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PubMed2005

[The effects of chloride channel blockers on thrombocytic cytoplasmic free calcium concentration and platelet aggregation].

OBJECTIVE: To explore the effects of chloride channels on the regulation of platelet cytoplasmic free calcium concentration ([Ca2+]i) and platelet aggregation (PAG). METHODS: Freshly separated platelets were activated by thrombin. Chloride channel blockers DIDS or NFA and calcium channel blockers SK&F96365 or nifedipine were added to study the effects on platelet [Ca2+]i and PAG by a single reagent or the combination of reagents and find out the interactions among DIDS, NFA, SK&F96365 and nifedipine. RESULTS: Both DIDS and NFA could inhibit the thrombin (1 U/ml) induced PAG in a dose-dependent manner, whereas had little effect on resting [Ca2+]i. As compared with the control group, DIDS, SK&F96365 and Nifedipine could significantly reduce the PAG, Ca2+ release and Ca2+ influx in thrombin activated platelet (P < 0.05). The combination of DIDS and SK&F96365 had greater effects in reducing the PAG, Ca2+ release and Ca2+ influx than either reagent alone (P < 0.05). The combination of DIDS and nifedipine also had greater effect than each alone in reducing Ca2+ release (P < 0.05). The combination of NFA and SK&F96365 weakened each other's effect on Ca2+ release (P < 0.05), while NFA and nifedipine weakened each other's effects on PAG, Ca2+ release and Ca2+ influx in thrombin activated platelet (P < 0.05). CONCLUSION: DIDS and NFA have no effect on the resting [Ca2+]i and the leak calcium influx of platelet. DIDS can inhibit the Ca2+ release, Ca2+ influx and PAG of platelet induced by thrombin, while NFA can only inhibit the Ca2+ release. The chloride channel and calcium channel blockers have interactions in affecting resting [Ca2+]i and PAG of platelet. The opening of chloride channel can influence the cellular calcium movement of platelet.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1999

Diversification of Drosophila chloride channel gene by multiple posttranscriptional mRNA modifications.

We have identified and analyzed a Drosophila melanogaster gene that encodes a chloride channel subunit (DrosGluCl-alpha) previously shown to function as a glutamate-gated chloride channel in an in vitro expression system. Sequence analysis of several cDNAs corresponding to the gene revealed sequence diversity in their open reading frames at seven specific sites. Site-specific A-to-G variations between cDNA and genomic sequences, consistent with RNA editing, were detected at five nucleotide positions. In addition, sequence variations among cDNA clones consistent with alternative splicing of mRNA were found at two different sites. In the 5' region, two small adjacent exons, containing similar but distinct modular sequences, are alternatively incorporated into the mature mRNA. In the 3' region, alternative splicing generates a variant encoding a protein with four additional amino acids just upstream of the fourth transmembrane domain. Combinations of RNA editing and alternative splicing can lead to extensive diversification of transcripts. These results give the first example of RNA editing in neurotransmitter-gated chloride channel genes or of alternative splicing in a glutamate-gated chloride channel gene of Drosophila.

Alternative Splicing↗
PubMed2001

Assessment of CFTR chloride channel openers in intact normal and cystic fibrosis murine epithelia.

1. A method is described for the detection of CFTR chloride channel openers (ClCOs) and blockers. Murine colonic epithelia were used throughout, but the method is applicable to other epithelia and biopsy material. 2. The principle was to render the epithelial basolateral membranes electrically transparent so that the apical membrane alone could be voltage clamped. This was achieved by potassium depolarization on the basolateral side. Imposition of an apical to basolateral chloride gradient allowed the effects of ClCOs on an outward chloride current and on apical membrane conductance to be measured. 3. 1-ethyl-2-benzimidazolone (EBIO), forskolin, chlorzoxazone, and genistein all showed ClCO activity. In cystic fibrosis (CF) epithelia, either from CF null or CF Delta F508 mice, EBIO showed only a minor effect, indicating that CFTR was the target in wild type tissues. 4. 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) was shown to block CFTR chloride channels. The blockade was pH and voltage-dependent and indicated that while the charged form was the active moiety, movement into the cell depended on the unionized drug. It is concluded NPPB blocks CFTR from the cytosolic side and that the agent preferentially blocks at potentials opposing the inflow of chloride ions. No significant blockade was seen with either N-phenylanthranilic acid (DPC) or with glibenclamide, under standard conditions. 5. The method described can be used to examine compounds reported to increase the trafficking of Delta F508 CFTR to the membrane or those capable of opening Delta F508 CFTR chloride channels and to differentiate between them. Further, the method distinguishes between chloride channel openers and those acting indirectly to increase the flux through CFTR chloride channels by indirect means, for example, hyperpolarization.

Animals↗
PubMed1994

Phosphatase inhibitors activate normal and defective CFTR chloride channels.

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is regulated by phosphorylation and dephosphorylation at multiple sites. Although activation by protein kinases has been studied in some detail, the dephosphorylation step has received little attention. This report examines the mechanisms responsible for the dephosphorylation and spontaneous deactivation ("rundown") of CFTR chloride channels excised from transfected Chinese hamster ovary (CHO) and human airway epithelial cells. We report that the alkaline phosphatase inhibitors bromotetramisole, 3-isobutyl-1-methylxanthine, theophylline, and vanadate slow the rundown of CFTR channel activity in excised membrane patches and reduce dephosphorylation of CFTR protein in isolated membranes. It was also found that in unstimulated cells, CFTR channels can be activated by exposure to phosphatase inhibitors alone. Most importantly, exposure of mammalian cells to phosphatase inhibitors alone activates CFTR channels that have disease-causing mutations, provided the mutant channels are present in the plasma membrane (R117H, G551D, and delta F508 after cooling). These results suggest that CFTR dephosphorylation is dynamic and that membrane-associated phosphatase activity may be a potential therapeutic target for the treatment of cystic fibrosis.

1-Methyl-3-isobutylxanthine↗
PubMed1993

On the existence of epithelial chloride channels in intact frogs and crayfish.

Chloride channels in isolated amphibian skins have been shown to be open (high PCl) when the transepithelial potential (TEP) across the skin is > 30 mV, inside positive, and [Cl-] in the external medium is high. In the absence of either of these signals PCl is much lower. Using Cl- efflux as a criterion, it is shown here that when the same cues are presented to intact frogs, channels open as in the in vitro preparation. The augmented efflux is partially inhibited by high I- in both preparations. However, salt-depleted frogs do not show channel opening, differing in this regard from isolated skins. Elevating external [Cl-] resulted in an even larger efflux stimulation in crayfish than in frogs. The TEP in these animals is usually small and sometimes blood negative; it does not appear to play a role in augmenting the efflux. Gluconate and NO3- also caused Cl efflux to increase markedly. Salt-depletion reduced the stimulation by high [Cl-] but did not abolish it as in frogs. Iodide had no effect on chloride efflux in crayfish. The results may indicate that gated channels exist in these animals, but interpretation is complicated by the possibility that the augmented efflux is due to exchange diffusion.

Animals↗
PubMed2006

[Effects of chloride channel blockers on excitatory junction potentials in smooth muscle cells of cochlear spiral modiolar artery in guinea pigs].

Chloride channels have been identified in vascular smooth muscle cells (SMCs). It has been shown that these channels are involved in myogenic tone regulation and neuromuscular transmission in various vascular beds. However, whether the chloride channels are responsible for the formation of excitatory junction potentials (EJPs) of SMCs in the spiral modiolar artery (SMA) remains unelucidated. In the present study, the effects of chloride channel blockers (niflumic acid, NFA; indanyloxyacetic acid 94, IAA-94; disodium 4, 4'-diisothiocyanatostilbene-2, 2'-disulfonate, DIDS) on EJP were explored in guinea pigs, using intracellular recording techniques on acutely isolated SMA. It was found that EJP was evoked in the majority of the SMCs (75%, n=49) with an adequate electronic stimulation. The amplitude of the EJP was partially blocked (30% approximately 80%) by combined application of alpha(1) receptor antagonist (prazosin) and alpha(2) receptor antagonist (idazoxan) at concentration of up to 1 micromol/L, and P(2x) receptor antagonist (PPADS, 10 approximately 100 micromol/L). NFA (100 micromol/L) could further inhibit the residual EJP in the presence of alpha(1), alpha(2)-adrenergic and P(2x) receptor antagonists. IAA-94 or DIDS not only inhibited the amplitude but also shortened the duration of EJP. Decrease of extracellular chloride concentration from 135.6 mmol/L to 60 mmol/L would enhance EJP. Moreover, IAA-94 (100 micromol/L) and DIDS (200 mumol/L) could reverse the enhancement of EJP by low extracellular Cl(-). NFA (100 micromol/L) could also block the residual depolarizations evoked by norepinephrine (NE, 1 approximately 50 micromol/L). Based on these results, it is inferred that NE could activate a novel adrenoceptor to open the chloride channel on the membrane of the SMCs, leading to a transmembrane Cl(-) current. This current is involved, at least partially, in the formation of EJP.

Adrenergic alpha-Antagonists↗
PubMed2000

Expression and canalicular localization of two isoforms of the ClC-3 chloride channel from rat hepatocytes.

The molecular identities of functional chloride channels in hepatocytes are largely unknown. We examined the ClC-3 chloride channel in rat hepatocytes and found that mRNA for two different isoforms is present. A short form is identical to the previously reported sequence for rat ClC-3, and a long form contains a 176-bp insertion immediately upstream of the translation initiation site. This predicts a 58-amino acid NH(2) terminal insertion. Both long and short form mRNA was expressed in diverse tissues of the rat. Transient transfection of the long form in CHO-K1 cells resulted in currents with an I(-) > B(-) > Cl(-) selectivity sequence, outward rectification, and inactivation at positive voltages. Short form currents had identical ionic selectivity but displayed a more extreme outward rectification and showed no voltage-dependent inactivation. Immunofluorescence and immunoblots localized native ClC-3 preferentially but not exclusively to the canalicular membrane. We have therefore identified a new isoform of rat ClC-3 and shown that expression of both isoforms produces functional channels. In hepatocytes, ClC-3 is located in association with the canalicular membrane.

Animals↗
PubMed1994

Rapid endocytosis of the cystic fibrosis transmembrane conductance regulator chloride channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is found at the apical region of exocrine epithelial cells, both at the cell surface and in an apically localized intracellular compartment. To determine if this internal pool was due to endocytosis, a technique was developed that allows the rate of CFTR internalization from the cell surface to be monitored. A two-step periodate/hydrazide biotinylation procedure was used to derivatize cell surface glycoconjugates. Because both of these steps are required for derivatization and are conducted at 4 degrees C, the inclusion of a 37 degrees C incubation between the treatments resulted in an assay for the internalization of cell surface glycoconjugates. CFTR was found to be targeted to a rapidly recycling endocytic pathway, as approximately 50% of cell surface CFTR was internalized within minutes and unavailable for biotinylation. In contrast, the major glycoproteins of the apical surface were not significantly endocytosed during even longer incubations at 37 degrees C. Elevating cAMP levels either by forskolin or cAMP analogs, which has been shown to activate CFTR chloride channel activity, inhibited CFTR internalization. However, cAMP did not affect the internalization of G551D CFTR, a naturally occurring Gly-551-->Asp mutant that is expressed at the cell surface but lacks normal ion-channel function. In addition, the inhibition by cAMP of CFTR was not observed when cells were depleted of cellular chloride. The presence of CFTR in epithelial cells had previously been shown to confer a cAMP-mediated inhibition on the rate of fluid-phase endocytosis. This effect was not seen in chloride-depleted cells, suggesting that CFTR's ion-channel function and localization to incipient endosomes may be responsible for the observed inhibition. The finding that CFTR is targeted to the endocytic pathway may provide insight into the role of CFTR in normal exocrine function. In addition, these findings suggest that the expression of a regulated ion channel in a membranous subcellular compartment provides a mechanism by which a cell can regulate vesicular trafficking through that compartment.

Animals↗
PubMed1987

Characteristics of apical chloride channels in human colon cells (HT29).

Recent studies have demonstrated that active chloride secretion in mammalian colon and other epithelia, is dependent on the induction of an increase of apical chloride conductance. Since the physical characteristics of apical chloride channels in man have not been elucidated, patch clamp analysis of human colon cells (HT29), in culture, was performed, after stimulation with db-cAMP 10(-4) mol/l. In excised inside out patches of apical membranes two types of channels were found. The smaller and less frequent channel had a mean conductance of 15 +/- 1 pS (n = 9). This type of channel showed identical I/V curves in NaCl and KCl solutions. It was inhibited by a chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). The more frequently observed larger conductance channel was selective for anions and was impermeable to Na+ and K+. Regarding anion selectivity, the channel was similarly permeable to Cl-, Br-, I-, and NO3-, but was impermeable to gluconate. The channel was completely inhibited by the potent Cl- channel blocker NPPB (10(-6) mol/l). This channel exhibited rectification: The conductance was 50 +/- 4 pS at positive clamp potentials (sign referred to bath with respect to pipette interior) and 32 +/- 3 (n = 33) pS at negative voltages. Moreover, the open state probability was doubled when the clamp potential was increased from -20 to +20 mV. These results demonstrate the existence of chloride channels in the apical membrane of db-cAMP treated colonic carcinoma cells.

Chlorides↗
PubMed2001

Non-CFTR chloride channels likely contribute to secretion in the murine small intestine.

While most cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-knockout animals die due to intestinal obstruction before or at the time of weaning, a subpopulation of these animals are long living and exhibit a milder phenotype. The decreased severity of intestinal disease in these mildly affected CF mice is related to the expression of non-CFTR genetic modifiers. The identity of these genetic modifiers is not known, but we hypothesize that they may complement CFTR function as a chloride channel in this tissue. To assess the contribution of non-CFTR chloride channels to chloride secretion across the small intestine of CF mice with mild disease, we measured the basal transepithelial potential difference across this tissue as well as the secretory response to agonists of the cAMP and the calcium-mediated signaling pathways. Chloride secretion across the small intestine of mildly affected CF mice was not stimulated by forskolin or by carbachol. The absence of CFTR is thus not compensated by the activity of a distinct, cAMP- or calcium-activated chloride channel at the apical surface of the intestinal epithelium. On the other hand, a basal chloride secretion across the intestinal epithelium was present in these animals, and we hypothesize that this activity may be linked to improved survival of these animals.

Angiogenesis Inhibitors↗
PubMed2005

The functioning of mammalian ClC-2 chloride channel in Saccharomyces cerevisiae cells requires an increased level of Kha1p.

The mammalian chloride channel ClC-2 is a member of the CLC voltage-gated chloride channels family. This broadly expressed protein shows diverse cellular locations and despite numerous studies, its precise function is poorly understood. Disruption of ClC-2-encoding gene in mouse leads to retinal and testicular degeneration and mutations in CLC2 (gene encoding the ClC-2 channel) are associated with idiopathic generalized epilepsies. ClC-2 may also be responsible for Cl- transport in mouse salivary glands. The only CLC homologue of the yeast Saccharomyces cerevisiae, Gef1p, exhibits CLC activity. We expressed the mammalian ClC-2 protein in S. cerevisiae devoid of Gef1p in an attempt to identify yeast proteins influencing the functioning of ClC-2. The presence of such proteins in yeast could indicate the existence of their homologues in mammalian cells and would greatly aid their identification. Expression of ClC-2 in yeast required optimization of the sequence context of the AUG translation initiation codon. After obtaining an efficient translation, we found that rat ClC-2 cannot directly substitute for yeast Gef1p. Functional substitution for Gef1p was, however, achieved in the presence of an increased level of intact or C-terminally truncated yeast Kha1 protein. Based on the deduced amino acid sequence, the Kha1 protein can be classified as a Na+/H+ transporter since it has a large N-terminal domain similar to the family of NHEs (Na+/H+ exchangers). This suggests that the Kha1p may take part in the regulation of intracellular cation homoeostasis and pH control. We have established that Kha1p is localized in the same cellular compartment as Gef1p and yeast-expressed ClC-2: the Golgi apparatus. We propose that Kha1p may aid ClC-2-dependent suppression of the Deltagef1-associated growth defects by keeping the Golgi apparatus pH in a range suitable for ClC-2 activity. The approach employed in the present study may be of general applicability to the characterization of poorly understood proteins by their functional expression in yeast.

Animals↗
PubMed2004

Identification of an N-terminal amino acid of the CLC-3 chloride channel critical in phosphorylation-dependent activation of a CaMKII-activated chloride current.

CLC-3, a member of the CLC family of chloride channels, mediates function in many cell types in the body. The multifunctional calcium-calmodulin-dependent protein kinase II (CaMKII) has been shown to activate recombinant CLC-3 stably expressed in tsA cells, a human embryonic kidney cell line derivative, and natively expressed channel protein in a human colonic tumour cell line T84. We examined the CaMKII-dependent regulation of CLC-3 in a smooth muscle cell model as well as in the human colonic tumour cell line, HT29, using whole-cell voltage clamp. In CLC-3-expressing cells, we observed the activation of a Cl(-) conductance following intracellular introduction of the isolated autonomous CaMKII into the voltage-clamped cell via the patch pipette. The CaMKII-dependent Cl(-) conductance was not observed following exposure of the cells to 1 microm autocamtide inhibitory peptide (AIP), a selective inhibitor of CaMKII. Arterial smooth muscle cells express a robust CaMKII-activated Cl(-) conductance; however, CLC-3(-/-) cells did not. The N-terminus of CLC-3, which contains a CaMKII consensus sequence, was phosphorylated by CaMKII in vitro, and mutation of the serine at position 109 (S109A) abolished the CaMKII-dependent Cl(-) conductance, indicating that this residue is important in the gating of CLC-3 at the plasma membrane.

Animals↗
PubMed1992

Modification of gating of an airway epithelial chloride channel by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB).

5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) has been shown to produce a reduction in channel open probability in a number of epithelial chloride channels. We have investigated its action on a calcium-dependent airway epithelial chloride channel, which shows voltage-dependent gating following incorporation into planar phospholipid bilayers. Since the channel demonstrates a distinct subconductance state at approximately one-third of the fully open level, both 50 and 20% threshold analysis (TA) have been used to describe channel kinetics. Lifetime analysis using 50% TA in the absence of NPPB showed that two open and three closed lifetimes provided the optimal fit. Similar analysis using 20% TA required only two closed lifetimes and allowed for tentative assignment of tau values to either substate or fully closed events. NPPB (10-100 microM) produced a concentration-related shift of the normal voltage dependence of gating, with more negative holding potentials being required to produce a given channel open probability than in the absence of NPPB. Ten microM NPPB produced a similar shift in lifetime values. However, addition of 50 microM NPPB produced a unique, single open lifetime. No evidence was found for NPPB acting as a direct voltage-dependent blocker of chloride conductance.

Animals↗
PubMed1991

The nucleotide sequence of a voltage-gated chloride channel from the electric organ of Torpedo californica.

The cDNA encoding the voltage-gated chloride channel from the electric organ of Torpedo californica has been isolated and sequenced. The 2.7 kilobase pair cDNA encodes an 810 amino acid polypeptide which is highly homologous at both the DNA (97%) and amino acid (97%) levels to the voltage-gated chloride channel from the electric organ of T. marmorata. The majority of the 24 amino acid differences between the T. californica and T. marmorata voltage-gated chloride channels are clustered in two putative cytoplasmic domains with six differences located between residues 10-92 and 14 differences occurring between residues 576 to 708. Only one amino acid difference occurs in one of the predicted transmembrane domains. The most dramatic difference is an insertion of Asp-Val-Pro-Gly in a large cytoplasmic domain at amino acid residue 627 of the T. californica channel.

Amino Acid Sequence↗
PubMed1989

Properties of single chloride channels in primary neuronal cultures of Drosophila.

An anion channel from Drosophila neurons had the selectivity sequence: NO3- (1.97) greater than Br- (1.12) approximately equal to I- (1.03) approximately equal to Cl- (1) greater than F- (0.32) much greater than glutamate (less than 0.02) as estimated by the permeability ratio based on the reversal potential measurement. 4-Acetoamido-4'-isothiocyanostilbene-2,2'-disulfonic acid applied internally at 10 microM blocked the channel. We suggest that the chloride channel identified here may provide a pathway for Cl- in the resting membrane of Drosophila neurons.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed2000

Biophysical properties of ClC-3 differentiate it from swelling-activated chloride channels in Chinese hamster ovary-K1 cells.

ClC-3 is a highly conserved voltage-gated chloride channel, which together with ClC-4 and ClC-5 belongs to one subfamily of the larger group of ClC chloride channels. Whereas ClC-5 is localized intracellularly, ClC-3 has been reported to be a swelling-activated plasma membrane channel. However, recent studies have shown that native ClC-3 in hepatocytes is primarily intracellular. Therefore, we reexamined the properties of ClC-3 in a mammalian cell expression system and compared them with the properties of endogenous swelling-activated channels. Chinese hamster ovary (CHO)-K1 cells were transiently transfected with rat ClC-3. The resulting chloride currents were Cl(-) > I(-) selective, showed extreme outward rectification, and lacked inactivation at positive voltages. In addition, they were insensitive to the chloride channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and were not inhibited by phorbol esters or activated by osmotic swelling. These properties are identical to those of ClC-5 but differ from those previously attributed to ClC-3. In contrast, nontransfected CHO-K1 cells displayed an endogenous swelling-activated chloride current, which was weakly outward rectifying, inactivated at positive voltages, sensitive to NPPB and DIDS, and inhibited by phorbol esters. These properties are identical to those previously attributed to ClC-3. Therefore, we conclude that when expressed in CHO-K1 cells, ClC-3 is an extremely outward rectifying channel with similar properties to ClC-5 and is neither activated by cell swelling nor identical to the endogenous swelling-activated channel. These data suggest that ClC-3 cannot be responsible for the swelling-activated chloride channel under all circumstances.

Animals↗
PubMed1996

Blockade of swelling-induced chloride channels by phenol derivatives.

1. In NIH3T3 fibroblasts, the chloride channel involved in regulatory volume decrease (RVD) was identified as ICln, a protein isolated from a cDNA library derived from Madin Darby canine Kidney (MDCK) cells. ICln expressed in Xenopus laevis oocytes gives rise to an outwardly rectifying chloride current, sensitive to the extracellular addition of nucleotides and the known chloride channel blockers, DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) and NPPB (5-nitro-2-(3-phenylpropylamino)-benzoic acid). We set out to study whether substances structurally similar to NPPB are able to interfere with RVD. 2. RVD in NIH3T3 fibroblasts and MDCK cells is temperature-dependent. 3. RVD, the swelling-dependent chloride current and the depolarization seen after reducing extracellular osmolarity can be blocked by gossypol and NDGA (nordihydroguaiaretic acid), both structurally related to NPPB. 4. The cyclic AMP-dependent chloride current elicited in CaCo cells is less sensitive to the two substances tested while the calcium-activated chloride current in fibroblasts is insensitive. 5. The binding site for the two phenol derivatives onto ICln seems to be distinct but closely related to the nucleotide binding site identified as G x G x G, a glycine repeat located at the predicted outer mouth of the ICln channel protein.

3T3 Cells↗
PubMed2000

Fast and slow gating relaxations in the muscle chloride channel CLC-1.

Gating of the muscle chloride channel CLC-1 involves at least two processes evidenced by double-exponential current relaxations when stepping the voltage to negative values. However, there is little information about the gating of CLC-1 at positive voltages. Here, we analyzed macroscopic gating of CLC-1 over a large voltage range (from -160 to +200 mV). Activation was fast at positive voltages but could be easily followed using envelope protocols that employed a tail pulse to -140 mV after stepping the voltage to a certain test potential for increasing durations. Activation was biexponential, demonstrating the presence of two gating processes. Both time constants became exponentially faster at positive voltages. A similar voltage dependence was also seen for the fast gate time constant of CLC-0. The voltage dependence of the time constant of the fast process of CLC-1, tau(f), was steeper than that of the slow one, tau(s) (apparent activation valences were z(f) approximately -0. 79 and z(s) approximately -0.42) such that at +200 mV the two processes became kinetically distinct by almost two orders of magnitude (tau(f) approximately 16 micros, tau(s) approximately 1 ms). This voltage dependence is inconsistent with a previously published gating model for CLC-1 (Fahlke, C., A. Rosenbohm, N. Mitrovic, A.L. George, and R. Rüdel. 1996. Biophys. J. 71:695-706). The kinetic difference at 200 mV allowed us to separate the steady state open probabilities of the two processes assuming that they reflect two parallel (not necessarily independent) gates that have to be open simultaneously to allow ion conduction. Both open probabilities could be described by Boltzmann functions with gating valences around one and with nonzero "offsets" at negative voltages, indicating that the two "gates" never close completely. For comparison with single channel data and to correlate the two gating processes with the two gates of CLC-0, we characterized their voltage, pH(int), and [Cl](ext) dependence, and the dominant myotonia inducing mutation, I290M. Assuming a double-barreled structure of CLC-1, our results are consistent with the identification of the fast and slow gating processes with the single-pore and the common-pore gate, respectively.

Animals↗