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Non-myelinating Schwann cells (NMSC) play important roles in peripheral nervous system formation and function. However, the molecular identity of these cells remains poorly defined. We provide evidence that Kir4.1, an inward-rectifying K+ channel encoded by the KCNJ10 gene, is specifically expressed and active in NMSC. Immunostaining revealed that Kir4.1 is present in terminal/perisynaptic SCs (TPSC), synaptic glia at neuromuscular junctions (NMJ), but not in myelinating SCs (MSC) of adult mice. To further examine the expression pattern of Kir4.1, we generated BAC transgenic Kir4.1-CreER

The repeated evolution of tetrodotoxin (TTX) resistance provides a model for testing hypotheses about the mechanisms of convergent evolution. This poison is broadly employed as a potent antipredator defence, blocking voltage-gated sodium channels (Na

Cellular effects of nanosecond-pulsed electric field exposures can be attenuated by an electric field reversal, a phenomenon called bipolar pulse cancellation. Our investigations of this phenomenon in neuroendocrine adrenal chromaffin cells show that a single 2-ns, 16 MV/m unipolar pulse elicited a rapid, transient rise in intracellular Ca2+ levels due to Ca2+ influx through voltage-gated calcium channels. The response was eliminated by a 2-ns bipolar pulse with positive and negative phases of equal duration and amplitude and fully restored (unipolar-equivalent response) when the delay between each phase of the bipolar pulse was 30 ns. Longer interphase intervals evoked Ca2+ responses that were greater in magnitude than those evoked by a unipolar pulse (stimulation). Cancellation was also observed when the amplitude of the second (negative) phase of the bipolar pulse was half that of the first (positive) phase but progressively lost as the amplitude of the second phase was incrementally increased above that of the first phase. When the amplitude of the second phase was twice that of the first phase, there was stimulation. By comparing the experimental results for each manipulation of the bipolar pulse waveform with analytical calculations of capacitive membrane charging/discharging, also known as accelerated membrane discharge mechanism, we show that the transition from cancellation to unipolar-equivalent stimulation broadly agrees with this model. Taken as a whole, our results demonstrate that electrostimulation of adrenal chromaffin cells with ultrashort pulses can be modulated with interphase intervals of tens of nanoseconds, a prediction of the accelerated membrane discharge mechanism not previously observed in other bipolar pulse cancellation studies. Such modulation of Ca2+ responses in a neural-type cell is promising for the potential use of nanosecond bipolar pulse technologies for remote electrostimulation applications for neuromodulation.

This study explored the mechanism by which Ca2+-activated Cl−channels (CaCCs) encoded by the Tmem16a gene are regulated by calmodulin-dependent protein kinase II (CaMKII) and protein phosphatases 1 (PP1) and 2A (PP2A). Ca2+-activated Cl−currents ( IClCa) were recorded from HEK-293 cells expressing mouse TMEM16A. IClCawere evoked using a pipette solution in which free Ca2+concentration was clamped to 500 nM, in the presence (5 mM) or absence of ATP. With 5 mM ATP, IClCadecayed to ClCarundown seen with ATP-containing pipette solution was greatly diminished by omitting ATP. IClCarecorded after 20 min of cell dialysis with 0 ATP were more than twofold larger than those recorded with 5 mM ATP. Intracellular application of autocamtide-2-related inhibitory peptide (5 µM) or KN-93 (10 µM), two specific CaMKII inhibitors, produced a similar attenuation of TMEM16A rundown. In contrast, internal application of okadaic acid (30 nM) or cantharidin (100 nM), two nonselective PP1 and PP2A blockers, promoted the rundown of TMEM16A in cells dialyzed with 0 ATP. Mutating serine 528 of TMEM16A to an alanine led to a similar inhibition of TMEM16A rundown to that exerted by either one of the two CaMKII inhibitors tested, which was not observed for three putative CaMKII consensus sites for phosphorylation (T273, T622, and S730). Our results suggest that TMEM16A-mediated CaCCs are regulated by CaMKII and PP1/PP2A. Our data also suggest that serine 528 of TMEM16A is an important contributor to the regulation of IClCaby CaMKII.

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

In whole-cell voltage clamped bovine adrenal chromaffin cells maintained at a holding potential of -70 mV, a single 5 ns, 5 MV/m pulse elicited an inward current carried mainly by Na

Hypertension is a major cause of cardiovascular morbidity and mortality, despite the availability of antihypertensive drugs with different targets and mechanisms of action. Here, we provide evidence that pharmacological inhibition of TMEM16A (ANO1), a calcium-activated chloride channel expressed in vascular smooth muscle cells, blocks calcium-activated chloride currents and contraction in vascular smooth muscle in vitro and decreases blood pressure in spontaneously hypertensive rats. The acylaminocycloalkylthiophene TMinh-23 fully inhibited calcium-activated TMEM16A chloride current with nanomolar potency in Fischer rat thyroid cells expressing TMEM16A, and in primary cultures of rat vascular smooth muscle cells. TMinh-23 reduced vasoconstriction caused by the thromboxane mimetic U46619 in mesenteric resistance arteries of wild-type and spontaneously hypertensive rats, with a greater inhibition in spontaneously hypertensive rats. Blood pressure measurements by tail-cuff and telemetry showed up to a 45-mmHg reduction in systolic blood pressure lasting for four-six hours in spontaneously hypertensive rats after a single dose of TMinh-23. A minimal effect on blood pressure was seen in wild-type rats or mice treated with TMinh-23. Five-day twice daily treatment of spontaneously hypertensive rats with TMinh-23 produced sustained reductions of 20-25 mmHg in daily mean systolic and diastolic blood pressure. TMinh-23 action was reversible, with blood pressure returning to baseline in spontaneously hypertensive rats by three days after treatment discontinuation. Thus, our studies provide validation for TMEM16A as a target for antihypertensive therapy and demonstrate the efficacy of TMinh-23 as an antihypertensive with a novel mechanism of action.

Previously we reported that adrenal chromaffin cells exposed to a 5 ns, 5 MV/m pulse release the catecholamines norepinephrine (NE) and epinephrine (EPI) in a Ca(2+)-dependent manner. Here we determined that NE and EPI release increased with pulse number (one versus five and ten pulses at 1 Hz), established that release occurs by exocytosis, and characterized the exocytotic response in real-time. Evidence of an exocytotic mechanism was the appearance of dopamine-β-hydroxylase on the plasma membrane, and the demonstration by total internal reflection fluorescence microscopy studies that a train of five or ten pulses at 1 Hz triggered the release of the fluorescent dye acridine orange from secretory granules. Release events were Ca(2+)-dependent, longer-lived relative to those evoked by nicotinic receptor stimulation, and occurred with a delay of several seconds despite an immediate rise in Ca(2+). In complementary studies, cells labeled with the plasma membrane fluorescent dye FM 1-43 and exposed to a train of ten pulses at 1 Hz underwent Ca(2+)-dependent increases in FM 1-43 fluorescence indicative of granule fusion with the plasma membrane due to exocytosis. These results demonstrate the effectiveness of ultrashort electric pulses for stimulating catecholamine release, signifying their promise as a novel electrostimulation modality for neurosecretion.

Knowledge of the dielectric properties of biological cells plays an important role in numerical models aimed at understanding how high intensity ultrashort nanosecond electric pulses affect the plasma membrane and the membranes of intracellular organelles. To this end, using electrical impedance spectroscopy, the dielectric properties of isolated, neuroendocrine adrenal chromaffin cells were obtained. Measured impedance data of the cell suspension, acquired between 1 kHz and 20 MHz, were fit into a combination of constant phase element and Cole-Cole models from which the effect of electrode polarization was extracted. The dielectric spectrum of each cell suspension was fit into a Maxwell-Wagner mixture model and the Clausius-Mossotti factor was obtained. Lastly, to extract the cellular dielectric parameters, the cell dielectric data were fit into a granular cell model representative of a chromaffin cell, which was based on the inclusion of secretory granules in the cytoplasm. Chromaffin cell parameters determined from this study were the cell and secretory granule membrane specific capacitance (1.22 and 7.10 μF/cm2, respectively), the cytoplasmic conductivity, which excludes and includes the effect of intracellular membranous structures (1.14 and 0.49 S/m, respectively), and the secretory granule milieu conductivity (0.35 S/m). These measurements will be crucial for incorporating into numerical models aimed at understanding the differential poration effect of nanosecond electric pulses on chromaffin cell membranes.

Previously we reported that adrenal chromaffin cells exposed to a 5 ns, 5 MV/m pulse release the catecholamines norepinephrine (NE) and epinephrine (EPI) in a Ca2+-dependent manner. Here we determined that NE and EPI release increased with pulse number (one versus five and ten pulses at 1 Hz), established that release occurs by exocytosis, and characterized the exocytotic response in real-time. Evidence of an exocytotic mechanism was the appearance of dopamine-β-hydroxylase on the plasma membrane, and the demonstration by total internal reflection fluorescence microscopy studies that a train of five or ten pulses at 1 Hz triggered the release of the fluorescent dye acridine orange from secretory granules. Release events were Ca2+-dependent, longer-lived relative to those evoked by nicotinic receptor stimulation, and occurred with a delay of several seconds despite an immediate rise in Ca2+. In complementary studies, cells labeled with the plasma membrane fluorescent dye FM 1-43 and exposed to a train of ten pulses at 1 Hz underwent Ca2+-dependent increases in FM 1-43 fluorescence indicative of granule fusion with the plasma membrane due to exocytosis. These results demonstrate the effectiveness of ultrashort electric pulses for stimulating catecholamine release, signifying their promise as a novel electrostimulation modality for neurosecretion.

Nanosecond-duration electric pulses (NEPs) can permeabilize the endoplasmic reticulum (ER), causing release of Ca2+ into the cytoplasm. This study used experimentation coupled with numerical modeling to understand the lack of Ca2+ mobilization from Ca2+-storing organelles in catecholamine-secreting adrenal chromaffin cells exposed to 5-ns pulses. Fluorescence imaging determined a threshold electric (E) field of 8 MV/m for mobilizing intracellular Ca2+ whereas whole-cell recordings of membrane conductance determined a threshold E-field of 3 MV/m for causing plasma membrane permeabilization. In contrast, a 2D numerical model of a chromaffin cell, which was constructed with internal structures representing a nucleus, mitochondrion, ER, and secretory granule, predicted that exposing the cell to the same 5-ns pulse electroporated the plasma and ER membranes at the same E-field amplitude, 3–4 MV/m. Agreement of the numerical simulations with the experimental results was obtained only when the ER interior conductivity was 30-fold lower than that of the cytoplasm and the ER membrane permittivity was twice that of the plasma membrane. A more realistic intracellular geometry for chromaffin cells in which structures representing multiple secretory granules and an ER showed slight differences in the thresholds necessary to porate the membranes of the secretory granules. We conclude that more sophisticated cell models together with knowledge of accurate dielectric properties are needed to understand the effects of NEPs on intracellular membranes in chromaffin cells, information that will be important for elucidating how NEPs porate organelle membranes in other cell types having a similarly complex cytoplasmic ultrastructure.

Exposing adrenal chromaffin cells to single 150 to 400 ns electric pulses triggers a rise in intracellular Ca2+ ([Ca2+]i) that is due to Ca2+ influx through voltage-gated Ca2+ channels (VGCC) and plasma membrane electropores. Immediate delivery of a second pulse of the opposite polarity in which the duration and amplitude were the same as the first pulse (a symmetrical bipolar pulse) or greater than the first pulse (an asymmetrical bipolar pulse) had a stimulatory effect, evoking larger Ca2+ responses than the corresponding unipolar pulse. Progressively decreasing the amplitude of the opposite polarity pulse while also increasing its duration converted stimulation to attenuation, which reached a maximum of 43% when the positive phase was 150 ns at 3.1 kV/cm, and the negative phase was 800 ns at 0.2 kV/cm. When VGCCs were blocked, Ca2+ responses evoked by asymmetrical and even symmetrical bipolar pulses were significantly reduced relative to those evoked by the corresponding unipolar pulse under the same conditions, indicating that attenuation involved mainly the portion of Ca2+ influx attributable to membrane electropermeabilization. Thus, by tuning the shape of the bipolar pulse, Ca2+ entry into chromaffin cells through electropores could be attenuated while preserving Ca2+ influx through VGCCs.

TMEM16A (Transmembrane protein 16A or Anoctamin1) is a calcium-activated chloride channel. (CaCC),that exerts critical roles in epithelial secretion. However, its localization, function, and regulation in intestinal chloride (Cl−) secretion remain obscure. Here, we show that TMEM16A protein abundance correlates with Cl− secretion in different regions of native intestine activated by the Ca2+-elevating muscarinic agonist carbachol (CCH). Basal, as well as both cAMP- and CCH-stimulated Isc, was largely reduced in Ano1 ± mouse intestine. We found CCH was not able to increase Isc in the presence of apical to serosal Cl− gradient, strongly supporting TMEM16A as primarily a luminal Cl− channel. Immunostaining demonstrated apical localization of TMEM16A where it colocalized with NHERF1 in mouse colonic tissue. Cellular depletion of NHERF1 in human colonic T84 cells caused a significant reduction of both cAMP- and CCH-stimulated Isc. Immunoprecipitation experiments revealed that NHERF1 forms a complex with TMEM16A through a PDZ-based interaction. We conclude that TMEM16A is a luminal Cl− channel in the intestine that functionally interacts with CFTR via PDZ-based interaction of NHERF1 for efficient and specific cholinergic stimulation of intestinal Cl− secretion., Highlights • TMEM16A express apically and operate Cl− secretion in mouse intestinal tissue. • TMEM16A potentially interacts with NHERF1 via its C-terminal PDZ binding motif. • TMEM16A-NHERF1 complex is requisite for cAMP and Ca2+ mediated apical Cl− secretion.

Patch clamp electrophysiology serves as a powerful method for studying changes in plasma membrane ion conductance induced by externally applied high-intensity nanosecond electric pulses (NEPs). This paper describes an enhanced monitoring technique that minimizes the length of time between pulse exposure and data recording in a patch-clamped excitable cell. Whole-cell membrane currents were continuously recorded up to 11 ms before and resumed 8 ms after delivery of a 5-ns, 6 MV/m pulse by a pair of tungsten rod electrodes to a patched adrenal chromaffin cell maintained at a holding potential of -70 mV. This timing was achieved by two sets of relay switches. One set was used to disconnect the patch pipette electrode from the pre-amplifier and connect it to a battery to maintain membrane potential at -70 mV, and also to disconnect the reference electrode from the amplifier. The other set was used to disconnect the electrodes from the pulse generator until the time of NEP/sham exposure. The sequence and timing of both sets of relays were computer-controlled. Using this procedure, we observed that a 5-ns pulse induced an instantaneous inward current that decayed exponentially over the course of several minutes, that a second pulse induced a similar response, and that the current was carried, at least in part, by Na+. This approach for characterizing ion conductance changes in an excitable cell in response to NEPs will yield information essential for assessing the potential use of NEP stimulation for therapeutic applications.

Exposing adrenal chromaffin cells to single 150 to 400 ns electric pulses triggers a rise in intracellular Ca2+ ([Ca2+]i) that is due to Ca2+ influx through voltage-gated Ca2+ channels (VGCC) and plasma membrane electropores. Immediate delivery of a second pulse of the opposite polarity in which the duration and amplitude were the same as the first pulse (a symmetrical bipolar pulse) or greater than the first pulse (an asymmetrical bipolar pulse) had a stimulatory effect, evoking larger Ca2+ responses than the corresponding unipolar pulse. Progressively decreasing the amplitude of the opposite polarity pulse while also increasing its duration converted stimulation to attenuation, which reached a maximum of 43% when the positive phase was 150 ns at 3.1 kV/cm, and the negative phase was 800 ns at 0.2 kV/cm. When VGCCs were blocked, Ca2+ responses evoked by asymmetrical and even symmetrical bipolar pulses were significantly reduced relative to those evoked by the corresponding unipolar pulse under the same conditions, indicating that attenuation involved mainly the portion of Ca2+ influx attributable to membrane electropermeabilization. Thus, by tuning the shape of the bipolar pulse, Ca2+ entry into chromaffin cells through electropores could be attenuated while preserving Ca2+ influx through VGCCs.

Exposing adrenal chromaffin cells to 5 ns electric pulses (nsPEF) causes a rapid rise in intracellular Ca2+(Ca2+i) that is solely the result of Ca2+influx through voltage-gated Ca2+channels (VGCCs). This study explored the effect of longer duration nsPEF onCa2+i. Single 150, 200, or 400 ns pulses at 3.1 kV/cm evoked rapid increases inCa2+i, the magnitude of which increased linearly with pulse width and electric field amplitude. Recovery ofCa2+ito prestimulus levels was faster for 150 ns exposures. Regardless of pulse width, no rise inCa2+ioccurred in the absence of extracellular Ca2+, indicating that the source of Ca2+was from outside the cell. Ca2+responses evoked by a 150 ns pulse were inhibited to varying degrees byω-agatoxin IVA,ω-conotoxin GVIA, nitrendipine or nimodipine, antagonists of P/Q-, N-, and L-type VGCCs, respectively, and by 67% when all four types of VGCCs were blocked simultaneously. The remaining Ca2+influx insensitive to VGCC inhibitors was attributed to plasma membrane nanoporation, which comprised theE-field sensitive component of the response. Both pathways of Ca2+entry were inhibited by 200 μM Cd2+. These results demonstrate that, in excitable chromaffin cells, single 150–400 ns pulses increased the permeability of the plasma membrane to Ca2+in addition to causing Ca2+influx via VGCCs.

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 [Ca2+], 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.

The mechanisms governing maintenance of quiescence during pregnancy remain largely unknown. The current study characterizes a stretch-activated, tetraethylammonium-insensitive K+ current in smooth muscle cells isolated from pregnant human myometrium. This study hypothesizes that these K+ currents can be attributed to TREK-1 and that upregulation of this channel during pregnancy assists with the maintenance of a negative cell membrane potential, conceivably contributing to uterine quiescence until full term. The results of this study demonstrate that, in pregnant human myometrial cells, outward currents at 80 mV increased from 4.8 ± 1.5 to 19.4 ± 7.5 pA/pF and from 3.0 ± 0.8 to 11.8 ± 2.7 pA/pF with application of arachidonic acid (AA) and NaHCO3, respectively, causing intracellular acidification. Similarly, outward currents were inhibited following application of 10 μM fluphenazine by 51.2 ± 9.8% after activation by AA and by 73.9 ± 4.2% after activation by NaHCO3. In human embryonic kidney (HEK-293) cells stably expressing TREK-1, outward currents at 80 mV increased from 91.0 ± 23.8 to 247.5 ± 73.3 pA/pF and from 34.8 ± 8.9 to 218.6 ± 45.0 pA/pF with application of AA and NaHCO3, respectively. Correspondingly, outward currents were inhibited 89.5 ± 2.3% by 10 μM fluphenazine following activation by AA and by 91.6 ± 3.4% following activation by NaHCO3. Moreover, currents in human myometrial cells were activated by stretch and were reduced by transfection with small interfering RNA or extracellular acidification. Understanding gestational regulation of expression and gating of TREK-1 channels could be important in determining appropriate maintenance of uterine quiescence during pregnancy.

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.

Pulmonary artery smooth muscle cells (PASMCs) are more depolarized and display higher Ca2+ levels in pulmonary hypertension (PH). Whether the functional properties and expression of Ca2+-activated Cl− channels (ClCa), an important excitatory mechanism in PASMCs, are altered in PH is unknown. The potential role of ClCa 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 Ca2+-activated Cl− 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 ICl(Ca) were accompanied by parallel changes in the expression of TMEM16A, a gene recently shown to encode for ClCa 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 Ca2+ channel blocker, and NFA (30 or 100 μM, with or without 10 μM indomethacin to inhibit cyclooxygenases) or T16AInh-A01 (10 μM), TMEM16A/ClCa channel inhibitors, than that of control animals. In conclusion, augmented ClCa/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.

Anoctamin-1 (ANO1) is a Ca2+-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 Ca2+ concentration ([Ca2+]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 [Ca2+]i. Increasing [Ca2+]i reduced the potency of this blocker. Similar Ca2+-dependent effects were also observed with benzbromarone. Experiments on excised, inside-out patches showed that the diminished potency of the blockers caused by intracellular Ca2+ 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 [Ca2+]i depends on the splice variant expressed. These experiments demonstrate that the efficacy of ANO1 antagonists depends on [Ca2+]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.

The main purpose of this study was to characterize the stimulation of Ca2+-activated Cl–(ClCa) by store-operated Ca2+entry (SOCE) channels in rabbit pulmonary arterial smooth muscle cells (PASMCs) and determine if this process requires reverse-mode Na+/Ca2+exchange (NCX). In whole-cell voltage clamped PASMCs incubated with 1 μmol/L nifedipine (Nif) to inhibit Ca2+channels, 30 μmol/L cyclopiazonic acid (CPA), a SERCA pump inhibitor, activated a nonselective cation conductance permeable to Na+(ISOC) during an initial 1–3 s step, ranging from–120 to +60 mV, and Ca2+-activated Cl–current (ICl(Ca)) during a second step to +90 mV that increased with the level of the preceding hyperpolarizing step. Niflumic acid (100 μmol/L), a ClCachannel blocker, abolished ICl(Ca)but had no effect on ISOC, whereas the ISOCblocker SKF-96365 (50 μmol/L) suppressed both currents. Dual patch clamp and Fluo-4 fluorescence measurements revealed the appearance of CPA-induced Ca2+transients of increasing magnitude with increasing hyperpolarizing steps, which correlated with ICl(Ca)amplitude. The absence of Ca2+transients at positive potentials following a hyperpolarizing step combined with the observation that SOCE-stimulated ICl(Ca)was unaffected by the NCX blocker KB-R7943 (1 μmol/L) suggest that the SOCE/ClCainteraction does not require reverse-mode NCX in our conditions.

Background and purpose: Ca2+-activated Cl- currents (ICl(Ca)) in arterial smooth muscle cells are inhibited by phosphorylation. The Ca2+-activated Cl- channel (ClCa) blocker niflumic acid (NFA) produces a paradoxical dual effect on ICl(Ca), causing stimulation or inhibition at potentials below or above 0 mV respectively. We tested whether the effects of NFA on ICl(Ca) were modulated by phosphorylation. Experimental approach: ICl(Ca) was elicited with 500 nM free internal Ca2+ 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 µM). Key results: Dephosphorylation enhanced the ability of 100 µM NFA to inhibit ICl(Ca). This effect was attributed to a large negative shift in the voltage-dependence of block, which was converted to stimulation at potentials 1 µM 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 ClCa 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 (KCa1.1). The goal of the present study was to ascertain whether blockers of KCa1.1 inhibited calcium-activated Cl- currents (IClCa) and if the pharmacological overlap between KCa1.1 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 [Ca2+]. Key results: The selective KCa1.1 blocker paxilline (1 µM) inhibited IClCa by ∼90%, whereas penitrem A (1 µM) and iberiotoxin (100 and 300 nM) reduced the amplitude of IClCa by ∼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 Ca2+-binding domain of murine KCa1.1 had no effect on IClCa while inhibiting spontaneous KCa1.1 currents. Structurally different modulators of small and intermediate conductance calcium-activated K+ channels (KCa2.1 and KCa2.3), namely 1-EBIO, (100 µM); NS309, (1 µM); TRAM-34, (10 µM); UCL 1684, (1 µM) had no effect on IClCa. Conclusions and implications: These data show that the selective KCa1.1 blockers also reduce IClCa considerably. However, the pharmacological overlap that exists between CaCCs and KCa1.1 does not extend to the calcium-binding domain or to other calcium-gated K+ channels.

Bestrophins form Ca 2+ -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.

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).