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Calcium-activated chloride channels (CaCC) provide a depolarizing stimulus to a variety of tissues through chloride efflux in response to a rise in internal Ca 2+ and voltage. One of these channels, Anoctamin-1 (ANO1 or TMEM16A) is now recognized to play a central role in promoting smooth muscle tone in various types of blood vessels. Its role in hypertension, and thus the therapeutic promise of targeting ANO1, is less straightforward. This review gives an overview of our current knowledge about the potential role ANO1 may play in hypertension within the systemic, portal, and pulmonary vascular systems and the importance of this information when pursuing potential treatment strategies. While the role of ANO1 is well-established in several forms of pulmonary hypertension, its contributions to both the generation of vascular tone and its role in hypertension within the systemic and portal systems are much less clear. This, combined with ANO1's various roles throughout a multitude of tissues throughout the body, command caution when targeting ANO1 as a therapeutic target and may require tissue-selective strategies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Ca 2+ -activated Cl - channels (CaCCs) perform a multitude of functions including the control of cell excitability, regulation of cell volume and ionic homeostasis, exocrine and endocrine secretion, fertilization, amplification of olfactory sensory function, and control of smooth muscle cell contractility. CaCCs are the translated products of two members (ANO1 and ANO2, also known as TMEM16A and TMEM16B) of the Anoctamin family of genes comprising ten paralogs. This review focuses on recent progress in understanding the molecular mechanisms involved in the regulation of ANO1 by cytoplasmic Ca 2+ , post-translational modifications, and how the channel protein interacts with membrane lipids and protein partners. After first reviewing the basic properties of native CaCCs, we then present a brief historical perspective highlighting controversies about their molecular identity in native cells. This is followed by a summary of the fundamental biophysical and structural properties of ANO1. We specifically address whether the channel is directly activated by internal Ca 2+ or indirectly through the intervention of the Ca 2+ -binding protein Calmodulin (CaM), and the structural domains responsible for Ca 2+ - and voltage-dependent gating. We then review the regulation of ANO1 by internal ATP, Calmodulin-dependent protein kinase II-(CaMKII)-mediated phosphorylation and phosphatase activity, membrane lipids such as the phospholipid phosphatidyl-(4,5)-bisphosphate (PIP 2 ), free fatty acids and cholesterol, and the cytoskeleton. The article ends with a survey of physical and functional interactions of ANO1 with other membrane proteins such as CLCA1/2, inositol trisphosphate and ryanodine receptors in the endoplasmic reticulum, several members of the TRP channel family, and the ancillary Κ + channel β subunits KCNE1/5.

Hypertension is the leading risk factor for the development of heart diseases and stroke. Many hypertensive patients experience undesirable side effects to conventional antihypertensive pharmacotherapy. Cil et al. documented the antihypertensive profile of a novel molecule, TM inh -23 (2-bromodifluoroacetylamino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylic acid o-tolylamide), in the spontaneously hypertensive rat model of systemic hypertension. They showed that this agent reduces blood pressure by inhibiting transmembrane member 16A-encoded calcium-activated chloride channels in vascular myocytes from resistance arteries. If validated, TM inh -23 could become a useful clinical tool., (Copyright © 2021 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Anoctamin-1 (ANO1) is a Ca(2+)-activated Cl(-) channel expressed in many types of cells. Splice variants of ANO1 have been shown to influence the biophysical properties of conductance. It has been suggested that several new antagonists of ANO1 with relatively high affinity and selectivity might be useful for experimental and, potentially, therapeutic purposes. We investigated the effects of intracellular Ca(2+) concentration ([Ca(2+)]i) at 100-1,000 nM, a concentration range that might be achieved in cells during physiological activation of ANO1 channels, on blockade of ANO1 channels expressed in HEK-293 cells. Whole cell and excised patch configurations of the patch-clamp technique were used to perform tests on a variety of naturally occurring splice variants of ANO1. Blockade of ANO1 currents with aminophenylthiazole (T16Ainh-A01) was highly dependent on [Ca(2+)]i Increasing [Ca(2+)]i reduced the potency of this blocker. Similar Ca(2+)-dependent effects were also observed with benzbromarone. Experiments on excised, inside-out patches showed that the diminished potency of the blockers caused by intracellular Ca(2+) might involve a competitive interaction for a common binding site or repulsion of the blocking drugs by electrostatic forces at the cytoplasmic surface of the channels. The degree of interaction between the channel blockers and [Ca(2+)]i depends on the splice variant expressed. These experiments demonstrate that the efficacy of ANO1 antagonists depends on [Ca(2+)]i, suggesting a need for caution when ANO1 blockers are used to determine the role of ANO1 in physiological functions and in their use as therapeutic agents., (Copyright © 2016 the American Physiological Society.)

Background and Purpose: Calcium-activated chloride channels (CaCCs) are key depolarizing mechanisms that have an important role in vascular smooth muscle contraction. Here, we investigated whether these channels are regulated by phosphatidylinositol (4,5) bisphosphate [P(4,5)P2 ], a known regulator of various ion channels., Experimental Approach: Calcium-activated Cl(-) currents (IClCa ) were recorded by patch clamp electrophysiology of rat isolated pulmonary artery smooth muscle cells. TMEM16A protein-phosphoinositide interaction was studied by co-immunoprecipitation and phosphoinositide binding arrays on protein lysates from whole pulmonary arteries and HEK293 cells overexpressing TMEM16A, the molecular correlate., Key Results: PI(4,5)P2 and other phospholipids were shown to bind directly to TMEM16A isolated from whole pulmonary artery (PA) and TMEM16A-eGFP expressed in HEK293 cells. Agents that reduced PI(4,5)P2 levels through different routes [PLC activation, PI4K inhibition, PI(4,5)P2 scavenging and absorption] all increased IClCa evoked by solutions containing clamped-free [Ca(2+) ], whereas enrichment of activating solutions with PI(4,5)P2 inhibited IClca in PA smooth muscle cells with approximately 50% reduction at 1 μM., Conclusions and Implications: These data are the first to show a negative regulation of TMEM16A-encoded CaCCs by PI(4,5)P2 and propose that control of PI(4,5)P2 levels is a key determinant of arterial physiology., (© 2014 The British Pharmacological Society.)

Background and Purpose: T16A(inh) -A01 is a recently identified inhibitor of the calcium-activated chloride channel TMEM16A. The aim of this study was to test the efficacy of T16A(inh) -A01 for inhibition of calcium-activated chloride channels in vascular smooth muscle and consequent effects on vascular tone., Experimental Approach: Single 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 Results: In rabbit pulmonary artery myocytes T16A(inh) -A01 (1-30 μM) inhibited single calcium (Ca(2+) )-activated chloride (Cl(-) ) channels and whole cell currents activated by 500 nM free Ca(2+) . Similar effects were observed for single Ca(2+) -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, F(act) (10 μM), increased single channel and whole cell Ca(2+) -activated Cl(-) currents in rabbit pulmonary arteries. In isometric tension studies, T16A(inh) -A01 relaxed mouse thoracic aorta pre-contracted with methoxamine with an IC(50) of 1.6 μM and suppressed the methoxamine concentration-effect curve. T16A(inh) -A01 did not affect the maximal contraction produced by 60 mM KCl and the relaxant effect of 10 μM T16A(inh) -A01 was not altered by incubation of mouse thoracic aorta in a cocktail of potassium (K(+) ) channel blockers. T16A(inh) -A01 (10 μM) also relaxed human visceral adipose arteries by 88 ± 3%., Conclusions and Implications: T16A(inh) -A01 blocks calcium-activated chloride channels in vascular smooth muscle cells and relaxes murine and human blood vessels., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

Pulmonary artery smooth muscle cells (PASMCs) are more depolarized and display higher Ca(2+) levels in pulmonary hypertension (PH). Whether the functional properties and expression of Ca(2+)-activated Cl- 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(2+)-activated Cl(-) current [I(Cl(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 μM), an l-type Ca(2+) channel blocker, and NFA (30 or 100 μM, with or without 10 μM indomethacin to inhibit cyclooxygenases) or T16A(Inh)-A01 (10 μM), 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.

The main purpose of this study was to characterize the stimulation of Ca(2+)-activated Cl(-) (Cl(Ca)) by store-operated Ca(2+) entry (SOCE) channels in rabbit pulmonary arterial smooth muscle cells (PASMCs) and determine if this process requires reverse-mode Na(+)/Ca(2+) exchange (NCX). In whole-cell voltage clamped PASMCs incubated with 1 μmol/L nifedipine (Nif) to inhibit Ca(2+) channels, 30 μmol/L cyclopiazonic acid (CPA), a SERCA pump inhibitor, activated a nonselective cation conductance permeable to Na(+) (I(SOC)) during an initial 1-3 s step, ranging from-120 to +60 mV, and Ca(2+)-activated Cl(-) current (I(Cl(Ca))) during a second step to +90 mV that increased with the level of the preceding hyperpolarizing step. Niflumic acid (100 μmol/L), a Cl(Ca) channel blocker, abolished I(Cl(Ca)) but had no effect on I(SOC), whereas the I(SOC) blocker SKF-96365 (50 μmol/L) suppressed both currents. Dual patch clamp and Fluo-4 fluorescence measurements revealed the appearance of CPA-induced Ca(2+) transients of increasing magnitude with increasing hyperpolarizing steps, which correlated with I(Cl(Ca)) amplitude. The absence of Ca(2+) transients at positive potentials following a hyperpolarizing step combined with the observation that SOCE-stimulated I(Cl(Ca)) was unaffected by the NCX blocker KB-R7943 (1 μmol/L) suggest that the SOCE/Cl(Ca) interaction does not require reverse-mode NCX in our conditions.

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.

Aims: Calcium-activated chloride channels (CACCs) share common pharmacological properties with Kcnma1-encoded large conductance K(+) channels (BK(Ca) or K(Ca)1.1) and it has been suggested that they may co-exist in a macromolecular complex. As K(Ca)1.1 channels are known to localize to cholesterol and caveolin-rich lipid rafts (caveolae), the present study investigated whether Ca(2+)-sensitive Cl(-) currents in vascular myocytes were affected by the cholesterol depleting agent methyl-beta-cyclodextrin (M-betaCD)., Methods and Results: Calcium-activated chloride and potassium currents were recorded from single murine portal vein myocytes in whole cell voltage clamp. Western blot was undertaken following sucrose gradient ultracentrifugation using protein lysates from whole portal veins. Ca(2+)-activated Cl(-) currents were augmented by 3 mg mL(-1) M-betaCD with a rapid time course (t(0.5) = 1.8 min). M-betaCD had no effect on the bi-modal response to niflumic acid or anthracene-9-carboxylate but completely removed the inhibitory effects of the K(Ca)1.1 blockers, paxilline and tamoxifen, as well as the stimulatory effect of the K(Ca)1.1 activator NS1619. Discontinuous sucrose density gradients followed by western blot analysis revealed that the position of lipid raft markers caveolin and flotillin-2 was altered by 15 min application of 3 mg mL(-1) M-betaCD. The position of K(Ca)1.1 and the newly identified candidate for CACCs, TMEM16A, was also affected by M-betaCD., Conclusion: These data reveal that CACC properties are influenced by lipid raft integrity.

The purpose of this study was to determine whether extracellular matrix (ECM) composition through integrin receptors modulated the volume-sensitive osmolyte anion channels (VSOACs) in skeletal muscle-derived C2C12 cells. Cl(-) currents were recorded in whole cell voltage-clamped cells grown on laminin (LM), fibronectin (FN), or in the absence of a defined ECM (NM). Basal membrane currents recorded in isotonic media (300 mosmol/kg) were larger in cells grown on FN (3.8-fold at +100 mV) or LM (8.8-fold at +100 mV) when compared with NM. VSOAC currents activated by cell exposure to hypotonic solution were larger in cells grown on LM (1.72-fold at +100 mV) or FN (1.75-fold at +100 mV) compared with NM. Additionally, the kinetics of VSOAC activation was approximately 27% quicker on FN and LM. These currents were tamoxifen sensitive, displayed outward rectification, reversed at the equilibrium potential of Cl(-) and inactivated at potentials >+60 mV. Specific knockdown of beta(1)-integrin by short hairpin RNA interference strongly inhibited the VSOAC Cl(-) currents in cells plated on FN. In conclusion, ECM composition and integrins profoundly influence the biophysical properties and mechanisms of onset of VSOACs.

Ca(2+)-activated Cl-() channels (Cl(Ca)) represent an important excitatory mechanism in vascular smooth muscle cells. Active accumulation of Cl-() by several classes of anion transporters results in an equilibrium potential for this ion about 30 mV more positive than the resting potential. Stimulation of Cl(Ca) channels leads to membrane depolarization, which enhances Ca(2+) entry through voltage-gated Ca(2+) channels and leads to vasoconstriction. Cl(Ca) channels can be activated by distinct sources of Ca(2+) that include (1) mobilization from intracellular Ca(2+) stores (ryanodine or inositol 1,4,5-trisphosphate [InsP(3)]) and (2) Ca(2+) entry through voltage-gated Ca(2+) channels or reverse-mode Na(+)/Ca(2+) exchange. The present study was undertaken to determine whether Ca(2+) influx triggered by store depletion (store-operated calcium entry, SOCE) activates Cl(Ca) channels in rabbit pulmonary artery (PA) smooth muscle. Classical store depletion protocols involving block of sarcoplasmic reticular Ca(2+) reuptake with thapsigargin (TG; 1 microM) or cyclopiazonic acid (CPA; 30 microM) led to a consistent nifedipine-insensitive contraction of intact PA rings and rise in intracellular Ca(2+) concentration in single PA myocytes that required the presence of extracellular Ca(2+). In patch clamp experiments, TG or CPA activated a time-independent nonselective cation current (I (SOC)) that (1) reversed between -10 and 0 mV; (2) displayed the typical "N"-shaped current-voltage relationship; and (3) was sensitive to the (I (SOC)) blocker by SKF-96365 (50 microM). In double-pulse protocol experiments, the amplitude of I (SOC) was varied by altering membrane potential during an initial step that was followed by a second constant step to +90 mV to register Ca(2+)-activated Cl(-) current, I (Cl(Ca)). The niflumic acid-sensitive time-dependent I (Cl(Ca)) at +90 mV increased in proportion to the magnitude of the preceding hyperpolarizing step, an effect attributed to graded membrane potential-dependent Ca(2+) entry through I (SOC) and confirmed in dual patch clamp and Fluo-5 experiments to record membrane current and free intracellular Ca(2+) concentration simultaneously. Reverse-transcription polymerase chain reaction (RT-PCR) experiments confirmed the expression of several molecular determinants of SOCE, including transient receptor potential canonical (TRPC) 1, TRPC4, and TRPC6; stromal interacting molecule (STIM) 1 and 2; and Orai1 and 2, as well as the novel and probable molecular candidates thought to encode for Cl(Ca) channels transmembrane protein 16A (TMEM16A) Anoctamin 1 (ANO1) and B (ANO2). Ourpreliminary investigation provides new evidence for a Ca(2+) entry pathway consistent with store-operated Ca(2+) entry signaling that can activate Ca(2+)-activated Cl-() channels in rabbit PA myocytes. We hypothesize that this mechanism may be important in the regulation of membrane potential, Ca(2+) influx, and tone in these cells under physiological and pathophysiological conditions.

Background and Purpose: Ca(2+)-activated Cl(-) currents (I(Cl(Ca))) in arterial smooth muscle cells are inhibited by phosphorylation. The Ca(2+)-activated Cl(-) channel (Cl(Ca)) blocker niflumic acid (NFA) produces a paradoxical dual effect on I(Cl(Ca)), causing stimulation or inhibition at potentials below or above 0 mV respectively. We tested whether the effects of NFA on I(Cl(Ca)) were modulated by phosphorylation., Experimental Approach: I(Cl(Ca)) was elicited with 500 nM free internal Ca(2+) in rabbit pulmonary artery myocytes. The state of global phosphorylation was altered by cell dialysis with either 5 mM ATP or 0 mM ATP with or without an inhibitor of calmodulin-dependent protein kinase type II, KN-93 (10 microM)., Key Results: Dephosphorylation enhanced the ability of 100 microM NFA to inhibit I(Cl(Ca)). This effect was attributed to a large negative shift in the voltage-dependence of block, which was converted to stimulation at potentials <-50 mV, approximately 70 mV more negative than cells dialysed with 5 mM ATP. NFA dose-dependently blocked I(Cl(Ca)) in the range of 0.1-250 microM in cells dialysed with 0 mM ATP and KN-93, which contrasted with the stimulation induced by 0.1 microM, which converted to block at concentrations >1 microM when cells were dialysed with 5 mM ATP., Conclusions and Implications: Our data indicate that the presumed state of phosphorylation of the pore-forming or regulatory subunit of Cl(Ca) channels influenced the interaction of NFA in a manner that obstructs interaction of the drug with an inhibitory binding site.

Background and Purpose: Recent pharmacological studies have proposed there is a high degree of similarity between calcium-activated Cl(-) channels (CaCCs) and large conductance, calcium-gated K(+) channels (K(Ca)1.1). The goal of the present study was to ascertain whether blockers of K(Ca)1.1 inhibited calcium-activated Cl(-) currents (I(ClCa)) and if the pharmacological overlap between K(Ca)1.1 and CaCCs extends to intermediate and small conductance, calcium-activated K(+) channels., Experimental Approaches: Whole-cell Cl(-) and K(+) currents were recorded from murine portal vein myocytes using the whole-cell variant of the patch clamp technique. CaCC currents were evoked by pipette solutions containing 500 nM free [Ca(2+)]., Key Results: The selective K(Ca)1.1 blocker paxilline (1 microM) inhibited I(ClCa) by approximately 90%, whereas penitrem A (1 microM) and iberiotoxin (100 and 300 nM) reduced the amplitude of I(ClCa) by approximately 20%, as well as slowing channel deactivation. Paxilline also abolished the stimulatory effect of niflumic acid on the CaCC. In contrast, an antibody against the Ca(2+)-binding domain of murine K(Ca)1.1 had no effect on I(ClCa) while inhibiting spontaneous K(Ca)1.1 currents. Structurally different modulators of small and intermediate conductance calcium-activated K(+) channels (K(Ca)2.1 and K(Ca)2.3), namely 1-EBIO, (100 microM); NS309, (1 microM); TRAM-34, (10 microM); UCL 1684, (1 microM) had no effect on I(ClCa)., Conclusions and Implications: These data show that the selective K(Ca)1.1 blockers also reduce I(ClCa) considerably. However, the pharmacological overlap that exists between CaCCs and K(Ca)1.1 does not extend to the calcium-binding domain or to other calcium-gated K(+) channels.

Bestrophins form Ca2+-activated Cl- channels when they are expressed heterologously. Here we report the functional characterization of murine bestrophin 1 (mBest1). We isolated mBest1 transcript from mouse heart and analyzed the biophysical properties and expression of this channel protein using a tetracycline inducible system. mBest1 expression is localized at the membrane of transfected HEK cells, in agreement with its role as a channel. Whole-cell patch clamp experiments revealed a calcium sensitive, time independent chloride current. mBest1 current displayed slight voltage dependence, exhibited an anion permeability sequence of SCN- > I- > Cl- and was sensitive to DIDS and niflumic acid. Anion replacement studies were also performed on mBest2 and mBest3 and differences were observed in their relative permeability and slope conductance to SCN-. Our study provides the first characterization of the biophysical properties of mBest1 and a framework for the elucidation of the physiological role of bestrophins.

Bestrophins are a novel family of proteins that encode calcium-activated chloride channels. In this study we establish that Bestrophin transcripts are expressed in the mouse and human heart. Native mBest3 protein expression and localization in heart was demonstrated by using a specific polyclonal mBest3 antibody. Immunostaining of isolated cardiac myocytes indicates that mBest3 is present at the membrane. Using the patch-clamp technique, we characterized the biophysical and pharmacological properties of mBest3 cloned from heart. Whole cell chloride currents were evoked in both HEK293 and COS-7 cells expressing mBest3 by elevation of intracellular calcium. mBest3 currents displayed a K(D) for Ca(2+) of approximately 175 nM. The calcium-activated chloride current was found to be time and voltage independent and displayed slight outward rectification. The anion permeability sequence of the channel was SCN(-)>I(-)>Cl(-), and the current was inhibited by niflumic acid and DIDS in the micromolar range. In addition, we generated a site-specific mutation (F80L) in the putative pore region of mBest3 that significantly altered the ion conduction and pharmacology of this channel. Our functional and mutational studies examining the biophysical properties of mBest3 indicate that it functions as a pore-forming chloride channel that is activated by physiological levels of calcium. This study reports novel findings regarding the molecular expression, tissue localization, and functional properties of mBest3 cloned from heart.

Because chloride (Cl(-)) channel blockers such as niflumic acid enhance large-conductance Ca(2+)-activated potassium channels (BK(Ca)), the aim of this study was to determine whether there is a reciprocal modification of Ca(2+)-activated chloride Cl(-) currents (I(ClCa)) by two selective activators of BK(Ca). Single smooth muscle cells were isolated by enzymatic digestion from murine portal vein and rabbit pulmonary artery. The BK(Ca) activators NS1619 [1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl-)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one] and isopimaric acid (IpA) augmented macroscopic I(ClCa) elicited by pipette solutions containing [Ca(2+)](i) > 100 nM without any alteration in current kinetics. Enhanced currents recorded in the presence of NS1619 or IpA reversed at the theoretical Cl(-) equilibrium potential, which was shifted by approximately -40 mV upon replacement of the external anion with the more permeable thiocyanate anion. NS1619 increased the sensitivity of calcium-activated chloride channel (Cl(Ca)) to Ca(2+) (approximately 100 nM at +60 mV) and induced a leftward shift in their voltage dependence (approximately 80 mV with 1 micro Ca(2+)). Single-channel experiments revealed that NS1619 increased the number of open channels times the open probability of small-conductance (1.8-3.1 pS) Cl(Ca) without any alteration in their unitary amplitude or number of observable unitary levels of activity. These data, in addition to the established stimulatory effects of niflumic acid on BK(Ca), show that there is similarity in the pharmacology of calcium-activated chloride and potassium channels. Although nonspecific interactions are possible, one alternative hypothesis is that the channel underlying vascular I(ClCa) shares some structural similarity to the BK(Ca) or that the latter K(+) channel physically interacts with Cl(Ca).

Research into Ca2+-activated Cl- channels is hampered by the inability to decipher their molecular identity and the fact that all extant Cl- channel blockers have effects on other ion channels. Most notably, Cl- channel blockers such as the fenamates (e.g. niflumic acid and flufenamic acid) activate Ca2+-dependent K+ channels, although other pharmacological overlaps have been discovered. In this article, we highlight the complex pharmacology of Ca2+-activated Cl- channels and the caveats associated with using these blockers--a necessary requirement because many researchers use Cl- channel blockers as probes for Cl- channel activity. Moreover, we discuss the argument for a common structural motif between Ca2+-activated Cl- channels and Ca2+-dependent K+ channels, which has led to the possibility that the molecular identity of Cl- channels will be revealed by research in this new direction, in addition to the use of existing candidates such as the CLCA, Bestrophin and tweety genes.

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 (I(ClCa)) 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+, I(ClCa) was evoked immediately upon membrane rupture but then exhibited marked rundown to approximately 20% of initial values. In contrast, when phosphorylation was prohibited by using pipette solutions containing adenosine 5'-(beta,gamma-imido)-triphosphate (AMP-PNP) or with ATP omitted, the rundown was severely impaired, and after 20 min dialysis, I(ClCa) was approximately 100% of initial levels. I(ClCa) 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 I(ClCa) 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 I(ClCa) 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.

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.

Calcium-activated chloride channels (Cl(Ca)) are crucial regulators of vascular tone by promoting a depolarizing influence on the resting membrane potential of vascular smooth muscle cells. Niflumic acid (NFA), a potent blocker of Cl(Ca) in vascular myocytes, was shown recently to cause inhibition and paradoxical stimulation of sustained calcium-activated chloride currents [I(Cl(Ca))] in rabbit pulmonary artery myocytes. The aims of the present study were to investigate whether NFA produced a similar dual effect in coronary artery smooth muscle cells and to determine the concentration-dependence and dynamics of such a phenomenon. Sustained I(Cl(Ca)) evoked by intracellular Ca(2+) clamped at 500 nM were dose-dependently inhibited by NFA (IC(50) = 159 microM) and transiently augmented in a concentration-independent manner (10 microM to 1 mM) approximately 2-fold after NFA removal. However, the time to peak and duration of NFA-enhanced I(Cl(Ca)) increased in a concentration-dependent fashion. Moreover, the rate of recovery was reduced by membrane depolarization, suggesting the involvement of a voltage-dependent step in the interaction of NFA, leading to stimulation of I(Cl(Ca)). Computer simulations derived from a kinetic model involving low (K(i) = 1.25 mM) and high (K(i) < 30 microM) affinity sites could reproduce the properties of the NFA-modulated I(Cl(Ca)) fairly well.

The role of the Ca2+-dependent phosphatase calcineurin (CaN) in the modulation of Ca2+-dependent Cl- channels (ClCa) was studied in freshly isolated rabbit coronary arterial myocytes. Immunocytochemical experiments showed that calmodulin-dependent protein kinase II (CaMKII) and CaN were distributed evenly throughout the cytoplasm of coronary myocytes at rest and translocated to the plasmalemma when intracellular Ca2+ was increased. ClCa currents (ICl(Ca)) elicited by cell dialysis with fixed intracellular Ca2+ levels up to 500 nM were inhibited by 10 microM cyclosporin A (CsA), a specific inhibitor of CaN, in a voltage-dependent manner, whereas currents evoked by 1 microM Ca2+ were not affected. Inhibition of CaN with CsA also led to a significant reduction in Ca2+ sensitivity of the channel at +50 mV; half-maximal activation increased from 363 +/- 16 nM Ca2+ in control to 515 +/- 40 nM Ca2+ in the presence of CsA. Similar effects were observed on ICl(Ca) when a specific peptide fragment inhibitor of CaN (CaN-AF, 5 microM) was dialysed into the cell via the pipette (500 nM Ca2+). Application of KN-93 (10 microM), a specific inhibitor of CaMKII, enhanced ICl(Ca) in myocytes dialysed with 1 microM Ca2+ but produced no significant effect on this current when the cells were dialysed with 350 or 500 nM Ca2+. These results are consistent with the notion that in coronary arterial cells, the activity of ClCa is enhanced by dephosphorylation of the channel or a closely associated regulatory protein. Moreover the balance of CaN and CaMKII regulating ICl(Ca) is dependent on the level of Ca2+ used to activate ICl(Ca).

The present study investigated the effect of redox agents on Ca2+-activated Cl- currents ( ICl(Ca)) recorded in smooth muscle cells isolated from rabbit portal vein. In perforated-patch experiments on portal vein cells the amplitude of ICl(Ca) evoked by either spontaneous release of Ca2+ from internal stores or Ca2+ influx through voltage-dependent Ca2+ channels was markedly and irreversibly enhanced by the non-specific oxidant, diamide (10-200 microM). Diamide also prolonged the decay of both currents. The reductant dithiothreitol had no effect on control ICl(Ca) but reversed the increase of current amplitude produced by diamide. Diamide also increased global intracellular Ca2+ at rest but had no effect on the time-course of Ca "sparks" determined by confocal microscopy. Diamide and the endogenous oxidant hydrogen peroxide increased the amplitude and prolonged the kinetics of ICl(Ca)evoked by pipette solutions containing free Ca2+ clamped at 500 nM. Similar effects were observed with the hydrophilic thiol-reactants thimerosal and p-chloromercuriphenylsulphonic acid (PCMPS). Therefore, the gating and activation of Ca2+-activated Cl- conductance is sensitive to redox modification.

1. Ca(2+)-activated chloride currents (I(Cl(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 Ca(2+)-calmodulin-dependent kinase II (CaMKII) were compared between the three cell types. 2. In PA and CA myocytes dialysed and superfused with K+ -free media, pipette solutions containing fixed levels of free Ca(2+) in the range of 250 nM to 1 microM evoked well sustained, outwardly rectifying I(Cl(Ca)) currents in about 90 % of cells. The CaMKII inhibitor KN-93 (5 microM) increased the amplitude of I(Cl(Ca)) in PA and CA myocytes. However, the threshold intracellular Ca(2+) 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. 3. 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 I(Cl(Ca)) evoked by 500 nM Ca(2+). 4. In PV myocytes, I(Cl(Ca)) was evoked by pipette solutions containing up to 1 microM free Ca(2+) in less than 40 % of cells. Application of KN-93 to cells where I(Cl(Ca)) was sustained produced a small inhibition (approximately 25%) of the current in 70 % of the cells. 5. The present study shows that regulation of Ca(2+)-dependent Cl(-) channels by CaMKII differs between arterial and portal vein myocytes.

Background: Ca2+-activated (I(Cl(Ca))) and swelling-induced (I(Cl(swell))) Cl- channels have, respectively, been postulated to participate in the membrane depolarization and contraction mediated by activation of alpha1-adrenoceptors and vascular wall distension during pressurization. Their respective function in generating active force in pressurized arterioles during alpha1-adrenoceptor stimulation remains unsettled., Objectives: Experimental protocols were designed to: (1) assess the relative contribution of I(Cl(Ca)) to the pressure-dependence of lumen diameter of mesenteric arterioles at different states of activation of the alpha1-adrenoceptor, and (2) investigate the potential role of I(Cl(swell)) in spontaneous and agonist-mediated myogenic reactivity., Methods: Segments of endothelium-denuded rabbit mesenteric arterioles with a lumen diameter of approximately 70 microm were cannulated at both ends and studied under isobaric conditions at 36 degrees C. Steady-state lumen diameter at each pressure step investigated (0-100 mmHg, in 20-mmHg increments) was measured by a video-microscopy edge-detection technique., Results: Under control conditions, 23% of the arterioles developed nifedipine-sensitive spontaneous myogenic tone. In the presence of 1 mM tetraethylammonium chloride (TEA) to inhibit Ca2+-dependent K+ channels, the alpha1-agonist phenylephrine (PE) contracted the vessels in a concentration-dependent manner (0.1-10 microM) and potentiated myogenic reactivity. The contraction mediated by 1 microM PE/TEA was abolished by 1 microM nifedipine, indicating that Ca2+ entry through voltage-gated Ca2+ channels was a necessary step in the cascade leading to contraction. Niflumic acid (NfA, 100 microM), a relatively selective inhibitor of I(Cl(Ca)), had no effect on myogenic tone but reversed the PE-induced contraction, varying with the concentration of PE and transmural pressure. For PE concentrations between 0.1 and 1 microM, but not for 10 microM PE, the relaxing efficacy of NfA decreased as applied pressure was raised from 0 to 100 mmHg. At all pressure steps, the NfA-induced relaxation was inversely related to the concentration of PE. DIDS (200 microM), another Cl- channel blocker, inhibited spontaneous myogenic tone, and partially suppressed a component of contraction at elevated transmural pressures in arterioles incubated in 1 microM PE/1 mM TEA/100 microM NfA., Conclusions: Our data indicate that under low to moderate stimulation of the alpha1-adrenoceptor signaling pathway, I(Cl(Ca)) channels play an important role in the sustained contraction produced. Their declining contribution to contraction with increasing transmural pressure may be explained, at least in part, by a progressive enhancement of stretch-induced ionic conductances, possibly volume-sensitive Cl- channels.

The Ca(2+)-activated Cl(-) current [I(Cl(Ca))] contributes to the repolarization of the cardiac action potential under physiological conditions. I(Cl(Ca)) is known to be primarily activated by Ca(2+) release from the sarcoplasmic reticulum (SR). L-type Ca(2+) current [I(Ca(L))] represents the major trigger for Ca(2+) release in the heart. Recent evidence, however, suggests that Ca(2+) entry via reverse-mode Na(+)/Ca(2+) exchange promoted by voltage and/or Na(+) current (I(Na)) may also play a role. The purpose of this study was to test the hypothesis that I(Cl(Ca)) can be induced by I(Na) in the absence of I(Ca(L)). Macroscopic currents and Ca(2+) transients were measured using the whole cell patch-clamp technique in rabbit ventricular myocytes loaded with Indo-1. Nicardipine (10 microM) abolished I(Ca(L)) at a holding potential of -75 mV as tested in Na(+)-free external solution. In the presence of 131 mM external Na(+) and in the absence of I(Ca(L)), a 4-aminopyridine-resistant transient outward current was recorded in 64 of 81 cells accompanying a phasic Ca(2+) transient. The current reversed at -42. 0 +/- 1.3 mV (n = 6) and at +0.3 +/- 1.4 mV (n = 6) with 21 and 141 mM of internal Cl(-), respectively, similar to the predicted reversal potential with low intracellular Cl(-) concentration ([Cl(-)](i)) (-47.8 mV) and high [Cl(-)](i) (-1.2 mV). Niflumic acid (100 microM) inhibited the current without affecting the Ca(2+) signal (n = 8). Both the current and Ca(2+) transient were abolished by 10 mM caffeine (n = 6), 10 microM ryanodine (n = 3), 30 microM tetrodotoxin (n = 9), or removal of extracellular Ca(2+) (n = 6). These properties are consistent with those of I(Cl(Ca)) previously described in mammalian cardiac myocytes. We conclude that 1) I(Cl(Ca)) can be recorded in the absence of I(Ca(L)), and 2) I(Na)-induced SR Ca(2+) release mechanism is also present in the rabbit heart and may play a physiological role in activating the Ca(2+)-sensitive membrane Cl(-) conductance.