The role of sodium-calcium exchange at the sarcolemma in the release of calcium from cardiac sarcoplasmic reticulum was investigated in voltage-clamped, isolated cardiac myocytes. In the absence of calcium entry through voltage-dependent calcium channels, membrane depolarization elicited release of calcium from ryanodine-sensitive internal stores. This process was dependent on sodium entry through tetrodotoxin-sensitive sodium channels. Calcium release under these conditions was also dependent on extracellular calcium concentration, suggesting a calcium-induced trigger release mechanism that involves calcium entry into the cell by sodium-calcium exchange. This sodium current-induced calcium release mechanism may explain, in part, the positive inotropic effects of cardiac glycosides and the negative inotropic effects of a variety of antiarrhythmic drugs that interact with cardiac sodium channels. In response to a transient rise of intracellular sodium, sodium-calcium exchange may promote calcium entry into cardiac cells and trigger sarcoplasmic calcium release during physiologic action potentials.

Ca2+‐activated chloride currents (ICl(Ca)) were recorded from smooth muscle cells isolated from rabbit pulmonary (PA) and coronary artery (CA) as well as rabbit portal vein (PV). The characteristics and regulation by Ca2+‐calmodulin‐dependent kinase II (CaMKII) were compared between the three cell types. In PA and CA myocytes dialysed and superfused with K+‐free media, pipette solutions containing fixed levels of free Ca2+ in the range of 250 nm to 1 μm evoked well sustained, outwardly rectifying ICl(Ca) currents in about 90 % of cells. The CaMKII inhibitor KN‐93 (5 μm) increased the amplitude of ICl(Ca) in PA and CA myocytes. However, the threshold intracellular Ca2+ concentration for detecting this effect was different in the two arterial cell types. KN‐93 also enhanced the rate of activation of the time‐dependent current during depolarising steps, slowed the kinetics of the tail current following repolarisation, and induced a negative shift of the steady‐state activation curve. In PA myocytes, the effects of KN‐93 were not mirrored by its inactive analogue KN‐92 but were reproduced by the inclusion of autocamtide‐2‐related CaMKII inhibitory peptide (ARIP) in the pipette solution. Cell dialysis with constitutively active CaMKII (30 nm) significantly reduced ICl(Ca) evoked by 500 nm Ca2+. In PV myocytes, ICl(Ca) was evoked by pipette solutions containing up to 1 μm free Ca2+ in less than 40 % of cells. Application of KN‐93 to cells where ICl(Ca) was sustained produced a small inhibition (≈25 %) of the current in 70 % of the cells. The present study shows that regulation of Ca2+‐dependent Cl− channels by CaMKII differs between arterial and portal vein myocytes.

Calcium-activated chloride channels (ClCa) are ligand-gated anion channels as they have been shown to be activated by a rise in intracellular Ca2+ concentration in various cell types including cardiac, skeletal and vascular smooth muscle cells, endothelial and epithelial cells, as well as neurons. Because ClCa channels are normally closed at resting, free intracellular Ca2+ concentration (approximately 100 nmol/L) in most cell types, they have generally been considered excitatory in nature, providing a triggering mechanism during signal transduction for membrane excitability, osmotic balance, transepithelial chloride movements, or fluid secretion. Unfortunately, the genes responsible for encoding this class of ion channels is still unknown. This review centers primarily on recent findings on the properties of these channels in smooth muscle cells. The first section discusses the functional significance and biophysical and pharmacological properties of ClCa channels in smooth muscle cells, and ends with a description of 2 candidate gene families (i.e., CLCA and Bestrophin) that are postulated to encode for these channels in various cell types. The second section provides a summary of recent findings demonstrating the regulation of native ClCa channels in vascular smooth muscle cells by calmodulin-dependent protein kinase II and calcineurin and how their fine tuning by these enzymes may influence vascular tone.

Recently, overexpression of the genes TMEM16A and TMEM16B has been shown to produce currents qualitatively similar to native Ca(2+)-activated Cl(-) currents (I(ClCa)) in vascular smooth muscle. However, there is no information about this new gene family in vascular smooth muscle, where Cl(-) channels are a major depolarizing mechanism. Qualitatively similar Cl(-) currents were evoked by a pipette solution containing 500 nM Ca(2+) in smooth muscle cells isolated from BALB/c mouse portal vein, thoracic aorta, and carotid artery. Quantitative PCR using SYBR Green chemistry and primers specific for transmembrane protein (TMEM) 16A or the closely related TMEM16B showed TMEM16A expression as follows: portal vein > thoracic aorta > carotid artery > brain. In addition, several alternatively spliced variant transcripts of TMEM16A were detected. In contrast, TMEM16B expression was very low in smooth muscle. Western blot analysis with different antibodies directed against TMEM16A revealed a number of products with a consistent band at ∼120 kDa, except portal vein, where an 80-kDa band predominated. TMEM16A protein was identified in the smooth muscle layers of 4-μm-thick slices of portal vein, thoracic aorta, and carotid artery. In isolated myocytes, fluorescence specific to a TMEM16A antibody was detected diffusely throughout the cytoplasm, as well as near the membrane. The same antibody used in Western blot analysis of lysates from vascular tissues also recognized an ∼147-kDa mouse TMEM16A-green fluorescent protein (GFP) fusion protein expressed in HEK 293 cells, which correlated to a similar band detected by a GFP antibody. Patch-clamp experiments revealed that I(ClCa) generated by transfection of TMEM16A-GFP in HEK 293 cells displayed remarkable similarities to I(ClCa) recorded in vascular myocytes, including slow kinetics, steep outward rectification, and a response similar to the pharmacological agent niflumic acid. This study shows that TMEM16A expression is robust in murine vascular smooth muscle cells, consolidating the view that this gene is a viable candidate for the native Ca(2+)-activated Cl(-) channel in this cell type.

The aim of the present study was to provide a mechanistic insight into how phosphatase activity influences calcium-activated chloride channels in rabbit pulmonary artery myocytes. Calcium-dependent Cl− currents (IClCa) were evoked by pipette solutions containing concentrations between 20 and 1000 nM Ca2+ and the calcium and voltage dependence was determined. Under control conditions with pipette solutions containing ATP and 500 nM Ca2+, IClCa was evoked immediately upon membrane rupture but then exhibited marked rundown to ∼20% of initial values. In contrast, when phosphorylation was prohibited by using pipette solutions containing adenosine 5′-(β,γ-imido)-triphosphate (AMP-PNP) or with ATP omitted, the rundown was severely impaired, and after 20 min dialysis, IClCa was ∼100% of initial levels. IClCa recorded with AMP-PNP–containing pipette solutions were significantly larger than control currents and had faster kinetics at positive potentials and slower deactivation kinetics at negative potentials. The marked increase in IClCa was due to a negative shift in the voltage dependence of activation and not due to an increase in the apparent binding affinity for Ca2+. Mathematical simulations were carried out based on gating schemes involving voltage-independent binding of three Ca2+, each binding step resulting in channel opening at fixed calcium but progressively greater “on” rates, and voltage-dependent closing steps (“off” rates). Our model reproduced well the Ca2+ and voltage dependence of IClCa as well as its kinetic properties. The impact of global phosphorylation could be well mimicked by alterations in the magnitude, voltage dependence, and state of the gating variable of the channel closure rates. These data reveal that the phosphorylation status of the Ca2+-activated Cl− channel complex influences current generation dramatically through one or more critical voltage-dependent steps.

Background-Transient atrial contractile dysfunction ("atrial stunning") follows conversion of atrial fibrillation (AF) to sinus rhythm and has significant clinical implications; however, the underlying mechanisms are poorly understood. We investigated the hypothesis that rapid atrial activation (as during AF) impairs cellular contractility and affects cellular Ca 2ϩ handling. Methods and Results-Edge detection and indo 1 fluorescence techniques were used to measure unloaded cell shortening and intracellular Ca 2ϩ transients in atrial myocytes from control (Ctl) dogs and dogs subjected to atrial pacing at 400 bpm for 7 (P7) or 42 (P42) days. Atrial tachycardia reduced fractional cell shortening (0.

N. Increased TMEM16A-encoded calcium-activated chloride channel activity is associated with pulmonary hypertension. Pulmonary artery smooth muscle cells (PASMCs) are more depolarized and display higher Ca 2ϩ levels in pulmonary hypertension (PH). Whether the functional properties and expression of Ca 2ϩ -activated ClϪ channels (Cl Ca), an important excitatory mechanism in PASMCs, are altered in PH is unknown. The potential role of Cl Ca channels in PH was investigated using the monocrotaline (MCT)-induced PH model in the rat. Three weeks postinjection with a single dose of MCT (50 mg/kg ip), the animals developed right ventricular hypertrophy (heart weight measurements) and changes in pulmonary arterial flow (pulse-waved Doppler imaging) that were consistent with increased pulmonary arterial pressure and PH. Whole cell patch experiments revealed an increase in niflumic acid (NFA)-sensitive Ca 2ϩ -activated Cl Ϫ current [ICl(Ca)] density in PASMCs from large conduit and small intralobar pulmonary arteries of MCT-treated rats vs. aged-matched saline-injected controls. Quantitative RT-PCR and Western blot analysis revealed that the alterations in I Cl(Ca) were accompanied by parallel changes in the expression of TMEM16A, a gene recently shown to encode for Cl Ca channels. The contraction to serotonin of conduit and intralobar pulmonary arteries from MCT-treated rats exhibited greater sensitivity to nifedipine (1 M), an L-type Ca 2ϩ channel blocker, and NFA (30 or 100 M, with or without 10 M indomethacin to inhibit cyclooxygenases) or T16A Inh-A01 (10 M), TMEM16A/Cl Ca channel inhibitors, than that of control animals. In conclusion, augmented Cl Ca/TMEM16A channel activity is a major contributor to the changes in electromechanical coupling of PA in this model of PH. TMEM16A-encoded channels may therefore represent a novel therapeutic target in this disease. pulmonary arterial tone; TMEM16A; anoctamin-1; Ca 2ϩ -activated Cl Ϫ channel; patch-clamp technique PULMONARY HYPERTENSION (PH) is defined as a sustained high blood pressure (Ͼ25 mmHg at rest and Ͼ30 mmHg during exercise) in the main pulmonary artery (PA) that ultimately leads to failure of the right hand side of the heart and death (4). Characteristic pathophysiological manifestations of PH are enhanced vasoconstriction, thickening of the arterial muscle wall, and a propensity for thrombosis, as a result of changes in all layers of the blood vessel, but little is known about the molecular mechanisms that drive these pathological responses. It is well established that pulmonary arterial smooth muscle cells (PASMCs) from animal models of PH and human PH patients are more depolarized and exhibit a higher intracellular calcium concentration ([Ca 2ϩ ] i ) than cells from healthy individuals and several ionic conductances are altered in PASMCs from animal models of PH and PH patients (4,13,29,43,68,70). Except for one recent study carried out using the chronic hypoxic model of PH in the rat (58), there is little information regarding the potential role of Ca 2ϩ -activa...

BACKGROUND AND PURPOSET16Ainh-A01 is a recently identified inhibitor of the calcium-activated chloride channel TMEM16A. The aim of this study was to test the efficacy of T16Ainh-A01 for inhibition of calcium-activated chloride channels in vascular smooth muscle and consequent effects on vascular tone. EXPERIMENTAL APPROACHSingle channel and whole cell patch clamp was performed on single smooth muscle cells from rabbit pulmonary artery and mouse thoracic aorta. Isometric tension studies were performed on mouse thoracic aorta and mesenteric artery as well as human abdominal visceral adipose artery. KEY RESULTSIn rabbit pulmonary artery myocytes T16Ainh-A01 (1-30 mM) inhibited single calcium (Ca -activated Cl -channels in mouse thoracic aorta, and in both cell types, channel activity was abolished by two antisera raised against TMEM16A but not by a bestrophin antibody. The TMEM16A potentiator, Fact (10 mM), increased single channel and whole cell Ca 2+ -activated Cl -currents in rabbit pulmonary arteries. In isometric tension studies, T16Ainh-A01 relaxed mouse thoracic aorta pre-contracted with methoxamine with an IC50 of 1.6 mM and suppressed the methoxamine concentration-effect curve. T16Ainh-A01 did not affect the maximal contraction produced by 60 mM KCl and the relaxant effect of 10 mM T16Ainh-A01 was not altered by incubation of mouse thoracic aorta in a cocktail of potassium (K + ) channel blockers. T16Ainh-A01 (10 mM) also relaxed human visceral adipose arteries by 88 Ϯ 3%. CONCLUSIONS AND IMPLICATIONST16Ainh-A01 blocks calcium-activated chloride channels in vascular smooth muscle cells and relaxes murine and human blood vessels. Abbreviations