Zimniak L, Winters C J, Reeves W B, Andreoli T E
Department of Internal Medicine, University of Arkansas College of Medicine, Little Rock, Arkansas, USA.
Am J Physiol. 1996 Jun;270(6 Pt 2):F1066-72. doi: 10.1152/ajprenal.1996.270.6.F1066.
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.
本实验研究了兔外髓质的CIC家族氯离子通道cDNA即rbClC-Ka，是否编码一种介导髓袢升支粗段（MTAL）净氯离子吸收的基底外侧膜氯离子通道。MTAL细胞含有一种具有某些特性的氯离子通道，使其成为该节段基底外侧氯离子通道的合理候选者。这些特性中特别相关的一个事实是，胞质氯离子在2-25 mM范围内增加会激活这些氯离子通道。将培养的小鼠MTAL细胞在对rbCIC-Ka特异的反义寡核苷酸或与rbCIC-Ka无互补性的随机寡核苷酸存在下培养。通过从这些细胞制备的膜囊泡中氯离子通道掺入脂质双层的频率以及使用针对rbClC-ka蛋白COOH末端的抗血清进行蛋白质印迹分析来评估氯离子通道的丰度。使用未处理细胞或用随机寡核苷酸处理的细胞的囊泡时，氯离子通道分别在17%和16%的试验中掺入双层。然而，当从用反义寡核苷酸预处理的细胞制备囊泡时，氯离子通道掺入双层几乎完全消失，但对钾离子通道掺入频率没有影响。与氯离子通道掺入减少同时发生的是，蛋白质印迹上rbClC-Ka蛋白的丰度降低了约50%。最后，脂质双层中的氯离子通道暴露于rbClC-Ka抗血清导致通道活性受阻。这些结果支持了以下论点，即介导MTAL中净氯离子吸收的基底外侧氯离子通道由rbClC-Ka编码。
