闰细胞去极化激活电流的发现与鉴定

Recording and identification of depolarization-activated current in intercalated cells

本实验室首次在肾脏远端肾单位闰细胞记录到去极化激活电流,并根据电生理学和药理学特征鉴定其离子通道的类型。用Axon Multi Clamp 700B膜片钳系统记录C57BL/6J小鼠肾脏远端肾单位肾小管细胞全细胞电流,并观察钾通道抑制剂对闰细胞去极化激活电流的影响。此外,应用免疫荧光技术研究介导该电流的离子通道的具体定位。结果显示,当细胞外液为等钾溶液时,可在闰细胞记录到去极化激活电流,但该去极化激活电流未在主细胞观察到。在远端肾单位闰细胞记录到的去极化激活电流能被电压门控钾通道Kv4.1抑制剂阻断。Kv4.1蛋白免疫荧光只存在于闰细胞,未在主细胞观察到。Kv4.1蛋白免疫荧光可见于闰细胞的管腔膜和管周膜,但管腔膜的荧光强度高于管周膜。由此得出结论,闰细胞去极化激活电流由Kv4.1钾通道介导,该通道主要表达在闰细胞的管腔膜上。

The depolarization-activated current of intercalated cells in the distal nephron was detected for the first time,and the type of ion channel mediating the current was identified based on electrophysiological and pharmacological properties.The whole-cell current of distal nephron in kidney of C57BL/6J mice was recorded by Axon MultiClamp 700B patch-clamp system,and the effects of several K+channel inhibitors on the depolarization-activated current in intercalated cells were observed.In addition,the immunofluorescence technique was used to investigate the localization of the channel in intercalated cells.The results showed that when K+concentration of the bath solution was equal to intracellular fluid(140 mmol/L K+),the depolarization-activated current could be recorded in intercalated cells,but this current was not observed in the principal cells.The depolarization-activated current detected in the intercalated cells could be blocked by Kv4.1 inhibitors.The immunofluorescence experiment showed that the fluorescence of Kv4.1 protein was only present in intercalated cells and not observed in principal cells.Kv4.1 protein immunofluorescence was observed in the luminal and basolateral membrane of intercalated cells,but the fluorescence intensity of luminal membrane was higher than that of basolateral membrane.We conclude that the depolarization-activated current detected in intercalated cells is mediated by Kv4.1 and this channel is mainly expressed in the luminal membrane of intercalated cells.