Concentration and pH dependence of skeletal muscle chloride channel ClC‐1.

Sertoli cells from mammalian testis are key cells involved in development and maintenance of stem cell spermatogonia as well as secretion of a chloride-and potassium-rich fluid into the lumen of seminiferous tubules. Using whole-cell patch clamp experiments, a novel chloride current was identified. It is activated only in the presence of an extracellular acidic pH, with an estimated half-maximal activation at pH 5.5. The current is strongly outwardly rectifying, activated with a fast time-dependent onset of activation but a slow timedependent kinetic at depolarization pulses. The pH-activated chloride current was not detected at physiological or basic pH and is not sensitive to intracellular or extracellular Ca 2؉ variation. Diphenylamine-2-carboxylic acid and 4,4-diisothiocyanatostilbene-2,2-disulfonic acid blocked the induced currents, and its anionic selectivity sequence was Cl ؊ > Br ؊ > I ؊ > gluconate. We have performed a reverse transcription-PCR analysis to search for voltage-dependent chloride rClC channels in cultured rat Sertoli cells. Among the nine members of the family only rClC-2, rClC-3, rClC-6, and rClC-7 have been identified. The inwardly rectifying rClC-2 chloride current was activated by hyperpolarization but not by pH variation. A different depolarization-activated outwardly rectifying chloride current was activated only by hypotonic challenge and may correspond either to rClC-3 or rClC-6. Immunolocalization experiments demonstrate that rClC-7 resides in the intracellular compartment of Sertoli cells. This study provides the first functional identification of a native acid-activated chloride current. Based on our molecular analysis of rClC proteins, this new chloride current does not correspond to rClC-2, rClC-3, rClC-6, or rClC-7 channels. The potential physiological role of this native current in an epithelial cell from the reproductive system is discussed.

Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

A Novel Voltage-dependent Chloride Current Activated by Extracellular Acidic pH in Cultured Rat Sertoli Cells

Auzanneau

Thoreau

Kitzis

et al. 2003

“…From the pK a and Hill coefficient of such titration curves, we hoped to infer the most likely sort and number of residues participating in the block. Changes of pH alone have profound effects on the gating of hClC-1, whereas outward current amplitudes do not change considerably (20,21). Block of whole-cell currents was tested by applying repetitive pulses to ϩ50 mV (for maximum channel activation), followed by a hyperpolarizing step at Ϫ125 mV to monitor the deactivation time course (Fig.…”

Section: Dependence Of Chloride Current Block By Znmentioning

confidence: 99%

Identification of Three Cysteines as Targets for the Zn2+ Blockade of the Human Skeletal Muscle Chloride Channel

Kürz

Klink

Jakob

et al. 1999

Currents through the human skeletal muscle chloride channel hClC-1 can be blocked by external application of 1 mM Zn 2؉ or the histidine-reactive compound diethyl pyrocarbonate (DEPC). The current block by Zn 2؉ strongly depends on the external pH (pK a near 6.9), whereas the block by DEPC is rather independent of the pH in the range of 5.5 to 8.5. To identify the target sites of these reagents, we constructed a total of twelve cysteine-and/or histidine-replacement mutants, transfected tsA201 cells with them, and investigated the resulting whole-cell chloride currents. The majority of the mutants exhibited a similar sensitivity toward Zn 2؉ or DEPC as wild type (WT) channels. Block by 1 mM Zn 2؉was nearly absent only with the mutant C546A. Four mutants (C242A, C254A, H180A, and H451A) were slightly less sensitive to Zn 2؉ than WT. Tests with double, triple, and quadruple mutants yielded that, in addition to C546, C242 and C254 are also most likely participating in Zn 2؉ -binding.The main chloride channel of human skeletal muscle, hClC-1, 1 is a member of the ClC chloride channel family that is unrelated to any other known ion channels (1). The first membrane topology model of ClC proteins was derived from hydropathy analysis (2); several times it had to be revised on the grounds of firmer experimental evidence (3-5). Certain mutations in the human ClC-1 gene (reviewed in Ref. 6) lead to myotonia, a disease characterized by muscle stiffness. The study of such myotonia-causing mutations provided first insights into the relationships between the primary sequence and the functions of this channel (7-10). Strong evidence suggests regions between transmembrane segments D3 and the end of D5 to participate in the pore-forming structure (11). However, these results are not in agreement with the most recently postulated topology model of ClC-1 (5). Furthermore, studies of ClC-0 (reviewed in Ref. 1), a homologous chloride channel in the electric organ of Torpedo, led to the suggestion of additional protein parts being involved in forming the ion conducting pathway.ClC channels most likely consist of two subunits (12), and in the case of ClC-0, each subunit was proposed to contain a single pore (4,13,14). Recent evidence speaks against this "doublebarreled" channel model, at least in the case of ClC-1 (15). We have found earlier that the exposure of hClC-1, stably expressed in HEK-293 cells, to 1 mM Zn 2ϩ leads to a massive reduction of the conducted chloride current (16). The results were compatible with the presence of at least two extracellularly accessible zinc-binding sites and a direct effect on ion permeation, e.g. by obstruction of the pore. The histidyl-reactive diethyl pyrocarbonate (DEPC), applied from the outside, also reduced the currents through hClC-1. The aim of this study was to identify the target residues for these blockers and, thereby, to draw inferences regarding the membrane topology and the potential location of pore-forming structures. It is known from other proteins, that Zn 2ϩ binding sites are of...

“…Data were collected, filtered at 3 kHz, and analyzed on an IBM-compatible PC using pCLAMP v6.0 software (Axon Instruments). Parameters such as time constants, apparent open probability, and IC 50 were determined as described previously (16,17). …”

mentioning

confidence: 99%

Relevance of the D13 Region to the Function of the Skeletal Muscle Chloride Channel, ClC-1

Hryciw¹,

Rychkov²,

Hughes³

et al. 1998

Although hydropathy analysis of the skeletal muscle chloride channel protein, ClC-1, initially predicted 13 potential membrane spanning domains (D1 to D13), later topological studies have suggested that domain D4 is extracellular and that D13, conserved in all eukaryotic ClC channels, is located within the extensive cytoplasmic tail that makes up the carboxyl terminus of the protein. We have examined the effect of deleting D13 (⌬D13) and the function of the carboxyl tail by removing the final 72 (fs923X), 100 (fs895X), 125 (L869X), 398 (N596X), and 420 (Q574X) amino acids from rat ClC-1. Appropriate cDNA constructs were prepared and expressed using the baculovirus Sf9 insect cell system. Patch clamp analysis of chloride currents in Sf9 cells showed that only relatively insubstantial changes could be attributed to the expressed fs923X, fs895X, and ⌬D13 mutants compared with wild type rat ClC-1. For N596X and Q574X, however, adequate mRNA could be detected, but neither patch clamp nor polyacrylamide gel electrophoresis showed corresponding protein production. By contrast, expression of L869X was demonstrable by polyacrylamide gel electrophoresis, but no chloride conductance attributable to it could be detected. Overall, our results indicate that the domain D13 is dispensable, as are the final 100 amino acids, but not the final 125 amino acids or more, of the carboxyl tail. Some essential region of unknown significance, therefore, appears to reside in the 18 amino acids after D13, from Lys 877 to Arg 894 .In mammalian skeletal muscle, the voltage-gated chloride channel, ClC-1, is responsible for the greater proportion of the resting conductance, which acts to stabilize the membrane potential against unwanted perturbations. Its structure, as predicted by hydropathy analysis (1) and in common with other members of the large ClC family, includes 13 relatively hydrophobic domains (termed D1 to D13), most of which probably span the membrane. There is good evidence, however, that D4 and D13 are located entirely extracellularly and intracellularly, respectively (2, 3), with the implication that close to half the amino acids of human ClC-1 (hClC-1) 1 must form an extensive cytoplasmic tail (Gln 574 -Leu 988 ). The D13 domain, which is conserved among all eukaryotic ClC channels (4), is situated in this tail, about two-thirds of the way to the carboxyl terminus (Leu 840 -Ile 870 ). Mutations in ClC-1 have been associated with both dominant and recessive forms of myotonia (e.g. Refs. 5 and 6) characterized by abnormal, sustained firing of action potentials that result in prolonged involuntary muscle contractions. In one (R894X) of two myotonic mutations identified in the carboxyl tail (7, 8) the final 95 amino acids of the protein (8) are lost. Functional analysis of this mutant, expressed in Xenopus oocytes, showed a large reduction in chloride currents, which could account for the myotonic symptoms (9). In similar studies (4), chloride currents were totally lost from human ClC-1 truncated at Ser 720 (G721X) and at Gln 597 ...