A novel voltage-dependent chloride current activated by extracellular acidic pH in cultured rat Sertoli cells
A novel voltage-dependent chloride current activated by extracellular acidic pH in cultured rat Sertoli cells
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DOI：
10.1074/jbc.m301096200
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发表时间：
2003-05-23
影响因子：
4.8
通讯作者：
Becq, F
中科院分区：
文献类型：
--
作者：
Auzanneau, C;Thoreau, V;Becq, F
哺乳动物睾丸中的支持细胞是参与干细胞精原细胞发育和维持以及将富含氯和钾的液体分泌到生精小管腔中的关键细胞。通过全细胞膜片钳实验，发现了一种新的氯电流。只有在胞外酸性pH存在的情况下，它才被激活，估计在pH 5.5时激活的一半是最大的。电流是强烈的外向整流，在去极化脉冲下激活具有快速的依赖于时间的激活开始，但具有缓慢的依赖于时间的动力学。在生理或基础pH条件下未检测到pH激活的氯电流，且对细胞内或细胞外钙离子变化不敏感。二苯胺-2-羧酸和4，4‘-二异硫氰基二苯乙烯-2，2’-二磺酸阻断了诱导电流，其阴离子选择性顺序为Cl->；br->；i->；葡萄糖酸。我们进行了逆转录-聚合酶链式反应分析，以寻找培养的大鼠支持细胞中电压依赖的氯离子rClC通道。在该家族的9个成员中，仅鉴定出rClC-2、rClC-3、rClC-6和rClC-7。内向整流的rClC-2氯电流可被超极化激活，但不受pH变化的激活。一种不同的去极化激活的外向整流氯电流仅在低张刺激下被激活，并可能对应于rClC-3或rClC-6。免疫定位实验表明，rClC-7存在于支持细胞的胞内。这项研究首次提供了天然酸激活氯离子电流的功能鉴定。根据我们对rClC蛋白的分子分析，这种新的氯电流与rClC-2、rClC-3、rClC-6或rClC-7通道不对应。讨论了这种天然电流在生殖系统上皮细胞中的潜在生理作用。
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.