to the NaV1.5 C terminus, but not to junctophilin-2. In adult rat cardiac myocytes, Q7R-FGF12, but not wild-type FGF12, reduced Na(+) channel current density and availability without affecting Ca(2+) channel function. Furthermore, the mutant, but not wild-type FGF12, reduced action potential amplitude, which is consistent with a mutant-induced loss of Na(+) channel function. These multilevel investigations strongly suggest that Q7R-FGF12 is a disease-associated BrS mutation. Moreover, these data suggest for the first time that FHF effects on Na(+) and Ca(2+) channels are separable. Most significantly, this study establishes a new method to analyze effects of human arrhythmogenic mutations on cardiac ionic currents.

Quercetin induces insulin secretion by direct activation of L‐type calcium channels in pancreatic beta cells

Quercetin is a natural polyphenolic flavonoid that displays anti-diabetic properties in vivo. Its mechanism of action on insulin-secreting beta cells is poorly documented. In this work, we have analysed the effects of quercetin both on insulin secretion and on the intracellular calcium concentration ([Ca(2+)]i) in beta cells, in the absence of any co-stimulating factor. Experiments were performed on both INS-1 cell line and rat isolated pancreatic islets. Insulin release was quantified by the homogeneous time-resolved fluorescence method. Variations in [Ca(2+)]i were measured using the ratiometric fluorescent Ca(2+) indicator Fura-2. Ca(2+) channel currents were recorded with the whole-cell patch-clamp technique. Quercetin concentration-dependently increased insulin secretion and elevated [Ca(2+)]i. These effects were not modified by the SERCA inhibitor thapsigargin (1 μmol·L(-1)), but were nearly abolished by the L-type Ca(2+) channel antagonist nifedipine (1 μmol·L(-1)). Similar to the L-type Ca(2+) channel agonist Bay K 8644, quercetin enhanced the L-type Ca(2+) current by shifting its voltage-dependent activation towards negative potentials, leading to the increase in [Ca(2+)]i and insulin secretion. The effects of quercetin were not inhibited in the presence of a maximally active concentration of Bay K 8644 (1 μmol·L(-1)), with the two drugs having cumulative effects on [Ca(2+)]i. Taken together, our results show that quercetin stimulates insulin secretion by increasing Ca(2+) influx through an interaction with L-type Ca(2+) channels at a site different from that of Bay K 8644. These data contribute to a better understanding of quercetin's mechanism of action on insulin secretion.

Hydrogen peroxide‐induced inhibition of L‐type Ca channel current in A7r5 cells: Effect of ebselen, dithiothreitol and N‐acetylcysteine

L‐type Ca2+ channel current (ICa,L) is increased by H2O2 in some cardiac and smooth muscle cells by phosphorylation of the channel but is inhibited in some cell types. We studied the mechanism of the inhibition by recording whole cell Ba2+ current from A7r5 cells derived from fetal rat aorta. We examined the effects of ebselen (Ebs), a scavenger of H2O2, dithiothreitol (DTT, 2 mM), a reducing agent, and N‐acetylcysteine (NAC, 10 mM), a precursor of glutathione (GSH) on ICa,L. Ebs is a glutathione peroxidase (GPx) mimic that converts H2O2 and GSH to H2O and GSSG which induces protein glutathionylation. Ebs induced a dose‐dependent inhibition of ICa,L resulting in complete suppression at 3 μM in a few minutes. The inhibition induced by sub‐maximal concentrations was not reversible by washing out and was enhanced by H2O2. H2O2 induced ~40% inhibition of ICa,L at 1 mM with weak voltage‐dependence without affecting ICa,T. Both DTT and NAC markedly reduced inhibitory effect of Ebs and H2O2 and they induced partial recovery of the inhibition when applied after washout of H2O2 and Ebs. We propose that H2O2‐induced inhibition of ICa,L is due to glutathionylation of CaV1.2. NAC did not aggravate but reduced the inhibition probably by facilitating enzymatic reduction of GSH‐protein mixed disulfides via glutaredoxin‐catalyzed pathway. Supported by National Heart, Lung, and Blood Institute Grant RO1‐HL‐085352.

Endothelin-1 Stimulates the Expression of L-Type Ca2+ Channels in Neonatal Rat Cardiomyocytes via the Extracellular Signal–Regulated Kinase 1/2 Pathway

The cardiac L-type Ca(2+) channel current (I(Ca,L)) plays an important role in controlling both cardiac excitability and excitation-contraction coupling and is involved in the electrical remodeling during postnatal heart development and cardiac hypertrophy. However, the possible role of endothelin-1 (ET-1) in the electrical remodeling of postnatal and diseased hearts remains unclear. Therefore, the present study was designed to investigate the transcriptional regulation of I(Ca,L) mediated by ET-1 in neonatal rat ventricular myocytes using the whole-cell patch-clamp technique, quantitative RT-PCR and Western blotting. Furthermore, we determined whether the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway is involved. ET-1 increased I(Ca,L) density without altering its voltage dependence of activation and inactivation. In line with the absence of functional changes, ET-1 increased L-type Ca(2+) channel pore-forming α1C-subunit mRNA and protein levels without affecting the mRNA expression of auxiliary β- and α2/δ-subunits. Furthermore, an actinomycin D chase experiment revealed that ET-1 did not alter α1C-subunit mRNA stability. These effects of ET-1 were inhibited by the ETA receptor antagonist BQ-123 but not the ETB receptor antagonist BQ-788. Moreover, the effects of ET-1 on I(Ca,L) and α1C-subunit expression were abolished by the ERK1/2 inhibitor (PD98059) but not by the p38 MAPK inhibitor (SB203580) or the c-Jun N-terminal kinase inhibitor (SP600125). These findings indicate that ET-1 increased the transcription of L-type Ca(2+) channel in cardiomyocytes via activation of ERK1/2 through the ETA receptor, which may contribute to the electrical remodeling of heart during postnatal development and cardiac hypertrophy.

Rescue of lethal CaVβ2 knock‐out and identification of novel CaVβ2 variants

Disruption of the CaVβ2 gene in mice resulted in diminished L‐type Ca currents in cardiomyocytes. Thereby heart function was compromised and embryonic death occurred. Partial rescue of the Ca channel currents by Bayk8644 significantly postponed embryonic death in CaVβ2‐deficient mice. The predominant ancillary β subunit of cardiac Ca channels is the β2 subunit and three splice variants are expressed in the heart, β2aN4>; β2aN1>;>;β2aN5. β2aN1 appears to be mainly expressed in embryonic heart and the β2aN4 in adult heart. We now rescued the lethal β2−/− phenotype by expression of a mouse β2 transgene under the control of the mouse a‐MHC promoter and generated two mouse lines: one rescued with the β2aN1 of embryonic heart, one with the β2aN4 of adult heart. Characterization of the founders and the β2−/−/tg+/‐ mice by Southern blots and Western blots show that the β2 transgenes are expressed in heart only and that the extracardial β2‐deficiency persists. Comparing β2 protein expression in adult heart and various tissues from wild‐type and transgenic mice reveals that additional and novel CaVβ2 variants are expressed in wild‐type tissues. We will characterize the novel CaVβ2 variants and the impact of β2‐deficiency in extracardial tissues. The availability of mice carrying either the N4 or the N1 β2 variant only in cardiac myocytes allows us to study the roles of either variant on L‐type Ca channels in cardiomyocytes.

Catharanthine Dilates Small Mesenteric Arteries and Decreases Heart Rate and Cardiac Contractility by Inhibition of Voltage-Operated Calcium Channels on Vascular Smooth Muscle Cells and Cardiomyocytes

Catharanthine is a constituent of anticancer vinca alkaloids. Its cardiovascular effects have not been investigated. This study compares the in vivo hemodynamic as well as in vitro effects of catharanthine on isolated blood vessels, vascular smooth muscle cells (VSMCs), and cardiomyocytes. Intravenous administration of catharanthine (0.5-20 mg/kg) to anesthetized rats induced rapid, dose-dependent decreases in blood pressure (BP), heart rate (HR), left ventricular blood pressure, cardiac contractility (dP/dt(max)), and the slope of the end-systolic pressure-volume relationship (ESPVR) curve. Catharanthine evoked concentration-dependent decreases (I(max) >98%) in endothelium-independent tonic responses of aortic rings to phenylephrine (PE) and KCl (IC(50) = 28 µM for PE and IC(50) = 34 µM for KCl) and of third-order branches of the small mesenteric artery (MA) (IC(50) = 3 µM for PE and IC(50) = 6 µM for KCl). Catharanthine also increased the inner vessel wall diameter (IC(50) = 10 µM) and reduced intracellular free Ca(2+) levels (IC(50) = 16 µM) in PE-constricted MAs. Patch-clamp studies demonstrated that catharanthine inhibited voltage-operated L-type Ca(2+) channel (VOCC) currents in cardiomyocytes and VSMCs (IC(50) = 220 µM and IC(50) = 8 µM, respectively) of MA. Catharanthine lowers BP, HR, left ventricular systolic blood pressure, and dP/dt(max) and ESPVR likely via inhibition of VOCCs in both VSMCs and cardiomyocytes. Since smaller vessels such as the third-order branches of MAs are more sensitive to VOCC blockade than conduit vessels (aorta), the primary site of action of catharanthine for lowering mean arterial pressure appears to be the resistance vasculature, whereas blockade of cardiac VOCCs may contribute to the reduction in HR and cardiac contractility seen with this agent.

Pathogenic roles of antiganglioside antibodies in immune‐mediated neuropathies

AbstractRecent studies have shown that antiganglioside antibody‐mediated complement activation plays a key role in development of Guillain–Barré syndrome and related disorders. Additionally, complement‐independent nerve dysfunction is suggested by in vitro studies, showing that antiganglioside antibodies directly inhibit voltage‐gated Ca channel currents and change the integrity of lipid rafts. These pathogenic actions of antiganglioside antibodies might be governed by the avidity of the antibodies, which is influenced by specific localization of target gangliosides in the peripheral nervous system or glycolipid environment around the target antigens. The recent discovery of antibodies to ganglioside complexes has expedited the understanding of the mechanisms underlying antiganglioside antibody‐mediated nerve dysfunction in Guillain–Barré syndrome and related disorders. In chronic immune‐mediated neuropathy, some antiganglioside antibodies have also been identified as diagnostic markers, although their pathophysiological roles remain to be determined.

Effects of Astragaloside IV on Action Potentials and Ionic Currents in Guinea-Pig Ventricular Myocytes

Astragaloside IV (AS-IV) is one of the main active constituents of Astragalus membranaceus, which has various actions on the cardiovascular system. However, its electrophysiological mechanisms are not clear. In the present study, we investigated the effects of AS-IV on action potentials and membrane currents using the whole-cell patch clamp technique in isolated guinea-pig ventricular myocytes. AS-IV prolonged the action potential duration (APD) at all three tested concentrations. The peak effect was achieved with 1×10(-6) M, at which concentration AS-IV significantly prolonged the APD at 95% repolarization from 313.1±38.9 to 785.3±83.7 ms. AS-IV at 1×10(-6) M also enhanced the inward rectifier K(+) currents (I(K1)) and inhibited the delayed rectifier K(+) currents (I(K)). AS-IV (1×10(-6) M) strongly depressed the peak of voltage-dependent Ca(2+) channel current (I(CaL)) from -607.3±37.5 to -321.1±38.3 pA. However, AS-IV was not found to affect the Na(+) currents. Taken together, AS-IV prolonged APD of guinea-pig ventricular myocytes, which might be explained by its inhibition of I(K). AS-IV also influences Ca(2+) signaling through suppressing ICaL.

Electrophysiological Properties of Prion-Positive Cardiac Progenitors Derived From Murine Embryonic Stem Cells

The prion protein (PrP) has been reported to serve as a surface maker for isolation of cardiomyogenic progenitors from murine embryonic stem (ES) cells. Although PrP-positive cells exhibited automaticity, their electrophysiological characteristics remain unresolved. The aim of the present study was therefore to investigate the electrophysiological properties of PrP-positive cells in comparison with those of HCN4p-or Nkx2.5-positive cells. Differentiation of AB1, HCN5p-EGFP and hcgp7 ES cells into cardiac progenitors was induced by embryoid body (EB) formation. EBs were dissociated and cells expressing PrP, HCN4-EGFP and/or Nkx2.5-GFP were collected via flow cytometry. Sorted cells were subjected to reverse transcriptase-polymerase chain reaction, immunostaining and patch-clamp experiments. PrP-positive cells expressed mRNA of undifferentiation markers, first and second heart field markers, and cardiac-specific genes and ion channels, indicating their commitment to cardiomyogenic progenitors. PrP-positive cells with automaticity showed positive and negative chronotropic responses to isoproterenol and carbamylcholine, respectively. Hyperpolarization-activated cation current (I(f)) was barely detectable, whereas Na(+) and L-type Ca(2+) channel currents were frequently observed. Their spontaneous activity was slowed by inhibition of sarcoplasmic reticulum Ca(2+) uptake and release but not by blocking I(f). The maximum diastolic potential of their spontaneous firings was more depolarized than that of Nkx2.5-GFP-positive cells. PrP-positive cells contained cardiac progenitors that separated from the lineage of sinoatrial node cells. PrP can be used as a marker to enrich nascent cardiac progenitors.

Bile Acid Inhibition of N-type Calcium Channel Currents from Sympathetic Ganglion Neurons

Under some pathological conditions as bile flow obstruction or liver diseases with the enterohepatic circulation being disrupted, regurgitation of bile acids into the systemic circulation occurs and the plasma level of bile acids increases. Bile acids in circulation may affect the nervous system. We examined this possibility by studying the effects of bile acids on gating of neuronal (N)-type Ca2+ channel that is essential for neurotransmitter release at synapses of the peripheral and central nervous system. N-type Ca2+ channel currents were recorded from bullfrog sympathetic neuron under a cell-attached mode using 100 mM Ba2+ as a charge carrier. Cholic acid (CA, 10-6 M) that is relatively hydrophilic thus less cytotoxic was included in the pipette solution. CA suppressed the open probability of N-type Ca2+ channel, which appeared to be due to an increase in null (no activity) sweeps. For example, the proportion of null sweep in the presence of CA was ~40% at +40 mV as compared with ~8% in the control recorded without CA. Other single channel properties including slope conductance, single channel current amplitude, open and shut times were not significantly affected by CA being present. The results suggest that CA could modulate N-type Ca2+ channel gating at a concentration as low as 10-6 M. Bile acids have been shown to activate nonselective cation conductance and depolarize the cell membrane. Under pathological conditions with increased circulating bile acids, CA suppression of N-type Ca2+ channel function may be beneficial against overexcitation of the synapses.

Ca2+ channel currents of cortical neurons from pure and mixed cultures

Voltage-gated Ca(2+) channels (VGCCs) are key regulators of many neuronal functions, and involved in multiple central nervous system diseases. In the last 30years, a large number of injury and disease models have been established based on cultured neurons. Culture with serum develops a mixture of neurons and glial cells, while culture without serum develops pure neurons. Both of these neuronal-culture methods are widely used. However, the properties of Ca(2+) currents in neurons from these two cultures have not been compared. In this study, we cultured rat cortical neurons in serum-containing or -free medium and then recorded the Ca(2+) channel currents using patch-clamp technique. Our results showed that there were significant differences in the amplitude and activation properties of whole-cell Ca(2+) channel currents, and of non-L-type Ca(2+) channel currents between the neurons from these two culture systems. Our data suggested that the difference of whole-cell Ca(2+) currents may result from the differences in non-L-type currents. Understanding of these properties will considerably advance studies of VGCCs in neurons from pure or mixed culture.

BKCa and KV channels limit conducted vasomotor responses in rat mesenteric terminal arterioles

Intracellular Ca(2+) signals underlying conducted vasoconstriction to local application of a brief depolarizing KCl stimulus was investigated in rat mesenteric terminal arterioles (<40μm). Using a computer model of an arteriole segment comprised of coupled endothelial cells (EC) and vascular smooth muscle cells (VSMC) simulations of both membrane potential and intracellular [Ca(2+)] were performed. The "characteristic" length constant, λ, was approximated using a modified cable equation in both experiments and simulations. We hypothesized that K(+) conductance in the arteriolar wall limit the electrotonic spread of a local depolarization along arterioles by current dissipation across the VSMC plasma membrane. Thus, we anticipated an increased λ by inhibition of voltage-activated K(+) channels. Application of the BK(Ca) channel blocker iberiotoxin (100nM) onto mesenteric arterioles in vitro and inhibition of BK(Ca) channel current in silico increased λ by 34% and 32%, respectively. Similarly, inhibition of K(V) channels in vitro (4-aminopyridine, 1mM) or in silico increased λ by 41% and 21%, respectively. Immunofluorescence microscopy demonstrated expression of BK(Ca), Kv1.5, Kv2.1, but not Kv1.2, in VSMCs of rat mesenteric terminal arterioles. Our results demonstrate that inhibition of voltage-activated K(+) channels enhance vascular-conducted responses to local depolarization in terminal arterioles by increasing the membrane resistance of VSMCs. These data contribute to our understanding of how differential expression patterns of voltage-activated K(+) channels may influence conducted vasoconstriction in small arteriolar networks. This finding is potentially relevant to understanding the compromised microcirculatory blood flow in systemic vascular diseases such as diabetes mellitus and hypertension.

Ahnak1 is a tuneable modulator of cardiac Ca(v)1.2 calcium channel activity

Ahnak1 has been implicated in the beta-adrenergic regulation of the cardiac L-type Ca(2+) channel current (I (CaL)) by its binding to the regulatory Cavβ(2) subunit. In this study, we addressed the question whether ahnak1/Cavβ(2) interactions are essential or redundant for beta-adrenergic stimulation of I (CaL). Three naturally occurring ahnak1 variants (V5075M, G5242R, and T5796M) identified by genetic screening of cardiomyopathy patients did essentially not influence the in vitro Cavβ(2) interaction as assessed by recombinant proteins. But, we observed a robust increase in Cavβ(2) binding by mutating Ala at position 4984 to Pro which creates a PxxP consensus motif in the ahnak1 protein fragment. Surface plasmon resonance measurements revealed that this mutation introduced an additional Cavβ(2) binding site. The functionality of A4984P was supported by the specific action of the Pro-containing ahnak1-derived peptide (P4984) in beta-adrenergic regulation of I (CaL). Patch clamp recordings on cardiomyocytes showed that intracellular perfusion of P4984 markedly reduced I (CaL) response to the beta-adrenergic agonist, isoprenaline, while the Ala-containing counterpart failed to affect I (CaL). Interestingly, I (CaL) of ahnak1-deficient cardiomyocytes was not affected by peptide application. Moreover, I (CaL) of ahnak1-deficient cardiomyocytes showed intact beta-adrenergic responsiveness. Similarly isolated ahnak1-deficient mouse hearts responded normally to adrenergic challenge. Our results indicate that ahnak1 is not essential for beta-adrenergic up-regulation of I (CaL) and cardiac contractility in mice. But, tuning ahnak1/Cavβ(2) interaction provides a tool for modulating the beta-adrenergic response of I (CaL).

Opening of Small and Intermediate Calcium-Activated Potassium Channels Induces Relaxation Mainly Mediated by Nitric-Oxide Release in Large Arteries and Endothelium-Derived Hyperpolarizing Factor in Small Arteries from Rat

This study was designed to investigate whether calcium-activated potassium channels of small (SK(Ca) or K(Ca)2) and intermediate (IK(Ca) or K(Ca)3.1) conductance activated by 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309) are involved in both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF)-type relaxation in large and small rat mesenteric arteries. Segments of rat superior and small mesenteric arteries were mounted in myographs for functional studies. NO was recorded using NO microsensors. SK(Ca) and IK(Ca) channel currents and mRNA expression were investigated in human umbilical vein endothelial cells (HUVECs), and calcium concentrations were investigated in both HUVECs and mesenteric arterial endothelial cells. In both superior (∼1093 μm) and small mesenteric (∼300 μm) arteries, NS309 evoked endothelium- and concentration-dependent relaxations. In superior mesenteric arteries, NS309 relaxations and NO release were inhibited by both N(G),N(G)-asymmetric dimethyl-l-arginine (ADMA) (300 μM), an inhibitor of NO synthase, and apamin (0.5 μM) plus 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) (1 μM), blockers of SK(Ca) and IK(Ca) channels, respectively. In small mesenteric arteries, NS309 relaxations were reduced slightly by ADMA, whereas apamin plus an IK(Ca) channel blocker almost abolished relaxation. Iberiotoxin did not change NS309 relaxation. HUVECs expressed mRNA for SK(Ca) and IK(Ca) channels, and NS309 induced increases in calcium, outward current, and NO release that were blocked by apamin and TRAM-34 or charybdotoxin. These findings suggest that opening of SK(Ca) and IK(Ca) channels leads to endothelium-dependent relaxation that is mediated mainly by NO in large mesenteric arteries and by EDHF-type relaxation in small mesenteric arteries. NS309-induced calcium influx appears to contribute to the formation of NO.

Interaction of Bestrophin-1 and Ca2+ Channel β-Subunits: Identification of New Binding Domains on the Bestrophin-1 C-Terminus

Bestrophin-1 modulates currents through voltage-dependent L-type Ca2+ channels by physically interacting with the β-subunits of Ca2+ channels. The main function of β-subunits is to regulate the number of pore-forming CaV-subunits in the cell membrane and modulate Ca2+ channel currents. To understand the influence of full-length bestrophin-1 on β-subunit function, we studied binding and localization of bestrophin-1 and Ca2+ channel subunits, together with modulation of CaV1.3 Ca2+ channels currents. In heterologeous expression, bestrophin-1 showed co-immunoprecipitation with either, β3-, or β4-subunits. We identified a new highly conserved cluster of proline-rich motifs on the bestrophin-1 C-terminus between amino acid position 468 and 486, which enables possible binding to SH3-domains of β-subunits. A bestrophin-1 that lacks these proline-rich motifs (ΔCT-PxxP bestrophin-1) showed reduced efficiency to co-immunoprecipitate with β3 and β4-subunits. In the presence of ΔCT-PxxP bestrophin-1, β4-subunits and CaV1.3 subunits partly lost membrane localization. Currents from CaV1.3 subunits were modified in the presence of β4-subunit and wild-type bestrophin-1: accelerated time-dependent activation and reduced current density. With ΔCTPxxP bestrophin-1, currents showed the same time-dependent activation as with wild-type bestrophin-1, but the current density was further reduced due to decreased number of Ca2+ channels proteins in the cell membrane. In summary, we described new proline-rich motifs on bestrophin-1 C-terminus, which help to maintain the ability of β-subunits to regulate surface expression of pore-forming CaV Ca2+-channel subunits.

Cardiac ion channel current modulation by the CFTR inhibitor GlyH-101

The role in the heart of the cardiac isoform of the cystic fibrosis transmembrane conductance regulator (CFTR), which underlies a protein kinase A-dependent Cl(-) current (I(Cl.PKA)) in cardiomyocytes, remains unclear. The identification of a CFTR-selective inhibitor would provide an important tool for the investigation of the contribution of CFTR to cardiac electrophysiology. GlyH-101 is a glycine hydrazide that has recently been shown to block CFTR channels but its effects on cardiomyocytes are unknown. Here the action of GlyH-101 on cardiac I(Cl.PKA) and on other ion currents has been established. Whole-cell patch-clamp recordings were made from rabbit isolated ventricular myocytes. GlyH-101 blocked I(Cl.PKA) in a concentration- and voltage-dependent fashion (IC(50) at +100 mV=0.3 ± 1.5 μM and at -100 mV=5.1 ± 1.3 μM). Woodhull analysis suggested that GlyH-101 blocks the open pore of cardiac CFTR channels at an electrical distance of 0.15 ± 0.03 from the external membrane surface. A concentration of GlyH-101 maximally effective against I(Cl.PKA) (30 μM) was tested on other cardiac ion currents. Inward current at -120 mV, comprised predominantly of the inward-rectifier background K(+) current, I(K1), was reduced by ∼43% (n=5). Under selective recording conditions, the Na(+) current (I(Na)) was markedly inhibited by GlyH-101 over the entire voltage range (with a fractional block at -40 mV of ∼82%; n=8). GlyH-101 also produced a voltage-dependent inhibition of L-type Ca(2+) channel current (I(Ca,L)); fractional block at +10 mV of ∼49% and of ∼28% at -10 mV; n=11, with a ∼-3 mV shift in the voltage-dependence of I(Ca,L) activation. Thus, this study demonstrates for the first time that GlyH-101 blocks cardiac I(Cl.PKA) channels in a similar fashion to that reported for recombinant CFTR. However, inhibition of other cardiac conductances may limit its use as a CFTR-selective blocker in the heart.

Ginsenoside Rg3 enhances large conductance Ca2+-activated potassium channel currents: a role of Tyr360 residue.

Ginsenosides, active ingredients of Panax ginseng, are known to exhibit neuroprotective effects. Large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels are key modulators of cellular excitability of neurons and vascular smooth muscle cells. In the present study, we examined the effects of ginsenosides on rat brain BK(Ca) (rSlo) channel activity heterologously expressed in Xenopus oocytes to elucidate the molecular mechanisms how ginsenoside regulates the BK(Ca) channel activity. Ginsenoside Rg(3) (Rg(3)) enhanced outward BK(Ca) channel currents. The Rg(3)-enhancement of outward BK(Ca) channel currents was concentration-dependent, voltage-dependent, and reversible. The EC(50) was 15.1 ± 3.1 μM. Rg(3) actions were not desensitized by repeated treatment. Tetraetylammonium (TEA), a K(+) channel blocker, inhibited BK(Ca) channel currents. We examined whether extracellular TEA treatment could alter the Rg(3) action and vice versa. TEA caused a rightward shift of the Rg(3) concentration-response curve (i.e., much higher concentration of Rg(3) is required for the activation of BK(Ca) channel compared to the absence of TEA), while Rg(3) caused a rightward shift of the TEA concentration-response curve in wild-type channels. Mutation of the extracellular TEA binding site Y360 to Y360I caused a rightward shift of the TEA concentration-response curve and almost abolished both the Rg(3) action and Rg(3)-induced rightward shift of TEA concentration-response curve. These results indicate that Tyr360 residue of BK(Ca) channel plays an important role in the Rg(3)-enhancement of BK(Ca) channel currents.

Tuning in to the ‘right’ calcium channel regulation in experimental models of diabetes

Elucidation of cellular and molecular mechanisms underlying vascular disease is of fundamental importance to the development of pharmacological agents to target these pathways. Pinho et al. in this issue of the BJP provide highly compelling evidence that the δ isoform of phosphatidyl inositol 3-kinase (PI3K δ) was upregulated and accounted for the increase in L-type, voltage-gated, Ca channel current in aortic vascular smooth muscle (VSM) cells of a mouse model of type 1 diabetes. There are several key issues of broad fundamental significance to this work. Firstly, what is the ‘right’ answer about calcium channel regulation in diabetes? Conflicting reports of increased and decreased Ca channel current may be due to specificity of the vascular bed and species. Then, the time course of diabetic vasculopathy may influence the expression of contractile versus proliferative phenotypes of VSM. Also the metabolic characterization of diabetes may enlighten or confound any study of diabetic vascular disease. These issues need attention to move forward work in this area.

T-type ca(2+) channel blockers increase smooth muscle progenitor cells and endothelial progenitor cells in bone marrow stromal cells in culture by suppression of cell death.

To examine the expression patterns and roles of voltage-dependent Ca2+ channels in bone marrow stromal cells (BMSCs). Ca(2+) currents of BMSCs were measured by the whole-cell patch clamp method. The number and percentage of deaths of BMSCs cultured for 14 days with or without Ca(2+) channel blockers were evaluated using a MTT assay and an LDH assay, respectively. T-type Ca(2+) channel current was recorded in 0, 2, 10, and 4% of BMSCs on days 3, 10, 17, and 24 in culture, respectively. L-type Ca(2+) channel current was first recorded on day 24 in 6% of BMSCs. Addition of the T-type Ca(2+) channel blocker mibefradil but not the L-type Ca(2+) channel blocker nifedipine significantly increased the cell count. Immunocytochemical analysis revealed increases in the counts of smooth muscle progenitor cells (SMPCs) and endothelial progenitor cells (EPCs). Mibefradil but not nifedipine significantly decreased the rate of cell death. T-type Ca(2+) channel blockers increased the numbers of SMPCs and EPCs in cultured BMSCs, partly through suppression of cell death. Thus, T-type Ca(2+) channel blockers may have the potential to provide an increased number of both BMSC-derived SMCs and ECs of potential use in cell and gene therapy.