Anoctamin-like protein 1 regulates repolarization in Paramecium behavioral responses.

Paramecium exhibits responsive behavior to environmental changes, moving either closer to or further away from stimuli. Electrophysiological experiments have revealed that these behavioral responses are controlled by membrane potentials. Anoctamin, a Ca2+-activated Cl- channel, is involved in the regulation of membrane potential in mammals. However, it remains uncertain whether Cl- channels like anoctamin regulate Paramecium behavior. Herein, replacement of external Cl- ions with acetate ion and application of Cl- channel blocker niflumic acid (NFA, 0.1 µM) increased spontaneous avoiding reactions (sARs). Hence, we hypothesized that anoctamin is involved in the stabilization of membrane potential fluctuation. Paramecium cells in which the anoctamin-like protein 1 gene was knocked down displayed frequent sARs in the culture medium without external stimulation. Treatment of anoctamin-like protein 1-knockdown cells with the Ca2+ chelator BAPTA or Ca-channel blocker nicardipine reversed the increase in sARs. Electrophysiological experiments revealed extension of membrane depolarization when positive currents were applied to anoctamin-like protein 1-knockdown cells. We concluded that anoctamin-like protein 1 works as a Cl-channel and stabilizes the membrane potential oscillation, reducing sARs.

Compensation mechanism for membrane potential against hypoosmotic stress in the Onchidium neuron.

The effect of acute hypoosmotic stress on the neural response was investigated using the neurons identified in the abdominal ganglion of the amphibious mollusk Onchidium. The membrane potential of an identified neuron (Ip-1/2) was not significantly altered in 50% hypoosmotic artificial sea water. In isotonic 50% artificial seawater (ASW) with osmolarity that was compensated for using glycerol or urea, the membrane potentials of Ip-1/2 were also not altered compared to those in 50% hypoosmotic ASW. However, hyperpolarization was induced in isotonic 50% ASW when osmolarity was compensated for using sucrose or mannose. In the presence of volume-regulated anion channel (VRAC) inhibitors (niflumic acid and glibenclamide), the Ip-1/2 membrane potentials were hyperpolarized in 50% hypoosmotic ASW. These results suggest that there is a compensatory mechanism involving aquaglyceroporin and VRAC-like channels that maintains membrane potential under hypoosmotic conditions. Here, we detected the expression of aquaglyceroporin mRNA in neural tissues of Onchidium.

TMEM16A Ca2+-Activated Cl- Channel Regulates the Proliferation and Migration of Brain Capillary Endothelial Cells.

The blood-brain barrier (BBB) is essential for the maintenance of homeostasis in the brain. Brain capillary endothelial cells (BCECs) comprise the BBB, and thus a delicate balance between their proliferation and death is required. Although the activity of ion channels in BCECs is involved in BBB functions, the underlying molecular mechanisms remain unclear. In the present study, the molecular components of Ca2+-activated Cl- (ClCa) channels and their physiological roles were examined using mouse BCECs (mBCECs) and a cell line derived from bovine BCECs, t-BBEC117. Expression analyses revealed that TMEM16A was strongly expressed in mBCECs and t-BBEC117 cells. In t-BBEC117 cells, whole-cell Cl- currents were sensitive to the ClCa channel blockers, 100 µM niflumic acid and 10 µM T16Ainh-A01, and were also reduced markedly by small-interfering RNA (siRNA) knockdown of TMEM16A. Importantly, block of ClCa currents with ClCa channel blockers or TMEM16A siRNA induced membrane hyperpolarization. Moreover, treatment with TMEM16A siRNA caused an increase in resting cytosolic Ca2+ concentration ([Ca2+]cyt). T16Ainh-A01 reduced cell viability in a concentration-dependent manner. Either ClCa channel blockers or TMEM16A siRNA also curtailed cell proliferation and migration. Furthermore, ClCa channel blockers attenuated the trans-endothelial permeability. In combination, these results strongly suggest that TMEM16A contributes to ClCa channel conductance and can regulate both the resting membrane potential and [Ca2+]cyt in BCECs. Our data also reveal how these BCECs may be involved in the maintenance of BBB functions, as both the proliferation and migration are altered following changes in channel activity. SIGNIFICANCE STATEMENT: In brain capillary endothelial cells (BCECs) of the blood-brain barrier (BBB), TMEM16A is responsible for Ca2+-activated Cl- channels and can regulate both the resting membrane potential and cytosolic Ca2+ concentration, contributing to the proliferation and migration of BCECs. The present study provides novel information on the molecular mechanisms underlying the physiological functions of BCECs in the BBB and a novel target for therapeutic drugs for disorders associated with dysfunctions in the BBB.

Properties of single-channel and whole cell Cl- currents in guinea pig detrusor smooth muscle cells.

Multiple types of Cl- channels regulate smooth muscle excitability and contractility in vascular, gastrointestinal, and airway smooth muscle cells. However, little is known about Cl- channels in detrusor smooth muscle (DSM) cells. Here, we used inside-out single channel and whole cell patch-clamp recordings for detailed biophysical and pharmacological characterizations of Cl- channels in freshly isolated guinea pig DSM cells. The recorded single Cl- channels displayed unique gating with multiple subconductive states, a fully opened single-channel conductance of 164 pS, and a reversal potential of -41.5 mV, which is close to the ECl of -65 mV, confirming preferential permeability to Cl-. The Cl- channel demonstrated strong voltage dependence of activation (half-maximum of mean open probability, V0.5, ~-20 mV) and robust prolonged openings at depolarizing voltages. The channel displayed similar gating when exposed intracellularly to solutions containing Ca2+-free or 1 mM Ca2+. In whole cell patch-clamp recordings, macroscopic current demonstrated outward rectification, inhibitions by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and niflumic acid, and insensitivity to chlorotoxin. The outward current was reversibly reduced by 94% replacement of extracellular Cl- with I-, Br-, or methanesulfonate (MsO-), resulting in anionic permeability sequence: Cl->Br->I->MsO-. While intracellular Ca2+ levels (0, 300 nM, and 1 mM) did not affect the amplitude of Cl- current and outward rectification, high Ca2+ slowed voltage-step current activation at depolarizing voltages. In conclusion, our data reveal for the first time the presence of a Ca2+-independent DIDS and niflumic acid-sensitive, voltage-dependent Cl- channel in the plasma membrane of DSM cells. This channel may be a key regulator of DSM excitability.

Volume-regulated Cl- current: contributions of distinct Cl- channels and localized Ca2+ signals.

The swelling-activated chloride current (ICl,swell) is induced when a cell swells and plays a central role in maintaining cell volume in response to osmotic stress. The major contributor of ICl,swell is the volume-regulated anion channel (VRAC). Leucine-rich repeat containing 8A (LRRC8A; SWELL1) was recently identified as an essential component of VRAC, but the mechanisms of VRAC activation are still largely unknown; moreover, other Cl- channels, such as anoctamin 1 (ANO1), were also suggested to contribute to ICl,swell. In this present study, we investigated the roles of LRRC8A and ANO1 in activation of ICl,swell; we also explored the role of intracellular Ca2+ in ICl,swell activation. We used a CRISPR/Cas9 gene editing approach, electrophysiology, live fluorescent imaging, selective pharmacology, and other approaches to show that both LRRC8A and ANO1 can be activated by cell swelling in HEK293 cells. Yet, both channels contribute biophysically and pharmacologically distinct components to ICl,swell, with LRRC8A being the major component. Cell swelling induced oscillatory Ca2+ transients, and these Ca2+ signals were required to activate both the LRRC8A- and ANO1-dependent components of ICl,swell. Both ICl,swell components required localized rather than global Ca2+ for activation. Interestingly, while intracellular Ca2+ was necessary and sufficient to activate ANO1, it was necessary but not sufficient to activate LRRC8A-mediated currents. Finally, Ca2+ transients linked to the ICl,swell activation were mediated by the G protein-coupled receptor-independent PLC isoforms.

TMEM16A inhibition impedes capacitation and acquisition of hyperactivated motility in guinea pig sperm.

Ca2+ -activated Cl- channels (CaCCs) are anionic channels that regulate many important physiological functions associated with chloride and calcium flux in some somatic cells. The molecular identity of CaCCs was revealed to be TMEM16A and TMEM16B (also known as Anoctamin or ANO1 and ANO2, respectively) in all eukaryotes. A recent study suggests the presence of TMEM16A in human sperm and a relationship with the rhZP-induced acrosome reaction. However, to the best of our knowledge, little is known about the role of TMEM16A in other spermatic processes such as capacitation or motility. In this study, we evaluated the effects of two TMEM16A antagonists on capacitation, acrosome reaction, and motility in guinea pig sperm; these antagonists were T16Ainh-A01, belonging to a second generation of potent antagonists of TMEM16A, and niflumic acid (NFA), a well-known antagonist of TMEM16A (CaCCs). First of all, we confirmed that the absence of Cl- in the capacitation medium changes motility parameters, capacitation, and the progesterone-induced acrosome reaction. Using a specific antibody, TMEM16A was found as a protein band of ∼120 kDa, which localization was in the apical crest of the acrosome and the middle piece of the flagellum. Inhibition of TMEM16A by T16Ainh-A01 affected sperm physiology by reducing capacitation, blocking the progesterone-induced acrosome reaction under optimal capacitation conditions, inhibiting progressive motility, and the acquisition of hyperactivated motility, diminishing [Ca2+ ]i, and increasing [Cl- ]i. These changes in sperm kinematic parameters provide new evidence of the important role played by TMEM16A in the production of sperm capable of fertilizing oocytes.

A Potassium-Selective Current Affected by Micromolar Concentrations of Anion Transport Inhibitors.

BACKGROUND/AIMS: In the human genome, more than 400 genes encode ion channels, which are ubiquitously expressed and often coexist and participate in almost all physiological processes. Therefore, ion channel blockers represent fundamental tools in discriminating the contribution of individual channel types to a physiological phenomenon. However, unspecific effects of these compounds may represent a confounding factor. Three commonly used chloride channel inhibitors, i.e. 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS), 5-nitro-2-[(3-phenylpropyl) amino]benzoic acid (NPPB) and the anti-inflammatory drug niflumic acid were tested to identify the lowest concentration effective on Cl- channels and ineffective on K+ channels. METHODS: The activity of the above mentioned compounds was tested by whole cell patch-clamp on the swelling-activated Cl- current ICl,swell and on the endogenous voltage-dependent, outwardly rectifying K+ selective current in human kidney cell lines (HEK 293/HEK 293 Phoenix). RESULTS: Micromolar (1-10 µM) concentrations of DIDS and NPPB could not discriminate between the Cl- and K+ selective currents. Specifically, 1 µM DIDS only affected the K+ current and 10 µM NPPB equally affected the Cl- and K+ currents. Only relatively high (0.1-1 mM) concentrations of DIDS and prolonged (5 minutes) exposure to 0.1-1 mM NPPB preferentially suppressed the Cl- current. Niflumic acid preferentially inhibited the Cl- current, but also significantly affected the K+ current. The endogenous voltage-dependent, outwardly rectifying K+ selective current in HEK 293/HEK 293 Phoenix cells was shown to arise from the Kv 3.1 channel, which is extensively expressed in brain and is involved in neurological diseases. CONCLUSION: The results of the present study underscore that sensitivity of a given physiological phenomenon to the Cl- channel inhibitors NPPB, DIDS and niflumic acid may actually arise from an inhibition of Cl- channels but can also result from an inhibition of voltage-dependent K+ channels, including the Kv 3.1 channel. The use of niflumic acid as anti-inflammatory drug in patients with concomitant Kv 3.1 dysfunction may result contraindicated.

Colonic Hypermotility in a Rat Model of Irritable Bowel Syndrome Is Associated with Upregulation of TMEM16A in Myenteric Plexus.

BACKGROUND: Irritable bowel syndrome (IBS) is a common disease with intestinal dysmotility, whose mechanism remains elusive. TMEM16A is a calcium-activated chloride channel (CaCC) involved in intestinal slow-wave generation. AIMS: To investigate whether TMEM16A is involved in colonic dysmotility in IBS. METHODS: A rat model of IBS was established by chronic water avoidance stress (WAS). Colonic pathological alterations were evaluated histologically, and intestinal motility was assessed by intestinal transit time (ITT) and fecal water content (FWC). Visceral sensitivity was determined by visceromotor response (VMR) to colorectal distension (CRD). TMEM16A expression was evaluated by RT-PCR, Western blot, and immunofluorescence. Colonic muscle strip contractility was measured by isometric transducers, and the effect of niflumic acid (NFA), a CaCC antagonist, on colonic motility was examined. RESULTS: After 10 days of WAS exposure, ITT was decreased and FWC was elevated. Furthermore, VMR magnitude of WAS rats in response to CRD was significantly enhanced. Protein and mRNA levels of TMEM16A in colon were considerably increased after WAS. The percentage of TMEM16A-positive neurons in myenteric plexus (MP) of WAS rats was significantly higher than controls. Pharmacological blockade of TMEM16A activity by NFA considerably enhanced ITT, with concentration-dependent declines in FWC and VMR magnitude in NFA-treated rats. Further, spontaneous contraction of colonic strips of NFA-treated rats was significantly ameliorated in a concentration-dependent manner in vitro. CONCLUSIONS: Upregulation of TMEM16A in MP neurons may play an important role in chronic stress-induced colonic hypermotility, making CaCC-blocking drugs a putatively effective treatment method for colonic hypermotility in IBS.

Inhibitory effects of sulfonylureas and non-steroidal anti-inflammatory drugs on in vitro metabolism of canagliflozin in human liver microsomes.

Canagliflozin, used to treat type 2 diabetes mellitus (T2DM), is commonly co-administered with sulfonylureas. The objective of the present study was to evaluate the possible inhibitory effect of sulfonylureas and non-steroidal anti-inflammatory drugs (NSAIDs) on canagliflozin metabolism in vitro. Three sulfonylurea derivatives were evaluated as inhibitors: chlorpropamide, glimepiride and gliclazide. Two other NSAIDs were used as positive control inhibitors: niflumic acid and diclofenac. The rate of formation of canagliflozin metabolites was determined by HPLC analysis of in vitro incubations of canagliflozin as a substrate with and without inhibitors, using human liver microsomes (HLMs). Among sulfonylureas, glimepiride showed the most potent inhibitory effect against canagliflozin M7 metabolite formation, with an IC50 value of 88 µm, compared to chlorpropamide and gliclazide with IC50 values of more than 500 µm. Diclofenac inhibited M5 metabolite formation more than M7, with IC50 values of 32 µm for M5 and 80 µm for M7. Niflumic acid showed no inhibition activity against M5 formation, but had relatively selective inhibitory potency against M7 formation, which is catalysed by UGT1A9, with an IC50 value of 1.9 µm and an inhibition constant value of 0.8 µm. A clinical pharmacokinetic interaction between canagliflozin and sulfonylureas is unlikely. However, a possible clinically important drug interaction between niflumic acid and canagliflozin has been identified.

The Odorant Receptor-Dependent Role of Olfactory Marker Protein in Olfactory Receptor Neurons.

Olfactory receptor neurons (ORNs) in the nasal cavity detect and transduce odorants into action potentials to be conveyed to the olfactory bulb. Odorants are delivered to ORNs via the inhaled air at breathing frequencies that can vary from 2 to 10 Hz in the mouse. Thus olfactory transduction should occur at sufficient speed such that it can accommodate repetitive and frequent stimulation. Activation of odorant receptors (ORs) leads to adenylyl cyclase III activation, cAMP increase, and opening of cyclic nucleotide-gated channels. This makes the kinetic regulation of cAMP one of the important determinants for the response time course. We addressed the dynamic regulation of cAMP during the odorant response and examined how basal levels of cAMP are controlled. The latter is particularly relevant as basal cAMP depends on the basal activity of the expressed OR and thus varies across ORNs. We found that olfactory marker protein (OMP), a protein expressed in mature ORNs, controls both basal and odorant-induced cAMP levels in an OR-dependent manner. Lack of OMP increases basal cAMP, thus abolishing differences in basal cAMP levels between ORNs expressing different ORs. Moreover, OMP speeds up signal transduction for ORNs to better synchronize their output with high-frequency stimulation and to perceive brief stimuli. Last, OMP also steepens the dose-response relation to improve concentration coding although at the cost of losing responses to weak stimuli. We conclude that OMP plays a key regulatory role in ORN physiology by controlling multiple facets of the odorant response.

Growth inhibition of fungus Phycomyces blakesleeanus by anion channel inhibitors anthracene-9-carboxylic and niflumic acid attained through decrease in cellular respiration and energy metabolites.

Increasing resistance of fungal strains to known fungicides has prompted identification of new candidates for fungicides among substances previously used for other purposes. We have tested the effects of known anion channel inhibitors anthracene-9-carboxylic acid (A9C) and niflumic acid (NFA) on growth, energy metabolism and anionic current of mycelium of fungus Phycomyces blakesleeanus. Both inhibitors significantly decreased growth and respiration of mycelium, but complete inhibition was only achieved by 100 and 500 µM NFA for growth and respiration, respectively. A9C had no effect on respiration of human NCI-H460 cell line and very little effect on cucumber root sprout clippings, which nominates this inhibitor for further investigation as a potential new fungicide. Effects of A9C and NFA on respiration of isolated mitochondria of P. blakesleeanus were significantly smaller, which indicates that their inhibitory effect on respiration of mycelium is indirect. NMR spectroscopy showed that both A9C and NFA decrease the levels of ATP and polyphosphates in the mycelium of P. blakesleeanus, but only A9C caused intracellular acidification. Outwardly rectifying, fast inactivating instantaneous anionic current (ORIC) was also reduced to 33±5 and 21±3 % of its pre-treatment size by A9C and NFA, respectively, but only in the absence of ATP. It can be assumed from our results that the regulation of ORIC is tightly linked to cellular energy metabolism in P. blakesleeanus, and the decrease in ATP and polyphosphate levels could be a direct cause of growth inhibition.

Enhanced gap junctional channel activity between vascular smooth muscle cells in cerebral artery of spontaneously hypertensive rats.

The aim of the present study is to investigate the effects of hypertension on the gap junctions between vascular smooth muscle cells (VSMCs) in the cerebral arteries (CAs) of spontaneously hypertensive rats (SHRs). The functions of gap junctions in the CAs of VSMCs in SHRs and control normotensive Wistar-Kyoto (WKY) rats were studied using whole-cell patch clamp recordings and pressure myography, and the expression levels of connexins were analyzed using reverse transcription-quantitative polymerase chain reaction and Western blot analyses. Whole-cell patch clamp measurements revealed that the membrane capacitance and conductance of in situ VSMCs in the CAs were significantly greater in SHRs than in WKY rats, suggesting that gap junction coupling is enhanced between VSMCs in the CAs of SHRs. Application of the endothelium-independent vasoconstrictors KCl or phenylephrine (PE) stimulated a greater vasoconstriction in the CAs of SHRs than in those of WKY rats. The EC50 value of KCl was 24.9 mM (n = 14) and 36.9 mM (n=12) for SHRs and WKY rats, respectively. The EC50 value of PE was 0.9 µM (n = 7) and 2.2 µM (n = 7) for SHRs and WKY rats, respectively. Gap junction inhibitors 18ß-glycyrrhetinic acid (18ß-GA), niflumic acid (NFA), and 2-aminoethoxydiphenyl borate (2-APB) attenuated KCl-induced vasoconstriction in SHRs and WKY rats. The mRNA and protein expression levels of the gap junction protein connexin 45 (Cx45) were significantly higher in the CAs of SHRs than in those of WKY rats. Phosphorylated Cx43 protein expression was significantly higher in the CAs of SHRs than in those of WKY rats, despite the total Cx43 mRNA and protein expression levels in the cerebral artery (CA) exhibiting no significant difference between SHRs and WKY rats. Increases in the expression of Cx45 and phosphorylation of Cx43 may promote gap junction communication among VSMCs in the CAs of SHRs, which may enhance the contractile response of the CA to vasoconstrictors.

Modification of Calcium-Activated Chloride Currents in Cerebellar Purkinje Neurons.

The whole-cell voltage clamp technique was employed to record the total ionic currents in rat cerebellar Purkinje neurons. When intrapipette solution contained 120 mM KCl, replacement of the standard external physiological saline with Na-free solution resulted in appearance of inward tail current after the end of the depolarizing pulse. When intrapipette potassium ions were replaced for cesium ones, the tail currents were observed even in the presence of normal Na+ concentration (140 mM) in the external solution. Tail currents were not observed when external solution contained no Cl- and/or Ca2+ ions. Niflumic acid (25-100 µM) blocked these currents by 80-100%. Complete replacement of external Na+ for Tris ions pronouncedly augmented the amplitude and duration of the tail currents. These findings suggest that the tail transients in rat cerebellar Purkinje neurons are calcium-activated chloride currents whose amplitude and kinetics depend on ionic composition of the extracellular and intracellular solutions.

Comparative Proteomics of Ovarian Cancer Aggregate Formation Reveals an Increased Expression of Calcium-activated Chloride Channel Regulator 1 (CLCA1).

Ovarian cancer is a lethal gynecological disease that is characterized by peritoneal metastasis and increased resistance to conventional chemotherapies. This increased resistance and the ability to spread is often attributed to the formation of multicellular aggregates or spheroids in the peritoneal cavity, which seed abdominal surfaces and organs. Given that the presence of metastatic implants is a predictor of poor survival, a better understanding of how spheroids form is critical to improving patient outcome, and may result in the identification of novel therapeutic targets. Thus, we attempted to gain insight into the proteomic changes that occur during anchorage-independent cancer cell aggregation. As such, an ovarian cancer cell line, OV-90, was cultured in adherent and non-adherent conditions using stable isotope labeling with amino acids in cell culture (SILAC). Anchorage-dependent cells (OV-90AD) were grown in tissue culture flasks, whereas anchorage-independent cells (OV-90AI) were grown in suspension using the hanging-drop method. Cellular proteins from both conditions were then identified using LC-MS/MS, which resulted in the quantification of 1533 proteins. Of these, 13 and 6 proteins were up-regulated and down-regulated, respectively, in aggregate-forming cells compared with cells grown as monolayers. Relative gene expression and protein expression of candidates were examined in other cell line models of aggregate formation (TOV-112D and ES-2), which revealed an increased expression of calcium-activated chloride channel regulator 1 (CLCA1). Moreover, inhibitor and siRNA transfection studies demonstrated an apparent effect of CLCA1 on cancer cell aggregation. Further elucidation of the role of CLCA1 in the pathogenesis of ovarian cancer is warranted.

Expression of anoctamins in retinal pigment epithelium (RPE).

The anoctamin (ANO, TMEM16) family of Ca2+-activated Cl- channels consists of ten members with different cellular functions (ANO1-10). ANO1 is a Ca2+-activated Cl- channel in secretory epithelial cells of exocrine pancreas, salivary glands, or enterocytes. Expression of ANO1 also promotes cell proliferation and migration of tumor cells. ANO6 is essential for Ca2+-dependent scrambling of membrane phospholipids in platelets, red blood cells, and lymphocytes. ANO10 modulates Ca2+ signals in macrophages and has a role in cerebellar ataxia and other neurological disorders. All three anoctamins have been proposed to control intracellular Ca2+ signals. Anoctamins may also form the basolateral Ca2+-activated Cl- channel in the retinal pigment epithelium (RPE). We show that native human, bovine, porcine, and mouse RPEs express ANO1, ANO6, and ANO10. Growth arrested and confluent RPR cells expressed ANO1 in the plasma membrane, whereas ANO6 and ANO10 were found in the primary cilium. Ussing chamber experiments showed that the application of ATP to the apical (retinal) side of porcine RPE induced a Ca2+-activated Cl- secretion. Activation was inhibited by basolateral (choroidal) administration of the ANO inhibitors AO1, niflumic acid (NFA), and 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). The results suggest that ANO1 is responsible for basolateral Ca2+-dependent Cl- secretion in RPE, whereas ANO6 and ANO10 may have different functions, such as modulating Ca2+ signals.

Distinct pharmacological and molecular properties of the acid-sensitive outwardly rectifying (ASOR) anion channel from those of the volume-sensitive outwardly rectifying (VSOR) anion channel.

Expressed by many cell types, acid-sensitive outwardly rectifying (ASOR) anion channels are known to be activated by extracellular acidification and involved in acidotoxic necrotic cell death. In contrast, ubiquitously expressed volume-sensitive outwardly rectifying (VSOR) anion channels are activated by osmotic cell swelling and involved in cell volume regulation and apoptotic cell death. Distinct inhibitors to distinguish ASOR from VSOR anion channels have not been identified. Although leucine-rich repeats containing 8A (LRRC8A) was recently found to be an essential component of VSOR anion channels, the possibility of an LRRC8 family member serving as a component of ASOR anion channels has not been examined. In this study, we explored the effects of 12 known VSOR channel inhibitors and small interfering RNA (siRNA)-mediated knockdown of LRRC8 family members on ASOR and VSOR currents in HeLa cells. Among these inhibitors, eight putative VSOR blockers, including 4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxobutyric acid (DCPIB) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), were totally ineffective at blocking ASOR channel activity, whereas suramin, R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy] acetic acid (DIOA), arachidonic acid, and niflumic acid were found to be effective ASOR anion channel antagonists. In addition, gene-silencing studies showed that no LRRC8 family members are essentially involved in ASOR anion channel activity, whereas LRRC8A is involved in VSOR anion channel activity in HeLa cells.

Inhibitors of Calcium-Activated Anion Channels Modulate Hypnotic Ethanol Responses in Adult Sprague Dawley Rats.

BACKGROUND: Ethanol is widely known for its depressant effects; however, the underlying neurobiological mechanisms are not clear. Calcium-activated anion channels (CAACs) contribute to extracellular chloride levels and thus may be involved in regulating inhibitory mechanisms within the central nervous system. Therefore, we hypothesized that CAACs influence ethanol behavioral sensitivity by altering CAAC expression. METHODS: We assessed the role of CAACs in ethanol-induced loss of righting reflex (LORR) and locomotor activity using intracerebroventricular infusions of several nonselective CAAC blockers. CAAC expression was determined after ethanol exposure. RESULTS: Ethanol-induced LORR (4.0 g/kg, intraperitoneally [i.p.]) was significantly attenuated by all 4 CAAC blockers. Blocking CAACs did not impact ethanol's low-dose (1.5 g/kg, i.p.) locomotor-impairing effects. Biochemical analysis of CAAC protein expression revealed that cortical Bestrophin1 (Best1) and Tweety1 levels were reduced as early as 30 minutes following a single ethanol injection (3.5 g/kg, intraperitoneally [i.p.]) and remained decreased 24 hours later in P2 fractions. Cortical Best1 levels were also reduced following 1.5 g/kg. However, CAAC expression was unaltered in the striatum following a single ethanol exposure. Ethanol did not affect Tweety2 levels in either brain region. CONCLUSIONS: These results suggest that CAACs are a major target of ethanol in vivo, and the regulation of these channels contributes to select behavioral actions of ethanol.

Targeting kidney CLC-K channels: pharmacological profile in a human cell line versus Xenopus oocytes.

CLC-K chloride channels play a crucial role in kidney physiology and genetic mutations, affecting their function are responsible for severe renal salt loss in humans. Thus, compounds that selectively bind to CLC-Ka and/or CLC-Kb channels and modulate their activity may have a significant therapeutic potential. Here, we compare the biophysical and pharmacological behaviors of human CLC-K channels expressed either in HEK293 cells or in Xenopus oocytes and we show that CLC-K channel properties are greatly influenced by the biochemical environment surrounding the channels. Indeed, in HEK293 cells the potentiating effect of niflumic acid (NFA) on CLC-Ka/barttin and CLC-Kb/barttin channels seems to be absent while the blocking efficacy of niflumic acid and benzofuran derivatives observed in oocytes is preserved. The NFA block does not seem to involve the accessory subunit barttin on CLC-K1 channels. In addition, the sensitivity of CLC-Ks to external Ca(2+) is reduced in HEK293 cells. Based on our findings, we propose that mammalian cell lines are a suitable expression system for the pharmacological profiling of CLC-Ks.

Characterization of the effects of Cl⁻ channel modulators on TMEM16A and bestrophin-1 Ca²âº activated Cl⁻ channels.

The Ca(2+) activated Cl(-) channels (CaCCs) play a multitude of important physiological functions. A number of candidate proteins have been proposed to form CaCC, but only two families, the bestrophins and the TMEM16 proteins, recapitulate the properties of native CaCC in expression systems. Studies of endogenous CaCCs are hindered by the lack of specific pharmacology as most Cl(-) channel modulators lack selectivity and a systematic comparison of the effects of these modulators on TMEM16A and bestrophin is missing. In the present study, we studied seven Cl(-) channel inhibitors: niflumic acid (NFA), NPPB, flufenamic acid (FFA), DIDS, tannic acid, CaCCinh-A01 and T16Ainh-A01 for their effects on TMEM16A and bestrophin-1 (Best1) stably expressed in CHO (Chinese hamster ovary) cells using patch clamp technique. Among seven inhibitors studied, NFA showed highest selectivity for TMEM16A (IC50 of 7.40 ± 0.95 µM) over Best1 (IC50 of 102.19 ± 15.05 µM). In contrast, DIDS displayed a reverse selectivity inhibiting Best1 with IC50 of 3.93 ± 0.73 µM and TMEM16A with IC50 of 548.86 ± 25.57 µM. CaCCinh-A01 was the most efficacious blocker for both TMEM16A and Best1 channels. T16Ainh-A01 partially inhibited TMEM16A currents but had no effect on Best1 currents. Tannic acid, NPPB and FFA had variable intermediate effects. Potentiation of channel activity by some of these modulators and the effects on TMEM16A deactivation kinetics were also described. Characterization of Cl(-) channel modulators for their effects on TMEM16A and Best1 will facilitate future studies of native CaCCs.

Functional properties of submucosal venules in the rat stomach.

Venules in the stomach may have intrinsic properties for maintaining active microcirculation drainage even during gastric filling. Properties of spontaneous and nerve-mediated activity of submucosal venules in the rat stomach were investigated. Changes in vasodiameter and intracellular Ca(2+) in venular smooth muscle cells (SMCs) were monitored by video tracking and Fluo-8 Ca(2+) imaging, respectively. Venular SMCs developed synchronous spontaneous Ca(2+) transients and corresponding rhythmic constrictions of the venules. Nominally Ca(2+)-free solution or an L-type Ca(2+) channel blocker (1 µM nifedipine) disrupted the Ca(2+) transient synchrony and abolished spontaneous constrictions. Spontaneous constrictions were also prevented by inhibitors of sarcoplasmic reticulum Ca(2+)-ATPase (10 µM cyclopiazonic acid (CPA)), IP3 receptors (100 µM 2-APB) or Ca(2+)-activated Cl(-) channels (100 µM niflumic acid). Transmural nerve stimulation (TNS) induced a long-lasting venular constriction that was abolished by α-adrenoceptor antagonist (1 µM phentolamine), while TNS evoked a sympathetic transient constriction of arterioles that was abolished by a combination of phentolamine and a P2 purinoceptor antagonist (10 µM pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS)). Consistently, P2X1 purinoceptor immunoreactivity was detected in arteriolar but not venular SMCs. Primary afferent nerve stimulation (300 nM capsaicin) caused a venular dilatation by releasing calcitonin gene-related peptide. Thus, Ca(2+) release from the sarcoplasmic reticulum may play a fundamental role in the generation of spontaneous Ca(2+) transients, while electrical coupling amongst venular SMCs via L-type Ca(2+) channel activation appears to be critical for Ca(2+) transient synchrony as well as spontaneous contractions. Sympathetic venular constrictions appear to be exclusively mediated by noradrenaline due to the lack of P2X1 receptor in venular SMCs.