Winters C J, Zimniak L, Reeves W B, Andreoli T E
Department of Internal Medicine, University of Arkansas College of Medicine, Little Rock, USA.
Am J Physiol. 1997 Dec;273(6):F1030-8. doi: 10.1152/ajprenal.1997.273.6.F1030.
Cl- channels in the medullary thick ascending limb (MTAL) studied by either patch-clamp technique or reconstitution into lipid bilayers are activated by increases in intracellular Cl- concentrations. rbClC-Ka, a ClC Cl- channel, may represent this channel. We therefore evaluated the role of rbClC-Ka in transcellular MTAL Cl- transport in two separate ways. First, an antibody was raised against a fusion protein containing a 153-amino acid fragment of rbClC-Ka. Immunostaining of rabbit kidney sections with the antibody was localized to basolateral regions of MTAL and cortical thick ascending limb (CTAL) segments and also to the cytoplasm of intercalated cells in the cortical collecting duct. Second, Cl- uptake and efflux were measured in suspensions of mouse MTAL segments. Cl- uptake was bumetanide sensitive and was stimulated by treatment with a combination of vasopressin + forskolin + dibutyryl adenosine 3',5-cyclic monophosphate (DBcAMP). Cl- efflux was also increased significantly by vasopressin + forskolin + DBcAMP from 114 +/- 20 to 196 +/- 36 nmol.mg protein-1.45 s-1 (P = 0.003). Cl- efflux was inhibited by the Cl- channel blocker diphenylamine-2-carboxylate (154 +/- 26 vs. 70 +/- 21 nmol.mg protein-1.45 s-1, P = 0.003). An anti-rbClC-Ka antibody, which inhibits the activity of MTAL Cl- channels in lipid bilayers, reduced Cl- efflux from intact MTAL segments (154 +/- 28 vs. 53 +/- 14 nmol.mg protein-1.45 s-1, P = 0.02). These results support the view that rbClC-Ka is the basolateral membrane Cl- channel that mediates vasopressin-stimulated net Cl- transport in the MTAL segment.
采用膜片钳技术或重构成脂质双分子层的方法对髓袢升支粗段（MTAL）中的氯离子通道进行研究时发现，细胞内氯离子浓度升高可激活这些通道。rbClC-Ka，一种氯离子通道蛋白，可能代表该通道。因此，我们通过两种不同方法评估了rbClC-Ka在MTAL跨细胞氯离子转运中的作用。首先，制备了一种针对含有rbClC-Ka 153个氨基酸片段的融合蛋白的抗体。用该抗体对兔肾切片进行免疫染色，结果显示其定位于MTAL和皮质升支粗段（CTAL）节段的基底外侧区域，以及皮质集合管闰细胞的细胞质中。其次，在小鼠MTAL节段悬液中测量氯离子的摄取和流出。氯离子摄取对布美他尼敏感，并且血管加压素+福斯可林+二丁酰腺苷3',5-环磷酸（DBcAMP）联合处理可刺激其摄取。血管加压素+福斯可林+DBcAMP也使氯离子流出量从114±20显著增加至196±36 nmol·mg蛋白-1·45 s-1（P = 0.003）。氯离子通道阻滞剂二苯胺-2-羧酸盐可抑制氯离子流出（154±26与70±21 nmol·mg蛋白-1·45 s-1，P = 0.003）。一种抑制脂质双分子层中MTAL氯离子通道活性的抗rbClC-Ka抗体，可减少完整MTAL节段的氯离子流出（154±28与53±14 nmol·mg蛋白-1·45 s-1，P = 0.02）。这些结果支持了这样一种观点，即rbClC-Ka是基底外侧膜氯离子通道，介导血管加压素刺激的MTAL节段净氯离子转运。
