Tousson A, Alley C D, Sorscher E J, Brinkley B R, Benos D J
Department of Cell Biology and Anatomy, University of Alabama, Birmingham 35294.
J Cell Sci. 1989 Jun;93 ( Pt 2):349-62. doi: 10.1242/jcs.93.2.349.
The localization of amiloride-sensitive Na+ channels in Na+-transporting epithelia was examined using antibodies made against amiloride-binding Na+ channel protein purified from bovine kidney. The distribution of the channel protein was determined in thick frozen sections at the light-microscopic level using indirect immunofluorescence, and at the electron-microscopic level using immunogold labelling. In the cells of both the intact bovine collecting tubule and A6 confluent monolayers, only the luminal or apical-facing surface membranes showed staining. Sodium channel protein was characteristically localized on microvillar domains of the apical plasma membrane. Little or no basolateral membrane staining was evident. Channel protein was also absent from subapical vesicles and tight junctions, and was not found in bovine renal proximal tubules, cultured human secretory sweat coils, non-epithelial Chinese hamster ovary (CHO) cells or human skin fibroblasts. Trypsinization of intact A6 monolayers prior to cell fixation abolished specific staining with antibody. Pretreatment with amiloride protected against this loss of staining. Thus, our probes are specific for amiloride-binding Na+ channel protein, and this channel protein is largely or completely confined to the apical membrane of Na+-transporting epithelia. The level and distribution of specific immunostaining in A6 cells was unchanged by aldosterone treatment, although channel activity, as measured by short-circuit current, increased threefold. This result demonstrates that Na+ channel protein is ever present at the cell surface and exists in both an active and an inactive form. We find no evidence that stimulation of Na+ uptake by aldosterone involves recruitment of new channels from a cytoplasmic pool.
使用针对从牛肾中纯化的氨氯地平结合性钠离子通道蛋白制备的抗体，研究了氨氯地平敏感性钠离子通道在钠离子转运上皮细胞中的定位。通过间接免疫荧光在光学显微镜水平上，以及通过免疫金标记在电子显微镜水平上，确定了厚冰冻切片中通道蛋白的分布。在完整的牛集合小管细胞和A6汇合单层细胞中，只有腔面或顶面的表面膜呈现染色。钠离子通道蛋白典型地定位于顶质膜的微绒毛区域。基底外侧膜几乎没有或没有明显染色。亚顶端小泡和紧密连接中也没有通道蛋白，在牛肾近端小管、培养的人分泌性汗腺蟠管、非上皮性中国仓鼠卵巢（CHO）细胞或人皮肤成纤维细胞中也未发现。在细胞固定之前对完整的A6单层细胞进行胰蛋白酶消化消除了抗体的特异性染色。用氨氯地平预处理可防止这种染色损失。因此，我们的探针对于氨氯地平结合性钠离子通道蛋白具有特异性，并且这种通道蛋白主要或完全局限于钠离子转运上皮细胞的顶膜。尽管通过短路电流测量的通道活性增加了三倍，但醛固酮处理并未改变A6细胞中特异性免疫染色的水平和分布。这一结果表明，钠离子通道蛋白始终存在于细胞表面，并且以活性和非活性两种形式存在。我们没有发现证据表明醛固酮刺激钠离子摄取涉及从细胞质池中募集新的通道。
