Search

Enterotoxins elaborated by Vibrio cholerae and Escherichia coli cannot elicit fluid secretion in the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. After enterotoxin exposure, CFTR channels are rapidly recruited from endosomes and undergo exocytic insertion into the apical plasma membrane of enterocytes to increase the number of channels on the cell surface by at least fourfold. However, the molecular machinery that orchestrates exocytic insertion of CFTR into the plasma membrane is largely unknown. The present study used immunofluorescence, immunoblotting, surface biotinylation, glutathione S-transferase (GST) pulldown assays, and immunoprecipitation to identify components of the exocytic soluble N-ethylmaleimide (NEM)-sensitive factor attachment receptor (SNARE) vesicle fusion machinery in cyclic nucleotide-activated exocytosis of CFTR in rat jejunum and polarized intestinal [Caco-2.sub.BB]e cells. Syntaxin 3, an intestine-specific SNARE, colocalized with CFTR on the apical domain of enterocytes in rat jejunum and polarized [Caco- 2.sub.BB]e cells. Coimmunoprecipitation and GST binding studies confirmed that syntaxin 3 interacts with CFTR in vivo. Moreover, heat-stable enterotoxin (STa) activated exocytosis of both CFTR and syntaxin 3 to the surface of rat jejunum. Silencing of syntaxin 3 by short hairpin RNA (shRNA) interference abrogated cyclic nucleotide-stimulated exocytosis of CFTR in cells. These observations reveal a new and important role for syntaxin 3 in the pathophysiology of enterotoxin-elicited diarrhea. cystic fibrosis transmembrane conductance regulator; intestine; secretory diarrhea; soluble N-ethylmaleimide-sensitive factor attachment protein receptor doi: 10.1152/ajpcell.00029.2010.

We have previously shown an important role of the chloride channel C1C-2 in orchestrating repair of tight junctions in ischemia-injured mucosa. In this study, we examined the role of ClC-2 in regulating barrier function of normal murine intestinal mucosa. Ex vivo, ClC-2-/- ileal mucosa mounted in Ussing chambers had significantly higher transepithelial electrical resistance (TER) and reduced [[sup.3]H]mannitol mucosal-to-serosal flux compared with wild-type (WT) mouse mucosa. We also noted that ileum from ClC-2-/- mice had a significantly reduced in vivo [beta]H]mannitol blood-to-lumen clearance compared with WT animals. By scanning electron microscopy, fiat leaflike villi were found to have tapering, rounded apical tips in C1C-2-/mucosa. By transmission electron microscopy, the apical intercellular tight junctions in ClC-2-/- intestine revealed lateral membranes that were less well defined but closely aligned compared with electrondense and closely apposed tight junctions in WT mucosa. The width of apical tight junctions was significantly reduced in ClC-2-/intestine. Such an alteration in tight junction ultrastructure was also noted in the testicular tissue from ClC-2-/- mice. The ClC-2-/intestinal mucosa had reduced expression of phospho-myosin light chain (MLC), and inhibition of myosin light chain kinase (MLCK) in WT mucosa partially increased TER toward the TER in C1C-2-/intestine. Contrary to our prior work on the reparative role of C1C-2 in injured mucosa, this study indicates that C1C-2 reduces barrier function in normal mucosa. The mechanisms underlying these differing roles are not entirely clear, although ultrastructural morphology of tight junctions and MLCK appear to be important to the function of ClC-2 in normal mucosa. myosin light chain kinase; chloride channel; mannitol flux doi: 10.1152/ajpgi.00520.2009.

The chloride intracellular channel 5A (CLIC5A) protein, one of two isoforms produced by the CLIC5 gene, was isolated originally as part of a cytoskeletal protein complex containing ezrin from placental microvilli. Whether CLIC5A functions as a bona fide ion channel is controversial. We reported previously that a CLIC5 transcript is enriched ~800-fold in human renal glomeruli relative to most other tissues. Therefore, this study sought to explore CLIC5 expression and function in glomeruli. RT-PCR and Western blots show that CLIC5A is the predominant CLIC5 isoform expressed in glomeruli. Confocal immunofluorescence and immunogold electron microscopy reveal high levels of CLIC5A protein in glomerular endothelial cells and podocytes. In podocytes, CLIC5A localizes to the apical plasma membrane of foot processes, similar to the known distribution of podocalyxin and ezrin. Ezrin and podocalyxin colocalize with CLIC5A in glomeruli, and podocalyxin coimmunoprecipitates with CLIC5A from glomerular lysates. In glomeruli of jitterbug (jbg/jbg) mice, which lack the CLIC5A protein, ezrin and phospho-ERM levels in podocytes are markedly lower than in wild-type mice. Transmission electron microscopy reveals patchy broadening and effacement of podocyte foot processes as well as vacuolization of glomerular endothelial cells. These ultrastructural changes are associated with microalbuminuria at baseline and increased susceptibility to adriamycininduced glomerular injury compared with wild-type mice. Together, the data suggest that CLIC5A is required for the development and/or maintenance of the proper glomerular endothelial cell and podocyte architecture. We postulate that the interaction between podocalyxin and subjacent filamentous actin, which requires ezrin, is compromised in podocytes of CLIC5A-deficient mice, leading to dysfunction under unfavorable genetic or environmental conditions. chloride intracellular channel 5; actin; microvilli; endothelial; jitterbug doi: 10.1152/ajprenal.00030.2010.

Chloride intracellular channel 5 (CLIC5) and other CLIC isoforms have been implicated in a number of biological processes, but their specific functions are poorly understood. The association of CLIC5 with ezrin and the actin cytoskeleton led us to test its possible involvement in gastric acid secretion. Clic5 mutant mice exhibited only a minor reduction in acid secretion, Clic5 mRNA was expressed at only low levels in stomach, and Clic5 mutant parietal cells were ultrastructurally normal, negating the hypothesis that CLIC5 plays a major role in acid secretion. However, the mutants exhibited gastric hemorrhaging in response to fasting, reduced monocytes and granulocytes suggestive of immune dysfunction, behavioral and social disorders suggestive of neurological dysfunction, and evidence of a previously unidentified metabolic defect. Wild-type and mutant mice were maintained on normal and high-fat diets; plasma levels of various hormones, glucose, and lipids were determined; and body composition was studied by quantitative magnetic resonance imaging. Clic5 mutants were lean, hyperphagic, and highly resistant to diet-induced obesity. Plasma insulin and glucose levels were reduced, and leptin levels were very low; however, plasma triglycerides, cholesterol, phospholipids, and fatty acids were normal. Indirect calorimetry revealed increased peripheral metabolism and greater reliance on carbohydrate metabolism. Because Clic5 mutants were unable to maintain energy reserves, they also exhibited increased susceptibility to fasting-induced torpor, as indicated by telemetric measurements showing episodes of reduced body temperature and heart rate. These data reveal a requirement for CLIC5 in the maintenance of normal systemic energy metabolism. gastric hemorrhage; leptin doi: 10.1152/ajpregu.00849.2009.

Previous studies have shown that in proximal and distal tubule nephron segments, peritubular ANG II stimulates sodium chloride transport. However, ANG II inhibits chloride transport in the medullary thick ascending limb (mTAL). Because ANG II and catecholamines are both stimulated by a decrease in extracellular fluid volume, the purpose of this study was to examine whether there was an interaction between ANG II and catecholamines to mitigate the inhibition in chloride transport by ANG II. In isolated perfused rat mTAL, [10.sup.-8] M bath ANG II inhibited wansport (from a basal transport rate of 165.6 [+ or -] 58.8 to 58.8 [+ or -] 29.4 pmol*[mm.sup.-1]*[min.sup.-1]; p < 0.0l). Bath norepinephrine stimulated chloride transport (from a basal transport rate of 298.1 [+ or -] 31.7 to 425.2 [+ or -] 45.8 pmol*[mm.sup.-1]*[min.sup.-1]; P < 0.05) and completely prevented the inhibition in chloride transport by ANG II. The stimulation of chloride transport by norepinephrine was mediated entirely by its [beta]-adrenergic effect; however, both the [beta]- and [alpha]-adrenergic agonists isoproterenol and phenylephrine prevent the ANG II-mediated inhibition in chloride transport. In the presence of [10.sup.-5] M propranolol, the effect of norepinephrine to prevent the inhibition of chloride transport by ANG II was still present. These data are consistent with an interaction of both [alpha]- and [beta]-catecholamines and ANG II on net chloride transport in the mTAL. NKCC2; norepinephrine; isoproterenol doi: 10.1152/ajpregu.00758.2009.

CFTR is a highly regulated apical chloride channel of epithelial cells that is mutated in cystic fibrosis (CF). In this study, we characterized the apical stability and intracellular trafficking of wild-type and mutant CFTR in its native environment, i.e., highly differentiated primary human airway epithelial (HAE) cultures. We labeled the apical pool of CFTR and subsequently visualized the protein in intracellular compartments. CFTR moved from the apical surface to endosomes and then efficiently recycled back to the surface. CFTR endocytosis occurred more slowly in polarized than in nonpolarized HAE cells or in a polarized epithelial cell line. The most common mutation in CF, [DELTA]F508 CFTR, was rescued from endoplasmic reticulum retention by low-temperature incubation but transited from the apical membrane to endocytic compartments more rapidly and recycled less efficiently than wild-type CFTR. Incubation with small-molecule correctors resulted in [DELTA]F508 CFTR at the apical membrane but did not restore apical stability. To stabilize the mutant protein at the apical membrane, we found that the dynamin inhibitor Dynasore and the cholesterol-extracting agent cyclodextrin dramatically reduced internalization of [DELTA]F508, whereas the proteasomal inhibitor MG-132 completely blocked endocytosis of [DELTA]F508. On examination of intrinsic properties of CFTR that may affect its apical stability, we found that N-linked oligosaccharides were not necessary for transport to the apical membrane but were required for efficient apical recycling and, therefore, influenced the turnover of surface CFTR. Thus apical stability of CFTR in its native environment is affected by properties of the protein and modulation of endocytic traff~cking. cystic fibrosis; [DELTA]F508; protein trafficking; turnover; polarized cells doi:10.1152/ajplung.00016.2009

Renal tabular reabsorption is important for extracellular fluid homeostasis and much of this occurs via the receptor-mediated endocytic pathway. This pathway is disrupted in Dent's disease, an X-linked renal tubular disorder that is characterized by low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. Dent's disease is due to mutations of CLC-5, a chloride/proton antiporter, expressed in endosomes and apical membranes of renal tubules. Loss of CLC-5 function alters receptor-mediated endocytosis and trafficking of megalin and cubilin, although the underlying mechanisms remain to be elucidated. Here, we report that CLC-5 interacts with kinesin family member 3B (KIF3B), a heterotrimeric motor protein that facilitates fast anterograde translocation of membranous organelles. Using yeast two-hybrid, glutathione-S-transferase pull-down and coimmunoprecipitation assays, the COOH terminus of CLC-5 and the coiled-coil and globular domains of KIF3B were shown to interact. This was confirmed in vivo by endogenous coimmunoprecipitation of CLC-5 and KIF3B and codistribution with endosomal markers in mouse kidney fractions. Confocal live cell imaging in kidney cells further demonstrated association of CLC-5 and KIF3B, and transport of CLC-5-contalning vesicles along KIF3B microtubules. KIF3B overexpression and underexpression, using siRNA, had reciprocal effects on whole cell chloride current amplitudes, CLC-5 cell surface expression, and endocytosis of albumin and transferrin. [Clcn5.sup.Y/-] mouse kidneys and isolated proximal tubular polarized cells showed increased KIF3B expression, whose effects on albumin endocytosis were dependent on CLC-5 expression. Thus, the CLC-5 and KIF3B interaction is important for CLC-5 plasma membrane expression and for facilitating endocytosis and microtubular transport in the kidney. chloride/proton antiporter; kinesin family; proximal tubular reabsorption; hypercalciuric nephrolithiasis; receptor-mediated endocytosis doi: 10.1152/ajprenal.00038.2009

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.sub.D] for [Ca.sup.2+] of ~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.sup.-]>[I.sup.-]>[Cl.sup.-], 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. calcium-activated chloride current; chloride channels; patch clamp; mutagenesis

Human bestrophin-1 (hBest1), which is genetically linked to several kinds of retinopathy and macular degeneration in both humans and dogs, is the founding member of a family of [C1.sup.-1] ion channels that are activated by intracellular [Ca.sup.2+]. At present, the structures and mechanisms responsible for [Ca.sup.2+] sensing remain unknown. Here, we have used a combination of molecular modeling, density functional--binding energy calculations, mutagenesis, and patch clamp to identify the regions of hBest1 involved in [Ca.sup.2+] sensing. We identified a cluster of a five contiguous acidic amino acids in the C terminus immediately after the last transmembrane domain, followed by an EF hand and another regulatory domain that are essential tot [Ca.sup.2+] sensing by hBest1. The cluster of five amino acids (293-308) is crucial for normal channel gating by [Ca.sup.2+] because all but two of the 35 mutations we made in this region rendered the channel incapable of being activated by [Ca.sup.2+]. Using homology, models built on the crystal structure of calmodulin (CAM), an EF hand (EF1) was identified in hBest1. EF1 was predicted to bind [Ca.sup.2+] with a slightly higher affinity than the third EF hand of CaM and lower affinity than the second EF hand of troponin C. As predicted by the model, the D312G mutation in the putative [Ca.sup.2+]-binding loop (312-323) reduced the apparent [Ca.sup.2+] affinity by 20-fold. In addition, the D312G and D323N mutations abolished [Ca.sup.2+]-dependent rundown of the current. Furthermore, analysis of truncation mutants of hBest1 identified a domain adjacent to EF1 that is rich in acidic amino acids (350-390) that is required for [Ca.sup.2+] activation and plays a role in current rundown. These experiments identity a region of hBest1 (312-323) that is involved in the gating of hBest1 by [Ca.sup.2+] and suggest a model in which [Ca.sup.2+] binding to EF1 activates the channel in a process that requires the acidic domain (293-308) and another regulatory domain (350-390). Many of the ~100 disease-causing mutations in hBest1 are located in this region that we have implicated in [Ca.sup.2+] sensing, suggesting that these mutations disrupt hBestl channel gating by [Ca.sup.2+].

Calcium ([Ca.sup.2+])-activated chloride channels are fundamental mediators in numerous physiological processes including transepithelial secretion, cardiac and neuronal excitation, sensory transduction, smooth muscle contraction and fertilization. Despite their physiological importance, their [...]

Pannabecker TL, Henderson CS, Dantzler WH. Quantitative analysis of functional reconstructions reveals lateral and axial zonation in the renal inner medulla. Am J Physiol Renal Physiol 294: F1306--Fl314, 2008. First published April 16, 2008; doi:10.l152/ajprenal.00068.2008.--Three-dimensional functional reconstructions of descending thin limbs (DTLs) and ascending thin limbs (ATLs) of loops of Henle, descending vasa recta (DVR), ascending vasa recta (AVR), and collecting ducts (CDs) permit quantitative definition of lateral and axial zones of probable functional significance in rat inner medulla (IM). CD clusters form the organizing motif for loops of Henle and vasa recta in the initial 3.0-3.5 mm of the IM. Using Euclidean distance mapping, we defined the lateral boundary of each cluster by pixels lying maximally distant from any CD. DTLs and DVR lie almost precisely on this independently defined boundary, placing them in the intercluster interstitium maximally distant from any CD. ATLs and AVR lie in a nearly uniform pattern throughout intercluster and intracluster regions, which we further differentiated by a polygon around CDs in each cluster. Loops associated with individual CD clusters show a similar axial exponential decrease as all loops together in the IM. Because ~3.0-3.5 mm below the IM base CD clusters cease to form the organizing motif, all DTLs lack aquaporin 1 (AQP 1), and all vasa recta are fenestrated, we have designated the first 3.0-3.5 mm of the IM the 'outer zone' (OZ) and the final 1.5-2.0 mm the 'inner zone' (IZ). We further subdivided these into OZ-1, OZ-2, IZ-1, and IZ-2 on the basis of the presence of completely AQP1-null DTLs only in the first 1 mm and on broad transverse loop bends only in the final 0.5 ram. These transverse segments expand surface area for probable NaCl efflux around loop bends from ~40% to ~140% of CD surface area in the final 100 [micro]m of the papilla. countercurrent systems; concentrating mechanism; aquaporin; kidney-specific chloride channel; urea transporter B; three-dimensional reconstruction

In myotonic dystrophy (dystrophia myotonica [DM]), an increase in the excitability of skeletal muscle leads to repetitive action potentials, stiffness, and delayed relaxation. This constellation of features, collectively known as myotonia, is associated with abnormal alternative splicing of the muscle-specific chloride channel (ClC-1) and reduced conductance of chloride ions in the sarcolemma. However, the mechanistic basis of the chloride channelopathy and its relationship to the development of myotonia are uncertain. Here we show that a morpholino antisense oligonucleotide (AON) targeting the 3&apos; splice site of ClC-1 axon 7a reversed the defect of ClC-1 alternative splicing in 2 mouse models of DM. By repressing the inclusion of this axon, the AON restored the full-length reading frame in ClC-1 mRNA, exon the level of ClC-1 mRNA, increased the expression of ClC-1 protein in the surface membrane, normalized muscle ClC-1 current density and deactivation kinetics, and eliminated myotonic discharges. These observations indicate that the myotonia and chloride channelopathy observed in DM both result from abnormal alternative splicing of ClC-1 and that antisense-induced exon skipping offers a powerful method for correcting alternative splicing defects in DM., Introduction Dystrophia myotonica (myotonic dystrophy) type 1 (DM1), the most common muscular dystrophy affecting adults, is caused by expansion of a CTG repeat in the 3' untranslated region of the [...]

ClC-0 is a chloride channel whose gating is sensitive to voltage, chloride, and pH. In a previous publication, we showed that the K149C mutation causes a +70-mV shift in the voltage dependence of ClC-0 fast gating. In this paper we analyze the effects of a series of mutations at K149 on the voltage and chloride dependence of gating. By fitting our data to the previously proposed four-state model for ClC-0 fast gating, we show which steps in fast-gate opening are likely to be affected by these mutations. Computational analysis of mutant ClC-0 homology models show electrostatic contributions to chloride binding that may partially account tor the effects of K149 on gating. The analysis of gating kinetics in combination with the available structural information suggests some of the structural changes likely to underpin fast-gate opening.

ClC-0 is a chloride channel whose gating is sensitive to both voltage and chloride. Based on analysis of gating kinetics using single-channel recordings, a five-state model was proposed to describe the dependence of ClC-0 fast-gate opening on voltage and external chloride (Chen, T.-Y., and C. Miller. 1996. J. Gen. Physiol. 108:237-250). We aimed to use this five-state model as a starting point for understanding the structural changes that occur during gating. Using macroscopic patch recordings, we were able to reproduce the effects of voltage and chloride that were reported by Chen and Miller and to fit our opening rate constant data to the five-state model. Upon further analysis of both our data and those of Chert and Miller, we learned that in contrast to their conclusions, (a) the features in the data are not adequate to rule out a simpler four-state model, and (b) the chloride-binding step is voltage dependent. In order to be able to evaluate the effects of mutants on gating (described in the companion paper, see Engh et al. on p. 351 of this issue), we developed a method for determining the error on gating model parameters, and evaluated the sources of this error. To begin to mesh the kinetic model(s) with the known CLC structures, a model of ClC-0 was generated computationally based on the X-ray crystal structure of the prokaryotic homolog ClC-ecl. Analysis of pore electrostatics in this homology model suggests that at least two of the conclusions derived from the gating kinetics analysis are consistent with the known CLC structures: (1) chloride binding is necessary for channel opening, and (2) chloride binding to any of the three known chloride-binding sites must be voltage dependent.

Chloride ion has a stimulatory effect on the transport of organic anions across renal basolateral membranes. However, the exact mechanisms at molecular levels have been unclear as of yet. Human organic anion transporters hOAT1 and hOAT3 play important roles in renal basolateral membranes. In this study, the effects of C1 on the activities of these transporters were evaluated by using HEK293 cells stably expressing hOAT1 or hOAT3 (HEK-hOAT1 or HEK-hOAT3). The uptake of p-[[sup.14]C]aminohippu-rate by HEK-hOAT1 and [[sup.3]H]estrone sulfate by HEK-hOAT3 was greater in the presence of [Cl.sup.-] than in the presence of S[O.sup.2-.sub.4] or gluconate. Additionally, the uptake of various compounds by HEK-hOAT1 and HEK-hOAT3 was significantly higher in the [Cl.sup.-]-containing medium than the gluconate-containing medium, suggesting that the influences of [Cl.sup.-] are not dependent on substrate and that [Cl.sup.-] directly stimulates the functions of hOAT1 and hOAT3. The substitution of gluconate with [Cl.sup.-] did not change the [K.sub.m] value for the uptake of p-[[sup.14]C]aminohippurate by HEK-hOAT1 but caused an approximately threefold increase in the maximal uptake rate ([V.sub.max]) value. On the other hand, replacement of gluconate with C1 decreased the [K.sub.m] value for the uptake of [[sup.3]H]estrone sulfate and cefotiam by HEK-hOAT3 to about one-third, while it did not change the [V.sub.max] value. In summary, [Cl.sup.-] upregulates the activities of both hOAT1 and hOAT3, but its effects on transport kinetics differ between these transporters. It was suggested that [Cl.sup.-] participates in the trans-location process for hOAT1, and the substrate recognition process for hOAT3. renal secretion; basolateral membrane; SLC22A; HEK293 doi:10.1152/ajprenal.00376.2006

Transmembrane chloride ion conductance in skeletal muscle increases during early postnatal development. A transgenic mouse model of myotonic dystrophy type 1 (DM1) displays decreased sarcolemmal chloride conductance. Both effects result from modulation of chloride channel 1 (CLCN1) expression, but the respective contributions of transcriptional vs. posttranscriptional regulation are unknown. Here we show that alternative splicing of CLCN1 undergoes a physiological splicing transition during the first 3 wk of postnatal life in mice. During this interval, there is a switch to production of CLCN1 splice products having an intact reading frame, an upregulation of CLCN1 mRNA encoding full-length channel protein, and an increase of CLCN1 function, as determined by patch-clamp analysis of single muscle fibers. In a transgenic mouse model of DM1, however, the splicing transition does not occur, CLCN1 channel function remains low throughout the postnatal interval, and muscle fibers display myotonic discharges. Thus alternative splicing is a posttranscriptional mechanism regulating chloride conductance during muscle development, and the chloride channelopathy in a transgenic mouse model of DM1 results from a failure to execute a splicing transition for CLCN1. chloride channel 1; nonsense-mediated decay; alternative splicing; CUG repeats; developmental regulation; muscular dystrophy; ion channel

The CLC-family protein CLC-ec1, a bacterial homologue of known structure, stoichiometrically exchanges two [Cl.sup.-] for one [H.sup.+] via an unknown membrane transport mechanism. This study examines mutations at a conserved tyrosine residue, Y445, that directly coordinates a [Cl.sup.-] ion located near the center of the membrane. Mutations at this position lead to 'uncoupling,' such that the [H.sup.+]/[Cl.sup.-] transport ratio decreases roughly with the volume of the substituted side chain. The uncoupled proteins are still able to pump protons uphill when driven by a [Cl.sup.-] gradient, but the extent and rate of this [H.sup.+] pumping is weaker in the more uncoupled variants. Uncoupling is accompanied by conductive [Cl.sup.-] transport that is not linked to counter-movement of [H.sup.+], i.e., a 'leak.' The unitary [Cl.sup.-] transport rate, measured in reconstituted liposomes by both a conventional initial-velocity method and a novel Poisson dilution approach, is ~4,000 [s.sup.-1] for wild-type protein, and the uncoupled mutants transport [Cl.sup.-] at similar rates.

We have previously shown that stimulation of acid secretion in parietal cells causes rapid initial cell shrinkage, followed by [Na.sup.+]/[H.sup.+] exchange-mediated regulatory volume increase (RVI). The factors leading to the initial cell shrinkage are unknown. We therefore monitored volume changes in cultured rabbit parietal cells by confocal measurement of the cytoplasmic calcein concentration. Although blocking the presumably apically located [K.sup.+] channel KCNQ1 with chromanol 293b reduced both the forskolin- and carbachol-induced cell shrinkage, inhibition of [Ca.sup.2+]sensitive [K.sup.+] channels with charybdotoxin strongly inhibited the cell volume decrease after carbachol, but not after forskolin stimulation. The cell shrinkage induced by both secretagogues was partially inhibited by blocking [H.sup.+]-[K.sup.+]-ATPase with SCH28080 and completely absent after incubation with NPPB, which inhibits parietal cell anion conductances involved in acid secretion. The subsequent RVI was strongly inhibited with the [Na.sup.+]/[H.sup.+] exchanger 1 (NHE1)-specific concentration of HOE642 and completely by 500 [micro]M dimethylamiloride (DMA), which also inhibits NHE4. None of the above substances induced volume changes under baseline conditions. Our results indicate that cell volume decrease associated with acid secretion is dependent on the activation of [K.sup.+] and [Cl.sup.-] channels by the respective secretagogues. [K.sup.+], [Cl.sup.-], and water secretion into the secretory canaliculi is thus one likely mechanism of stimulationassociated cell shrinkage in cultured parietal cells. The observed RVI is predominantly mediated by NHE1. acid secretion; volume regulation; potassium channels; chloride channels

Killifish are euryhaline teleosts that adapt to rapid changes in the salinity of the seawater. It is generally accepted that acclimation to seawater is mediated by cortisol activation of the glucocorticoid receptor (GR), which stimulates CFTR mRNA expression and CFTR-mediated [Cl.sup.-] secretion by the gill. Because there is no direct evidence in killifish that the GR stimulates CFTR gene expression, quantitative PCR studies were conducted to test the hypothesis that cortisol activation of GR upregulates CFTR mRNA expression and that this response is required for acclimation to seawater. Inhibition of the GR by RU-486 prevented killifish from acclimating to increased salinity and blocked the increase in CFTR mRNA. In contrast, inhibition of the mineralocorticoid receptor by spironolactone had no effect on acclimation to seawater. Thus acclimation to increased salinity in killifish requires signaling via the GR and includes an increase in CFTR gene expression. Because arsenic, a toxic metalloid that naturally occurs in the aquatic environment, has been shown to disrupt GR transcriptional regulation in avian and mammalian systems, studies were also conducted to determine whether arsenic disrupts cortisol-mediated activation of CFTR gene expression in this in vivo fish model and thereby blocks the ability of killifish to acclimate to increased salinity. Arsenic prevented acclimation to seawater and decreased CFTR protein abundance. However, arsenic did not disrupt the GR-induced increase in CFTR mRNA. Thus arsenic blocks acclimation to seawater in killifish by a mechanism that does not disrupt GR-mediated induction of CFTR gene expression. cystic fibrosis transmembrane conductance regulator, chloride channel; ion transport; chloride secretion; environmental toxicant

We have previously shown that [K.sup.+]-selective TASK2 channels and swelling-activated [Cl.sup.-] currents are involved in a regulatory volume decrease (RVD; Barriere H, Belfodil R, Rubera I, Tauc M, Lesage F, Poujeol C, Guy N, Barhanin J, Poujeol P. J Gen Physiol 122: 177-190, 2003; Belfodil R, Barriere H, Rubera I, Tauc M, Poujeol C, Bidet M, Poujeol P. Am J Physiol Renal Physiol 284: F812-F828, 2003). The aim of this study was to determine the mechanism responsible for the activation of TASK2 channels during RVD in proximal cell lines from mouse kidney. For this purpose, the patch-clamp whole-cell technique was used to test the effect of pH and the buffering capacity of external bath on [Cl.sup.-] and [K.sup.+] currents during hypotonic shock. In the presence of a high buffer concentration (30 mM HEPES), the cells did not undergo RVD and did not develop outward [K.sup.+] currents (TASK2). Interestingly, the hypotonic shock reduced the cytosolic pH ([pH.sub.i]) and increased the external pH ([pH.sub.e]) in wild-type but not in [cftr.sup.-/-] cells. The inhibitory effect of DIDS suggests that the acidification of [pH.sub.i] and the alkalinization of pile induced by hypotonicity in wild-type cells could be due to an exit of HC[O.sup.-.sub.3]. In conclusion, these results indicate that [Cl.sup.-] influx will be the driving force for HC[O.sup.-.sub.3] exit through the activation of the [Cl.sup.-]/HC[O.sup.-.sub.3] exchanger. This efflux of HC[O.sup.-.sub.3] then alkalinizes pile, which in turn activates TASK2 channels. regulatory volume decrease; CFTR; KCNE5; potassium and chloride channels; external and internal pH

Research into [Ca.sup.2+]-activated [Cl.sup.-] channels is hampered by the inability to decipher their molecular identity and the fact that all extant [Cl.sup.-] channel blockers have effects on other ion channels. Most notably, [Cl.sup.-] channel blockers such as the fenamates (e.g. niflumic acid and flufenamic acid) activate [Ca.sup.2+]-dependent [K.sup.+] channels, although other pharmacological overlaps have been discovered. In this article, we highlight the complex pharmacology of [Ca.sup.2+]-activated [Cl.sup.-] channels and the caveats associated with using these blockers--a necessary requirement because many researchers use [Cl.sup.-] channel blockers as probes for [Cl.sup.-] channel activity. Moreover, we discuss the argument for a common structural motif between [Ca.sup.2+]-activated [Cl.sup.-] channels and [Ca.sup.2+]-dependent [K.sup.+] channels, which has led to the possibility that the molecular identity of [Cl.sup.-] 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.

Abstract: Live cell imaging is a powerful tool for understanding the function and regulation of membrane proteins. In this review, we briefly discuss 4 fluorescence-microscopy-based techniques for studying the transport [...]

Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC (ATP binding cassette) transporter family, is a chloride channel whose activity is controlled by protein kinase--dependent phosphorylation. Opening and closing (gating) of the phosphorylated CFTR is coupled to ATP binding and hydrolysis at CFTR's two nucleotide binding domains (NBD1 and NBD2). Recent studies present evidence that the open channel conformation reflects a head-to-tail dimerization of CFTR's two NBDs as seen in the NBDs of other ABC transporters (Vergani et al., 2005). Whether these two ATP binding sites play an equivalent role in the dynamics of NBD dimerization, and thus in gating CFTR channels, remains unsettled. Based on the crystal structures of NBDs, sequence alignment, and homology modeling, we have identified two critical aromatic amino acids (W401 in NBD1 and Y1219 in NBD2) that coordinate the adenine ring of the bound ATR Conversion of the W401 residue to glycine (W401G) has little effect on the sensitivity of the opening rate to [ATP], but the same mutation at the Y1219 residue dramatically lowers the apparent affinity for ATP by >50-fold, suggesting distinct roles of these two ATP binding sites in channel opening. The W401G mutation, however, shortens the open time constant. Energetic analysis of our data suggests that the free energy of ATP binding at NBD1, but not at NBD2, contributes significantly to the energetics of the open state. This kinetic and energetic asymmetry of CFTR's two NBDs suggests an asymmetric motion of the NBDs during channel gating. Opening of the channel is initiated by ATP binding at the NBD2 site, whereas separation of the NBD dimer at the NBD1 site constitutes the rate-limiting step in channel closing.

Ca-activated Cl channels are an important component of olfactory transduction. Odor binding to olfactory receptors in the cilia of olfactory sensory neurons (OSNs) leads to an increase of intraciliary Ca concentration by Ca entry through cyclic nucleotide-gated (CNG) channels. Ca activates a Cl channel that leads to an efflux of CI from the cilia, contributing to the amplification of the OSN depolarization. The molecular identity of this Cl channel remains elusive. Recent evidence has indicated that bestrophins are able to form Ca-activated Cl channels in heterologous systems. Here we have analyzed the expression of bestrophins in the mouse olfactory epithelium and demonstrated that only mouse bestrophin-2 (mBest2) was expressed. Single-cell RT-PCR showed that mBest2 was expressed in OSNs but not in supporting cells. Immunohistochemistry revealed that mBest2 was expressed on the cilia of OSNs, the site of olfactory transduction, and colocalized with the main CNGA2 channel subunit. Electrophysiological properties of Ca-activated CI currents from native channels in dendritic knob/cilia of mouse OSNs were compared with those induced by the expression of mBest2 in HEK-293 cells. We found the same anion permeability sequence, small estimated single-channel conductances, a Ca sensitivity difference of one order of magnitude, and the same side-specific blockage of the two Cl channel blockers commonly used to inhibit the odorant-induced Ca-activated CI current in OSNs, niflumic acid, and 4-acetamido-4'-isothiocyanato-stilben-2, 2'-disulfonate (SITS). Therefore, our data suggest that mBest2 is a good candidate for being a molecular component of the olfactory Ca-activated Cl channel. ion channel | olfaction | olfactory sensory neurons | patch-clamp | sensory coding

Barttin is an accessory subunit of a subgroup of ClC-type chloride channels expressed in renal and inner ear epithelia. In this study, we examined the effects of barttin on two ClC-K channel isoforms, rat ClC-K1 and human ClC-Kb, using heterologous expression, patch clamping, confocal imaging, and flow cytometry. In the absence of barttin, only a small percentage of rClC-K1 and hClC-Kb channels are inserted into the plasma membrane. Coexpression of barttin enhances surface membrane insertion and furthermore modifies permeation and gating of ClC-K channels, hClC-Kb channels are nonfunctional without barttin and require the coexpressed accessory subunit to become anion conducting. In contrast, rClC-K1 channels are active without barttin, but at the cost of reduced unitary conductance as well as altered voltage dependence of activation. We mapped the separate functions of barttin to structural domains by a deletion analysis. Whereas the transmembrane core is necessary and sufficient to promote ClC-K channel exit from the endoplasmic reticulum, a short cytoplasmic segment following the second transmembrane helix modifies the unitary conductance. The entire cytoplasmic carboxyl terminus affects the open probability of ClC-K channels. The multiple functions of barttin might be necessary for a tight adjustment of epithelial [Cl.sup.-] conductances to ensure a precise regulation of body salt content and endocochlear potential. chloride channels | gating | kidney | ClC family | accessory subunit

Extracellular acidic pH was found to induce an outwardly rectifying [Cl.sup.-] current ([I.sub.Cl,acid]) in mouse ventricular cells, with a half-maximal activation at pH 5.9. The current showed the permeability sequence for anions to be [SCN.sup.-] > [Br.sup.-] > [I.sup.-] > [Cl.sup.-] > [F.sup.-] > aspartate, while it exhibited a time-dependent activation tit large positive potentials. Similar currents were also observed in mouse atrial cells and in atrial and ventricular cells from guinea pig. Some [Cl.sup.-] channel blockers (DIDS, niflumic acid, and glibenclamide) inhibited [I.sub.Cl,acid], whereas tamoxifen had little effect on it. Unlike volume-regulated [Cl.sup.-] current ([I.sub.Cl,vol]) and CFTR [Cl.sup.-] current ([I.sub.Cl,CFTR]). [I.sub.Cl,acid] was independent of the presence of intracellular ATP. Activation of [I.sub.Cl,acid] appeared to be also independent of intracellular [Ca.sup.2+] and G protein. [I.sub.Cl,acid] and [I.sub.Cl,vol] could develop in an additive fashion in acidic hypotonic solutions. Isoprenaline-induced [I.sub.Cl,CFTR] was inhibited by acidification in a pH-dependent manner in guinea pig ventricular cells. Our results support the view that [I.sub.Cl,acid] and [I.sub.Cl,vol] stem from two distinct populations of anion channels and that the [I.sub.Cl,acid] channels are present in cardiac cells. [I.sub.Cl,acid] may play a role in the control of action potential duration or cell volume under pathological conditions, such as ischemia-related cardiac acidosis. acidosis; chloride channel; heart

Regulatory volume decrease (RVD) is a protective mechanism that allows mammalian cells to restore their volume when exposed to a hypotonic environment. A key component of RVD is the release of [K.sup.+], [Cl.sup.+], and organic osmolytes, such as taurine, which then drives osmotic water efflux. Previous experiments have indicated that caveolin-l, a coat protein of caveolae microdomains in the plasma membrane, promotes the swelling-induced [Cl.sup.+] current ([I.sub.CI,swell]) through volume-regulated anion channels. However, it is not known whether the stimulation by caveolin-1 is restricted to the release of [Cl.sup.-] or whether it also affects the swelling-induced release of other components, such as organic osmolytes. To address this problem, we have studied [I.sub.CI,swell] II and the hypotonicity-induced release of taurine and ATP in wild-type Caco-2 cells that are caveolin-I deficient and in stably transfected Caco-2 cells that express caveolin-1. Electrophysiological characterization of wild-type and stably transfected Caco-2 showed that caveolin-I promoted [I.sub.CI,swell], but not cystic fibrosis transmembrane conductance regulator currents. Furthermore, caveolin-I expression stimulated the hypotonicity-induced release of taurine and ATP in stably transfected Caco-2 cells grown as a monolayer. Interestingly, the effect of caveolin-I was polarized because only the release at the basolateral membrane, but not at the apical membrane, was increased. It is therefore concluded that caveolin-I facilitates the hypotonicity-induced release of [Cl.sup.-], taurine, and ATP, and that in polarized epithelial cells, the effect of caveolin-1 is compartmentalized to the basolateral membrane. caveolae; osmolyte; epithelial cell: chloride channel

The voltage-dependent ClC-2 chloride channel has been implicated in a variety of physiological functions, including fluid transport across specific epithelia. ClC-2 is activated by hyperpolarization, weakly acidic external pH, intracellular [Cl.sup.-], and cell swelling. To add more insight into the mechanisms involved in ClC-2 regulation, we searched for associated proteins that may influence ClC-2 activity. With the use of immunoprecipitation of ClC-2 from human embryonic kidney-293 cells stably expressing the channel, followed by electrophoretic separation of coimmunoprecipitated proteins and mass spectrometry identification, Hsp70 and Hsp90 were unmasked as possible ClC-2 interacting partners. Association of Hsp90 with ClC-2 was confirmed in mouse brain. Inhibition of Hsp90 by two specific inhibitors, geldanamycin or radicicol, did not affect total amounts of ClC-2 but did reduce plasma membrane channel abundance. Functional experiments using the whole cell configuration of the patch-clamp technique showed that inhibition of Hsp90 reduced ClC-2 current amplitude and impaired the intracellular Cl concentration [Cl-]-dependent rightward shift of the fractional conductance. Geldanamycin and radicicol increased both the slow and fast activation time constants in a chloride-dependent manner. Heat shock treatment had the opposite effect. These results indicate that association of Hsp90 with ClC-2 results in greater channel activity due to increased cell surface channel expression, facilitation of channel opening, and enhanced channel sensitivity to intracellular [[Cl.sup.-]]. This association may have important pathophysiological consequences, enabling increased ClC-2 activity in response to cellular stresses such as elevated temperature, ischemia, or oxidative reagents. heat shock; geldanamycin; cellular stress; channel trafficking; transepithelial chloride transport

Various CIC-type voltage-gated chloride channel isoforms display a double barrel topology, and their gating mechanisms are thought to be similar. However; we demonstrate in this work that the nearly ubiquitous C1C-2 shows significant differences in gating when compared with ClC-0 and ClC-1. To delineate the gating of C1C-2 in quantitative terms, we have determined the voltage ([V.sub.m]) and time dependence of the protopore ([P.sub.f]) and common ([P.sub.s]) gates that control the opening and closing of the double barrel, mClC-2 was cloned from mouse salivary glands, expressed in HEK 293 cells, and the resulting chloride currents ([I.sub.Cl]) were measured using whole cell patch clamp. WT channels had [I.sub.Cl] that showed inward rectification and biexponential time course. Time constants of fast and slow components were ~10-fold different at negative [V.sub.m] and corresponded to Pf and [P.sub.s], respectively. [P.sub.f] and [P.sub.s] were ~1 at -200 mV, while at [V.sub.m] [greater than or equal to] 0 mV, [P.sub.f] ~ 0 and [P.sub.s] ~ 0.6. Hence, [P.sub.f] dominated open kinetics at moderately negative [V.sub.m], while at very negative [V.sub.m] both gates contributed to gating. At [V.sub.m] [greater than or equal to] 0 mV, mClC-2 closes by shutting off [P.sub.f]. Three- and two-state models described the open-to-closed transitions of [P.sub.f] and [P.sub.s], respectively. To test these models, we mutated conserved residues that had been previously shown to eliminate or alter [P.sub.f] or [P.sub.s] in other CIC channels. Based on the time and [V.sub.m] dependence of the two gates in WT and mutant channels, we constructed a model to explain the gating of mC1C-2. In this model the E213 residue contributes to [P.sub.f], the dominant regulator of gating, while the C258 residue alters the [V.sub.m] dependence of [P.sub.f], probably by interacting with residue E213. These data provide a new perspective on C1C-2 gating, suggesting that the protopore gate contributes to both fast and slow gating and that gating relies strongly on the E213 residue.

Plasma membrane wound repair is an important but poorly understood process. We used femtosecond pulses from a Ti-Sapphire laser to make multiphoton excitation--induced disruptions of the plasma membrane while monitoring the membrane potential and resistance. We observed two types of wounds that depolarized the plasma membrane. At threshold light levels, the membrane potential and resistance returned to prewound values within seconds; these wounds were not easily observed by light microscopy and resealed in the absence of extracellular [Ca.sup.2+]. Higher light intensities create wounds that are easily visible by light microscopy and require extracellular [Ca.sup.2+] to reseal. Within a few seconds the membrane resistance is ~100-fold lower, while the membrane potential has depolarized from -80 to -30 mV and is now sensitive to the [Cl.sup.-] concentration but not to that of [Na.sup.+], [K.sup.+], or [H.sup.+]. We suggest that the chloride sensitivity of the membrane potential, after wound resealing, is due to the fusion of chloride-permeable intracellular membranes with the plasma membrane.

ClC chloride channels, which are ubiquitously expressed in mammals, have a unique double-barreled structure, in which each monomer forms its own pore. Identification of pore-lining elements is important for understanding the conduction properties and unusual gating mechanisms of these channels. Structures of prokaryotic ClC transporters do not show an open pore, and so may not accurately represent the open state of the eukaryotic ClC channels. In this study we used cysteine-scanning mutagenesis and modification (SCAM) to screen >50 residues in the intracellular vestibule of ClC-0. We identified 14 positions sensitive to the negatively charged thiol-modifying reagents sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) or sodium 4-acetamido-4'-maleimidylstilbene-2'2-disulfonic acid (AMS) and show that 11 of these alter pore properties when modified. In addition, two MTSES-sensitive residues, on different helices and in close proximity in the prokaryotic structures, can form a disulfide bond in ClC-0. When mapped onto prokaryotic structures, MTSES/AMS-sensitive residues cluster around bound chloride ions, and the correlation is even stronger in the ClC-0 homology model developed by Corry et al. (2004). These results support the hypothesis that both secondary and tertiary structures in the intracellular vestibule are conserved among ClC family members, even in regions of very low sequence similarity. KEY WORDS: ClC channel * MTS modification * rectification * pore * homology model

Primary brain tumors (gliomas) often present with peritumoral edema. Their ability to thrive in this osmotically altered environment prompted us to examine volume regulation in human glioma cells, specifically the relative contribution of C1--channels and transporters to this process. After a hyposmotic challenge, cultured astrocytes, D54-MG glioma cells, and glioma cells from human patient biopsies exhibited a regulatory volume decrease (RVD). Although astrocytes were not able to completely reestablish their original prechallenge volumes, glioma cells exhibited complete volume recovery, sometimes recovering to a volume smaller than their original volumes ([V.suv.POST]-RVD < [V.sub.baseline]). In glioma cells, RVD was largely inhibited by treatment with a combination of [Cl.sup.-1] channel inhibitors, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and [Cd.sup.2+] ([V.sub.post]-RVD > 1.4*[V.sub.baseline]). Volume regulation was also attenuated to a lesser degree by the addition of R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H- inden-5-yl)oxy]acetic acid (DIOA), a known [K.sup.1]-[Cl.sup.-] cotransporter (KCC) inhibitor. To dissect the relative contribution of channels vs. transporters in RVD, we took advantage of the comparatively high temperature dependence of transport processes vs. channel-mediated diffusion. Cooling D54-MG glioma cells to 15[degrees]C resulted in a loss of DIOA-sensitive volume regulation. Moreover, at 15[degrees]C, the channel blockers NPPB + [Cd.sup.2+] completely inhibited RVD and cells behaved like perfect osmometers. The calculated osmolyte flux during RVD under these experimental conditions suggests that the relative contribution of [Cl.sup.-] channels vs. transporters to this process is ~60-70% and ~30-40%, respectively. Finally, we identified several candidate proteins that may be involved in RVD, including the [Cl.sup.-] channels C1C-2, C1C-3, C1C-5, C1C-6, and C1C-7 and the transporters KCC1 and KCC3a. voltage-gated chloride channel family; potassium-chloride cotransporters; peritumoral edema

The CFTR chloride channel is activated by phosphorylation of serine residues in the regulatory (R) domain and then gated by ATP binding and hydrolysis at the nucleotide binding domains (NBDs). Studies of the ATP-dependent gating process in excised inside-out patches are very often hampered by channel rundown partly caused by membrane-associated phosphatases. Since the severed [DELTA]R-CFTR, whose R domain is completely removed, can bypass the phosphorylation-dependent regulation, this mutant channel might be a useful tool to explore the gating mechanisms of CFTR. To this end, we investigated the regulation and gating of the AR-CFTR expressed in Chinese hamster ovary cells. In the cell-attached mode, basal AR-CFTR currents were always obtained in the absence of cAMP agonists. Application of cAMP agonists or PMA, a PKC activator, failed to affect the activity, indicating that the activity of [DELTA]R-CFTR channels is indeed phosphorylation independent. Consistent with this conclusion, in excised inside-out patches, application of the catalytic subunit of PKA did not affect ATP-induced currents. Similarities of ATP-dependent gating between wild type and AR-CFTR make this phosphorylation-independent mutant a useful system to explore more extensively the gating mechanisms of CFFR. Using the [DELTA]R-CFTR construct, we studied the inhibitory effect of ADP on CFTR gating. The Ki for ADP increases as the [ATP] is increased, suggesting a competitive mechanism of inhibition. Single channel kinetic analysis reveals a new closed state in the presence of ADP, consistent with a kinetic mechanism by which ADP binds at the same site as ATP for channel opening. Moreover, we found that the open time of the channel is shortened by as much as 54% in the presence of ADP. This unexpected result suggests another ADP binding site that modulates channel closing. KEYWORDS: chloride channel * single-channel kinetics * ABC transporter * gating mode * phosphorylation

Previously, we demonstrated that ADP inhibits cystic fibrosis transmembrane conductance regulator (CFTR) opening by competing with ATP for a binding site presumably in the COOH-terminal nucleotide binding domain (NBD2). We also found that the open time of the channel is shortened in the presence of ADR To further study this effect of ADP on the open state, we have used two CFTR mutants (D1370N and E1371S); both have longer open times because of impaired ATP hydrolysis at NBD2. Single-channel kinetic analysis of [DELTA]R/D1370N-CFTR shows unequivocally that the open time of this mutant channel is decreased by ADR [DELTA]R/E1371S-CFTR channels can be locked open by millimolar ATP with a time constant of ~ 100 s, estimated from current relaxation upon nucleotide removal. ADP induces a shorter locked-open state, suggesting that binding of ADP at a second site decreases the locked-open time. To test the functional consequence of the occupancy of this second nucleotide binding site, we changed the [ATP] and performed similar relaxation analysis for [DELTA] E1371S-CFTR channels. Two locked-open time constants can be discerned and the relative distribution of each component is altered by changing [ATP] so that increasing [ATP] shifts the relative distribution to the longer locked-open state. Singlechannel kinetic analysis for [DELTA]R/E1371S-CFTR confirms an [ATP]-dependent shift of the distribution of two locked-open time constants. These results support the idea that occupancy of a second ATP binding site stabilizes the locked-open state. This binding site likely resides in the [NH.sub.2]-terminal nucleotide binding domain (NBD1) because introducing the K464A mutation, which decreases ATP binding affinity at NBD1, into E1371S-CFTR shortens the relaxation time constant. These results suggest that the binding energy of nucleotide at NBD1 contributes to the overall energetics of the open channel conformation. KEY WORDS: chloride channel * single-channel kinetics * ABC transporter * energetics * macroscopic relaxation

[Cl.sup.-] channels are proposed to play a role in gut pacemaker activity, but little is known about the characteristics of [Cl.sup.-] channels in interstitial cells of Cajal (ICC), the intestinal pacemaker cells. The objective of the present study was to identify whole cell [Cl.sup.-] currents in ICC associated with previously observed single-channel activity and to characterize its inward rectification. Whole cell patch-clamp studies showed that ICC express an inwardly rectifying [Cl.sup.-] current that was not sensitive to changes in cation composition of the extracellular solutions. Currents were not affected by replacing all cations with N-methyl-D-glucamine ([NMDG.sup.+]). Whole cell currents followed the [Cl.sup.-] equilibrium potential and were inhibited by DIDS and 9-anthracene carboxylic acid. Ramp protocols of single-channel activity showed that inward rectification was due to reduction in single-channel open probability, not a reduction in single-channel conductance. Single-channel data led to the hypothesis that strong cooperation exists between 30-pS channels that show less cooperation at potentials positive to the reversal potential. Hence, an inwardly rectifying [Cl.sup.-] channel plays a prominent role in determining pacemaker activity in the gut. pacemaking activity; gastrointestinal motility; interstitial cells of Cajal

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation- and ATP-dependent chloride channel that modulates salt and water transport across lung and gut epithelia. The relationship between CFTR and oxidized forms of glutathione is of potential interest because reactive glutathione species are produced in inflamed epithelia where they may be modulators or substrates of CFTR. Here we show that CFTR channel activity in excised membrane patches is markedly inhibited by several oxidized forms of glutathione (i.e., GSSG, GSNO, and glutathione treated with diamide, a strong thiol oxidizer). Three lines of evidence indicate that the likely mechanism for this inhibitory effect is glutathionylation of a CFTR cysteine (i.e., formation of a mixed disulfide with glutathione): (a) channels could be protected from inhibition by pretreating the patch with NEM (a thiol alkylating agent) or by lowering the bath pH; (b) inhibited channels could be rescued by reducing agents (e.g., DTT) or by purified glutaredoxins (Grxs; thiol disulfide oxidoreductases) including a mutant Grx that specifically reduces mixed disulfides between glutathione and cysteines within proteins; and (c) reversible glutathionylation of CFTR polypeptides in microsomes could be detected biochemically under the same conditions. At the single channel level, the primary effect of reactive glutathione species was to markedly inhibit the opening rates of individual CFTR channels. CFTR channel inhibition was not obviously dependent on phosphorylation state but was markedly slowed when channels were first 'locked open' by a poorly hydrolyzable ATP analogue (AMP-PNP). Consistent with the latter finding, we show that the major site of inhibition is cys-1344, a poorly conserved cysteine that lies proximal to the signature sequence in the second nucleotide binding domain (NBD2) of human CFTR. This region is predicted to participate in ATP-dependent channel opening and to be occluded in the nucleotide-bound state of the channel based on structural comparisons to related ATP binding cassette transporters. Our results demonstrate that human CFTR channels are reversibly inhibited by reactive glutathione species, and support an important role of the region proximal to the NBD2 signature sequence in ATP-dependent channel opening. KEY WORDS: cystic fibrosis transmembrane conductance regulator * ABC transporter * glutathione * glutaredoxin * redox

Generation of the action potentials (AP) necessary to activate skeletal muscle fibers requires that inward membrane currents exceed outward currents and thereby depolarize the fibers to the voltage threshold for AP generation. Excitability therefore depends on both excitatory Na+ currents and inhibitory [K.sup.+] and [Cl.sup.-] currents. During intensive exercise, active muscle loses K+ and extracellular [K.sup.+] ([[[K.sup.+]].sub.o]) increases. Since high [[[K.sup.+]].sub.o] leads to depolarization and ensuing inactivation of voltage-gated [Na.sup.+] channels and loss of excitability in isolated muscles, exercise-induced loss of K+ is likely to reduce muscle excitability and thereby contribute to muscle fatigue in vivo. Intensive exercise, however, also leads to muscle acidification, which recently was shown to recover excitability in isolated [K.sup.+]-depressed muscles of the rat. Here we show that in rat soleus muscles at 11 mM [K.sup.+], the almost complete recovery of compound action potentials and force with muscle acidification (C[O.sub..2 changed from 5 to 24%) was associated with reduced chloride conductance (1731 [plus or minus] 151 to 938 [plus or minus] 64 [micro]S/c[m.sup.2], P < 0.01 but not with changes in potassium conductance (405 [plus or minus] 20 to 455 [plus or minus] 30 [micro]S/c[m.sup.2], P < 0.16). Furthermore, acidification reduced the rheobase current by 26% at 4 mM [K.sup.+] and increased the number of excitable fibers at elevated [[[K.sup.+]].sub.o]. At 11 mM [K.sup.+] and normal pH, a recovery of excitability and force similar to the observations with muscle acidification could be induced by reducing extracellular [Cl.sup.-] or by blocking the major muscle [Cl.sup.-] channel, ClC-1, with 30 [micro]M 9-AC. It is concluded that recovery of excitability in K+-depressed muscles induced by muscle acidification is related to reduction in the inhibitory [Cl.sup.-+] currents, possibly through inhibition of ClC-1 channels, and acidosis thereby reduces the Na+ current needed to generate and propagate an AP. Thus short term regulation of [Cl.sup.-] channels is important for maintenance of excitability in working muscle. KEY WORDS: lactic acid * muscle fatigue * action potentials * [Na.sup.+] channels * [Cl.sup.1] channels

The distal convoluted tubule (DCT) is a heterogeneous segment subdivided into early (DCT1) and late (DCT2) parts, depending on the distribution of various transport systems. We do not have an exhaustive picture of the [C1.sup.-] channels on the basolateral side: the presence of C1C-K2 channels is generally accepted, whereas that of C1C-K1 remains controversial. We used here single-cell RT-PCR and patch clamp to probe [C1.sup.-] channel heterogeneity in microdissected mouse DCT at the molecular and functional levels. Our findings show that 63% of the DCT cells express C1C-K2 mRNA, either alone (type 1 cells: 47 and 23% in DCT1 and DCT2, respectively), or combined with C1C-K1, mostly in DCT2 (type 2 cells: 33%), but 37% of DCT1 and DCT2 cells do not express any C1C-K. Patch-clamp experiments revealed that a [C1.sup.-] channel, with 9-pS conductance and [C1.sup.-] > N[O.sub.3] = [Br.sup.-] anion selectivity sequence, is present in the DCT1 and DCT2 basolateral membranes (87 and 71% of the patches, respectively). This dominant channel is likely to be C1C-K2 in type 1 cells. In type 2 cells, it could be C1C-K2 and/or C1C-K1 homodimers, but also C1C-K1/C1C-K2 heterodimers, or a mixture of all combinations. A second, distinct [C1.sup.-] channel (13% of DCT1 patches, 29% of DCT2 patches) also displayed 9-pS conductance but had a completely different anion selectivity ([I.sup.-] > N[O.sup.-.sub.3] > Br > [Cl.sup.-]), which was not compatible with that of the C1C-Ks. This indicates that a [Cl.sup.-] channel that is unlikely to belong to the C1C family may also be involved in [C1.sup.-] absorption in the DCT2. kidney; C1C-K; sodium chloride absorption

CFTR involvement in nasal potential differences in mice and pigs studied using a thiazolidinone CFTR inhibitor. Am J Physiol Lung Cell Mol Physiol 287: L936-L943, 2004. First published July 9, 2004; doi: 10.1152/ajplung.00354.2003.--Nasal potential difference (PD) measurements have been used to demonstrate defective CFTR function in cystic fibrosis (CF) and to evaluate potential CF therapies. We used the selective thiazolidinone CFTR inhibitor [CFTR.sub.inh]-172 to define the involvement of CFTR in nasal PD changes in mice and pigs. In normal mice infused intranasally with a physiological saline solution containing amiloride, nasal PD was -4.7 [+ or -] 0.7 mV, hyperpolarizing by 15 [+ or -] 1 mV after a low-[Cl.sup.-] solution, and a further 3.9 [+ or -] 0.5 mV after forskolin. [CFTR.sub.inh]-172 produced 1.1 [+ or -] 0.9- and 4.3 [+ or -] 0.7-mV depolarizations when added after low [Cl.sup.-] and forskolin, respectively. Systemically administered [CFTR.sub.inh]-172 reduced the forskolin-induced hyperpolarization from 4.7 [+ or -] 0.4 to 0.9 [+ or -] 0.1 mV but did not reduce the low [Cl.sup.-]-induced hyperpolarization. Nasal PD was -12 [+ or -] 1 mV in CF mice after amiloride, changing by cystic fibrosis; cystic fibrosis transmembrane conductance regulator; nasal potential difference; chloride channels

Mechanism of activation of Xenopus CFTR by stimulation of PKC. Am J Physiol Cell Physiol 287: C1256-C1263, 2004. First published June 30, 2004; doi:10.1152/ajpcell.00229.2004.--PKA-mediated phosphorylation of the regulatory (R) domain plays a major role in the activation of the human cystic fibrosis transmembrane conductance regulator (hCFTR). In contrast, the effect of PKC-mediated phosphorylation is controversial, smaller than that of PKA, and dependent on the cell type. In the present study, we expressed Xenopus CFTR (XCFTR) and hCFTR in Xenopus oocytes and examined their responses (i.e., macroscopic membrane conductance) to maximal stimulation by PKC and PKA agonists. With XCFTR, the average response to PKC was approximately sixfold that of PKA stimulation. In contrast, with hCFTR, the response to PKC was ~90% of the response to PKA stimulation. The reason for these differences was the small response of XCFTR to PKA stimulation. Using the substituted cysteine accessibility method, we found no evidence for insertion of functional CFTR channels in the plasma membrane in response to PKC stimulation. The increase in macroscopic conductance in response to PKC stimulation of XCFTR was due to an approximately fivefold increase in single-channel open probability, with a minor (~30%) increase in single-channel conductance. The responses of XCFTR to PKC stimulation and of hCFTR to PKA stimulation were mediated by similar increases in [P.sub.o]. In both instances, there were no changes in the number of channels in the membrane. We speculate that in animals other than humans, PKC stimulation may be the dominant mechanism for activation of CFTR. chloride channel; channel regulation; cystic fibrosis transmembrane conductance regulator gating; cystic fibrosis; phosphorylation; protein kinase A

SPI-0211 activates T84 cell chloride transport and recombinant human ClC-2 chloride currents. Am J Physiol Cell Physiol 287: C1173-C1183, 2004. First published June 22, 2004; doi:10.1152/ajpcell. 00528.2003.--The purpose of this study was to determine the mechanism of action of SPI-0211 (lubiprostone), a novel bicyclic fatty acid in development for the treatment of bowel dysfunction. Adult rabbit intestine was shown to contain mRNA for ClC-2 using RT-PCR, Northern blot analysis, and in situ hybridization. T84 cells grown to confluence on permeable supports were shown to express ClC-2 channel protein in the apical membrane. SPI-0211 increased electrogenic [Cl.sup.-] transport across the apical membrane of T84 cells, with an E[C.sub.50] of ~18 nM measured by short-circuit current ([I.sub.sc]) after permeabilization of the basolateral membrane with nystatin. SPI-0211 effects on [Cl.sup.-] currents were also measured by whole cell patch clamp using the human embryonic kidney (HEK)-293 cell line stably transfected with either recombinant human ClC-2 or recombinant human cystic fibrosis transmembrane regulator (CFTR). In these studies, SPI-0211 activated ClC-2 [Cl.sup.-] currents in a concentration-dependent manner, with an E[C.sub.50] of ~17 nM, and had no effect in nontransfected HEK-293 cells. In contrast, SPI-0211 had no effect on CFTR [Cl.sup.-] channel currents measured in CFTR-transfected HEK-293 cells. Activation of ClC-2 by SPI-0211 was independent of PKA. Together, these studies demonstrate that SPI-0211 is a potent activator of ClC-2 [Cl.sup.-] channels and suggest a physiologically relevant role for ClC-2 [Cl.sup.-] channels in intestinal [Cl.sup.-] transport after SPI-0211 administration. cystic fibrosis transmembrane regulator; intestinal chloride channels; chloride channel opener

Inhibition of ENaC by intracellular [Cl.sup.-] in an MDCK clone with high ENaC expression. Am J Physiol Renal Physiol 287:F722-F731, 2004. First published May 25, 2004:10.1152/ajprenal.00135.2004.--We examined the effects of intracellular Cl concentration ([[[Cl.sup.-]].sub.i]) on the epithelial Na channel (ENaC) in a line of Madin-Darby canine kidney (MDCK) cells (FL-MDCK) with a high rate of [Na.sup.+] transport produced by stable retroviral transfection with rENaC subunits (Morris RG and Schafer JA. J Gen Physiol 120:71- 85, 2002). Treatment with cAMP (100 [micro]M 8-cpt-cAMP plus 100 [micro]M IBMX) stimulated ENaC-mediated [Na.sup.+] absorption as well as Cl secretion via cystic fibrosis transmembrane conductance regulator, which was characterized in [alpha]-toxin-permeabilized monolayers to have the anion selectivity sequence N[O.sup.-.sub.3] > Br > [Cl.sup.-] > [I.sup.-]. With the use of FL-MDCK monolayers in which the basolateral membrane was permeabilized by nystatin, the ENaC conductance of the apical membrane [determined from the amiloride-sensitive short-circuit current (AS-[I.sub.sc]) driven by an apical-to-basolateral [Na.sup.+] concentration gradient] was progressively inhibited by increasing the [[Cl.sup.-]] in the basolateral solution (and hence in the cytosol), but it was insensitive to the [[Cl.sup.-]] in the apical solution. This inhibitory effect of [[[Cl.sup.-].sub.i] occurred regardless of the presence or absence of net [Cl.sup.-] transport. However, from fluorometric measurements using the Cl -sensitive dye 6-methoxy-N-(3-sulfopropyl)-quinolinium in intact FL-MDCK monolayers on permeable supports, cAMP, which activates both [Na.sup.+] absorption and [Cl.sup.-] secretion, produced a decrease of [[[Cl.sup.-]].sub.i] from 76 [+ or -] 14 to 36 [+ or -] 8 mM (P = 0.03). Thus it might be expected that activation of Cl secretion by cAMP would lead to stimulation rather than inhibition of ENaC. In the nystatin-treated monolayers, an increase in [[[Cl.sup.-]].sub.i] from 15 to 145 mM decreased AS-[I.sub.sc] from 24.5 [+ or -] 1.0 to 10.2 [+ or -] 1.6 [micro]A/[cm.sup.2]. This inhibition of ENaC could be attributed to nearly proportional decreases in the density of ENaC in the apical membrane from 1.91 [+ or -] 0.16 to 1.32 [+ or -] 0.17 fmol/[cm.sup.2] and in the intrinsic channel activity (the average current per ENaC subunit) from 13.3 [+ or -] 1.2 to 8.2 [+ or -] 1.4 [micro]A/fmol. epithelial sodium channel surface density; chloride channels; cystic fibrosis transmembrane conductance regulator

Elevations of cytoplasmic free calcium concentrations ([[[Ca.sup.2+]].sub.i]) evoked by cholinergic agonists stimulate isotonic fluid secretion in salivary, acinar cells. This process is driven by the apical exit of [Cl.sup.-] through [Ca.sup.2+]-activated [Cl.sup.-] channels, while [Cl.sup.-] enters the cytoplasm against its electrochemical gradient via a loop diuretic-sensitive [Na.sup.2+] -[K.sup.+] -2[Cl.sup.-]-2[Cl.sup-] cotransporter (NKCC) and/or parallel operations of [Cl.sup.-] -HC[O.sup.-.sub.3] and [Na.sup.+][-H.sup.+] exchangers, located in the basolateral membrane. To characterize the contributions of those activities to net [Cl.sup.-] secretion, we analyzed carbachol (CCh)-activated [Cl.sup.-] currents in submandibular acinar cells using the 'gramicidin-perforated patch recording configuration.' Since the linear polypeptide antibiotic gramicidin creates monovalent cation-selective pores, CCh-activated CI currents in the gramicidin-perforated patch recording were carried by [Cl.sup.-] efflux via [Cl.sup.-] channels, dependent upon [Cl.sup.-] entry through [Cl.sup.-] transporters expressed in the acinar cells. CCh-evoked oscillatory CI currents were associated with oscillations of membrane potential. Bumetanide, a loop diuretic, decreased the CCh-activated C1 currents and hyperpolarized the membrane potential. In contrast, neither methazolamide, a carbonic anhydrase inhibitox; nor elimination of external HC[O.sup.-.sub.3] had significant effects, suggesting that the cotransporter rather than parallel operations of [Cl.sup.-] -HC[O.sup.-.sub.3] and [Na.sup.+][-H.sup.+] exchangers is the primary [Cl.sup.-] uptake pathway. Pharmacological manipulation of the activities of the [Ca.sup.2+]-activated [Cl.sup.-] channel and the NKCC revealed that the NKCC plays a substantial role in determining the amplitude of oscillatory [Cl.sup.-] currents, while adjusting to the rate imposed by the [Ca.sup.2+]-activated [Cl.sup.-] channel, in the gramicidin-perforated patch configuration. By concerting with and being controlled by the cation steps, the oscillatory form of secretory [Cl.sup.-] movements may effectively provide a driving force for fluid secretion in intact acinar cells. KEY WORDS: chloride secretion * exocrine glands * chloride channels * cation-chloride cotransporter * gramicidin

Byline: Shuo-Bin Jou (2), Ling-I Chang (1), Huichin Pan (1), Pei-Ru Chen (3), Kuang-Ming Hsiao (1,4) Keywords: CLCN1 mutations; myotonia congenita; Taiwan Abstract: Abstract. We have performed genetic screening on the skeletal muscle chloride channel gene (CLCN1) in Taiwanese population. A total of four patients with myotonia congenita (MC) together with 106 normal individuals were examined. All 23 exons of the CLCN1 gene were analysed by direct sequencing of PCR products to detect the nucleotide changes. Five mutations and three polymorphisms were identified in this study. Among these, three missense mutations (S471F, P575S, D644G) and one polymorphism (T736I) are novel and could be unique to the Taiwanese. In addition, a previously documented recessive G482R mutation was identified in a heterozygous patient and his nonsymptomatic father, indicating that this mutation might indeed function recessively or dominantly with incomplete penetrance. In conclusion, this is the first report of MC in Taiwan with proven CLCN1 gene mutations and showing high molecular heterogeneity in Taiwanese MC patients. Author Affiliation: (1) Department of Life Sciences, Chung Shan Medical University, Taichung, Taiwan (2) Department of Neurology, China Medical University Hospital, Taichung, Taiwan (3) Instrument Center, Chung Shan Medical University, Taichung, Taiwan (4) Department of Life Sciences, Chung Shan Medical University No.110, Sec.1, Chien-Kuo North Road, Taichung Taiwan 402, Republic of China Article History: Registration Date: 01/01/2004 Received Date: 07/04/2003 Accepted Date: 09/12/2003

Immunocytochemical localization of [Na.sup.+]-HC[O.sup.-.sub.3] cotransporters and carbonic anhydrase dependence of fluid transport in corneal endothelial cells. Am J Physiol Cell Physiol 286: C1434-C1442, 2004. First published February 11, 2004; 10.1152/ajpcell.00539.2003.--In corneal endothelium, there is evidence for basolateral entry of HC[O.sup.-.sub.3] into corneal endothelial cells via N[a.sup.+]--HC[O.sup.-.sub.3] cotransporter (NBC) proteins and for net HC[O.sup.-.sub.3] flux from the basolateral to the apical side. However, bow HC[O.sup.-.sub.3] exits the cells through the apical membrane is unclear. We determined that cultured corneal endothelial cells transport HC[O.sup.-.sub.3] similarly to fresh tissue. In addition, [Cl.sup.-] channel inhibitors decreased fluid transport by at most 16%, and inhibition of membrane-bound carbonic anhydrase IV by benzolamide or dextran-bound sulfonamide decreased fluid transport by at most 29%. Therefore, more than half of the fluid transport cannot be accounted for by anion transport through apical CI channels, C[O.sub.2] diffusion across the apical membrane, or a combination of these two mechanisms. However, immunocytochemistry using optical sectioning by confocal microscopy and cryosections revealed the presence of NBC transporters in both the basolateral and apical cell membranes of cultured bovine corneal endothelial cells and freshly isolated rabbit endothelia. This newly detected presence of an apical NBC transporter is consistent with its being the missing mechanism sought. We discuss discrepancies with other reports and provide a model that accounts for the experimental observations by assuming different stoichiometries of the NBC transport proteins at the basolateral and apical sides of the cells. Such functional differences might arise either from the expression of different isoforms or from regulatory factors affecting the stoichiometry of a single isoform. confocal microscopy; cryosections; stoichiometry; pH titration; chloride channels

Osmotic swelling-provoked release of organic osmolytes in human intestinal epithelial cells. Am J Physiol Cell Physiol 286: C1417-C1422, 2004. First published February 11, 2004; 10.1152/ajpcell.00468.2003.--Human Intestine 407 cells respond to osmotic cell swelling by the activation of [Cl.sup.-] and [K.sup.+] -selective ionic channels, as well as by stimulating an organic osmolyte release pathway readily permeable to taurine and phosphocholine. Unlike the activation of volume-regulated anion channels (VRAC), activation of the organic osmolyte release pathway shows a lag time of ~30-60 s, and its activity persists for at least 8-12 min. In contrast to VRAC activation, stimulation of organic osmolyte release did not require protein tyrosine phosphorylation, active p[21.sup.rho], or phosphatidylinositol 3-kinase activity and was insensitive to [Cl.sup.-] channel blockers. Treatment of the cells with putative organic anion transporter inhibitors reduced the release of taurine only partially or was found to be ineffective. The efflux was blocked by a subclass of organic cation transporter (OCT) inhibitors (cyanine-863 and decynium-22) but not by other OCT inhibitors (cimetidine, quinine, and verapamil). Brief treatment of the cells with phorbol esters potentiated the cell swelling-induced taurine efflux, whereas addition of the protein kinase C (PKC) inhibitor GF109203X largely inhibited the response, suggesting that PKC is involved. Increasing the level of intracellular [Ca.sup.2+] by using A-23187-or [Ca.sup.2+]-mobilizing hormones, however, did not affect the magnitude of the response. Taken together, the results indicate that the hypotonicity-induced efflux of organic osmolytes is independent of VRAC and involves a PKC-dependent step. regulatory volume decrease; taurine; volume-regulated anion channels; chloride channel; protein kinase C

CIC channels constitute a large family of [Cl.sup.-]-selective ion channels that are present in diverse organisms. The channels have a complex gating behavior, in which channel opening and closing is tightly coupled to ion permeation. Recent success in the structure determination of bacterial channels has revealed the overall architecture of this family and provided important insight into ion selectivity and gating. The CIC channels are homodimers, with each subunit containing an ion conduction pore. In the narrow selectivity filter of each pore, [Cl.sup.-] are bound to several sites through electrostatic interactions with helix dipoles and the partial charges of protein residues. In the closed conformation of the channel, a conserved glutamate residue occupies one of the ion-binding sites; on channel opening, this glutamate residue moves out of the binding site, thereby making room for an additional [Cl.sup.-] ion.

Bestrophins have recently been proposed to comprise a new family of [Cl.sup.-] channels. Our goal was to test whether mouse bestrophin-2 (mBest2) is a bona fide [Cl.sup.-] channel. We expressed mBest2 in three different mammalian cell lines, mBest2 was trafficked to the plasma membrane as shown by biotinylation and immunoprecipitation, and induced a [Ca.sup.2+]-activated [Cl.sup.-] current in all three cell lines (E[C.sub.50] for [Ca.sup.2+] = 230 nM). The permeability sequence was SC[N.sup.-]: [I.sup.-] : [Br.sup.-] : [Cl.sup.-] : [F.sup.-] (8.2: 1.9: 1.4: 1: 0.5). Although SC[N.sup.-] was highly permeant, its conductance was ~10% that of [Cl.sup.-] and SC[N.sup.-] blocked [Cl.sup.-] conductance (I[C.sub.50] = 12 mM). Therefore, SC[N.sup.-] entered the pore more easily than [Cl.sup.-], but bound more tightly than [Cl.sup.-]. Mutations in S79 altered the relative permeability and conductance for SC[N.sup.-] as expected if S79 contributed to an anion binding site in the channel. [P.sub.SCN]/[P.sub.Cl] = 8.2 [+ or -] 1.3 for wild-type and 3.9 [+ or -] 0.4 for S79C. [G.sub.SCN]/[G.sub.Cl] = 0.14 [+ or -] 0.03 for wild-type and 0.94 [+ or -] 0.04 for S79C. In the S79 mutants, SC[N.sup.-] did not block [Cl.sup.-] conductance. This suggested that the S79C mutation altered the affinity of an anion binding site for SC[N.sup.-]. Additional evidence that S79 was located in the conduction pathway was provided by the finding that modification of the sulfhydryl group in S79C with MTSE[T.sup.+] or MTSE[S.sup.-] increased conductance significantly. Because the effect of positively and negatively charged MTS reagents was similar, electrostatic interactions between the permeant anion and the channel at this residue were probably not critical in anion selectivity. These data provide strong evidence that mBest2 forms part of the novel [Cl.sup.-] conduction pathway in mBest2-transfected cells and that S79 plays an important role in anion binding in the pore of the channel. KEY WORDS: chloride channels * ion permeation * electrophysiology * mutagenesis * ion channel

The aim of the present study was to investigate the role of [Ca.sup.2+]-activated [Cl.sup.-] channels in the renal vasoconstriction elicited by angiotensin II (ANG II) and norepinephrine (NE). Renal blood flow (RBF) was measured in vivo using electromagnetic flowmetry. Ratiometric, photometry of fura 2 fluorescence was used to estimate iutracellular free [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) in isolated preglomerular vessels from rat kidneys. Renal arterial injection of ANG II (2-4 ng) and NE (20-40 ng) produced a transient decrease in RBF. Administration orANG II ([10.sup.-7] M) and NE (5 x [10.sup.-6] M) to the isolated preglomerular vessels caused a prompt increase in [[Ca.sup.2+].sub.i]. Renal preinfusion of DIDS (0.6 and 1.25 [micro]mol/min) attenuated the ANG II-induced vasoconstriction to ~35% of the control response, whereas the effects of NE were unaltered. Niflumic acid (0.14 and 0.28 [micro]mol/min) and 2-[(2-cyclopentenyl-6,7-dichloro-2,3 -dihydro-2-methyl-1-oxo-1H-inden-5-yl)oxy]acetic acid (IAA-94; 0.045 and 0.09 [micro]mol/min) did not affect the vasoconstrictive responses of these compounds. Pretreatment with niflumic acid (50 [micro]M) or IAA-94 (30 [micro]M) for 2 min decreased baseline [[[Ca.sup.2+]].sub.i] but did not change the magnitude of the [[[Ca.sup.2+]].sub.i] response to ANG II and NE in the isolated vessels. The present results do not support the hypothesis that [Ca.sup.2+]-activated [Cl.sup.-] channels play a crucial role in the hemodynamic effects of ANG II and NE in rat renal vasculature. microcirculation; vascular smooth muscle; 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid: niflumic acid; calcium channels