A Novel voltage-dependent chloride current activated by extracellular acidic pH in cultured rat Sertoli cells.
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 time-dependent 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 Ca2+ 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.
Auzanneau
Céline
C
Laboratoire des Biomembranes et Signalisation Cellulaire CNRS UMR 6558, Université de Poitiers, 40 Avenue du Recteur Pineau, Poitiers, France.
Thoreau
Vincent
V
Kitzis
Alain
A
Becq
Frédéric
F
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D000090007:CLC-2 Chloride Channels; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D018118:Chloride Channels / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D002712:Chlorides / Q000378:metabolism; D004553:Electric Conductivity; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007038:Hypotonic Solutions; D008297:Male; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012708:Sertoli Cells / Q000737:chemistry / Q000502:physiology*; D014162:Transfection; D014982:Xenopus laevis; D062367:ortho-Aminobenzoates / Q000494:pharmacology
D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels; D002712:Chlorides; C115376:ClC-3 channel; C490801:Clcn6 protein, rat; C490802:Clcn7 protein, rat; D007038:Hypotonic Solutions; D012333:RNA, Messenger; D062367:ortho-Aminobenzoates; C001209:fenamic acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium