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

Abstract

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.

Pharmacological profile of inhibition of the chloride channels activated by extracellular acid in cultured rat Sertoli cells

“…2C) in the presence of 100 µM glibenclamide. Previous work [18] has shown no-Ca 2+ dependent activation of ICl acid . Here, we tested the Ca 2+ effect on ICl acid inhibition.…”

“…The EGTA-calcium buffer was used to hold the intracellular free calcium concentration at 1 µM. The pipette solution was always hypotonic (with respect to the bath solution) to prevent cell swelling and activation of the volumesensitive chloride channels [18]. All experiments were conducted at room temperature (20 • C).…”

“…ns, no significant difference; *** P < 0.001; ND, not determined. and whole cell patch-clamp has revealed the presence of several other Cl − conductances [18]. A molecular and whole cell patch clamp analysis of rat Sertoli cells already identified the following voltagedependent Cl − channels: rClC-2, rClC-3, rClC-6, rClC-7 and a volume-activated Cl − current (referred as ICl swell ) [18].…”

“…Indeed Sertoli cells express a variety of membrane transporters such as Na + /H + and Na + -dependent Cl − /HCO − 3 exchangers and Na + /HCO − 3 co-transporters [9]. Electrophysiological and molecular analysis revealed the expression of several types of ionic channels, among them voltage-dependent Ca 2+ channels [15], Ca 2+ -dependent Cl − channels [16], CFTR Cl − channels [17] and voltage-dependent Cl − channels including rClC-2, rClC-3, rClC-6, rClC-7 [18]. In addition, we recently identified a voltage-dependent Cl − current activated by extracellular acidic pH (hereafter noted ICl acid ) in rat Sertoli cells [18].…”

“…Electrophysiological and molecular analysis revealed the expression of several types of ionic channels, among them voltage-dependent Ca 2+ channels [15], Ca 2+ -dependent Cl − channels [16], CFTR Cl − channels [17] and voltage-dependent Cl − channels including rClC-2, rClC-3, rClC-6, rClC-7 [18]. In addition, we recently identified a voltage-dependent Cl − current activated by extracellular acidic pH (hereafter noted ICl acid ) in rat Sertoli cells [18]. This novel acid-activated Cl − current may be of particular interest for the physiology of Sertoli cells considering the importance of endogenous production of lactate [13,14].…”