Cl- channels in basolateral renal medullary vesicles XI. rbClC-Ka cDNA encodes basolateral MTAL Cl- channels.
Cl- channels in basolateral renal medullary vesicles XI. rbClC-Ka cDNA encodes basolateral MTAL Cl- channels.
复制标题
基底外侧肾髓质囊泡中的 Cl- 通道 XI。
DOI：
10.1152/ajprenal.1996.270.6.f1066
复制
发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Andreoli,TE
中科院分区：
文献类型：
--
作者：
Zimniak,L;Winters,CJ;Reeves,WB;Andreoli,TE
本实验研究是否rbClC-Ka，CIC家族氯通道的cDNA从兔外髓质，编码基底外侧膜氯通道介导净髓厚升肢（MTAL）氯吸收。MTAL细胞含有Cl-通道，其具有某些特性，使其成为该节段中基底外侧Cl-通道的合理候选者。在性质中特别相关的是细胞溶质Cl-在2-25 mM范围内增加激活这些Cl-通道的事实。培养的小鼠MTAL细胞在rbCIC-Ka特异性反义寡核苷酸或与rbCIC-Ka无互补性的随机寡核苷酸存在下生长。通过将来自这些细胞制备的膜囊泡的Cl-通道掺入脂双层的频率以及使用rbClC-ka蛋白的COOH末端的抗血清进行蛋白质印迹分析来评估Cl-通道的丰度。使用来自未处理细胞或用随机寡核苷酸处理的细胞的囊泡，Cl-通道分别在17%和16%的试验中并入双层中。然而，当从用反义寡核苷酸预处理的细胞中制备囊泡时，Cl-通道掺入双层中的虚拟废除，但对K+通道掺入的频率没有影响。与Cl-通道掺入的减少平行，在Western印迹上rbClC-Ka蛋白的丰度减少约50%。最后，暴露于脂双层中的Cl-通道的rbClC-Ka抗血清导致通道活性的阻断。这些结果支持以下论点：介导MTAL中净Cl-吸收的基底外侧Cl-通道由rbClC-Ka编码。
The present experiments examined whether rbClC-Ka, a CIC family Cl-channel cDNA from rabbit outer medulla, encodes a basolateral membrane Cl- channel mediating net medullary thick ascending limb (MTAL) Cl- absorption. MTAL cells contain a Cl- channel having certain properties that make it a plausible candidate for the basolateral Cl- channel in that segment. Especially pertinent among properties is the fact that cytosolic Cl- increases in the range 2-25 mM activated these Cl- channels. Cultured mouse MTAL cells were grown in the presence of an antisense oligonucleotide specific for rbCIC-Ka or a random oligonucleotide with no complementarity to rbCIC-Ka. The abundance of Cl- channels was assessed by the frequency of incorporation of Cl- channels from membrane vesicles prepared from these cells into lipid bilayers and by Western blot analysis using an antiserum to the COOH terminus of the rbClC-ka protein. With the use of vesicles from untreated cells or cells treated with the random oligonucleotide, Cl- channels were incorporated into bilayers in 17% and 16% of trials, respectively. However, when vesicles were prepared from cells pretreated with antisense oligonucleotide, there was a virtual abolition of Cl- channel incorporation into bilayers but no effect on the frequency of K+ channel incorporation. In parallel with the reduction in Cl- channel incorporation, the abundance of rbClC-Ka protein was reduced approximately 50% on Western blots. Finally, exposure of Cl- channels in lipid bilayers to the rbClC-Ka antiserum resulted in a block in channel activity. These results support the contention that the basolateral Cl- channel mediating net Cl- absorption in the MTAL is encoded by rbClC-Ka.