Smith P R, Stoner L C, Viggiano S C, Angelides K J, Benos D J
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294, USA.
J Membr Biol. 1995 Sep;147(2):195-205. doi: 10.1007/BF00233547.
We have previously demonstrated that apical Na+ channels in A6 renal epithelial cells are associated with spectrin-based membrane cytoskeleton proteins and that the lateral mobility of these channels, as determined by fluorescence photobleach recovery (FPR) analysis, is severely restricted by this association (Smith et al., 1991. Proc. Natl. Acad. Sci. USA 88:6971-6975). Recent data indicate that the actin component of the cytoskeleton may play a role in modulating Na+ channel activity (Cantiello et al., 1991. Am. J. Physiol. 261:C882-C888); however, it is unknown if the Na+ channel's linkage to the spectrin-based membrane cytoskeleton is also involved in regulating channel activity. In this study, we have used FPR to examine if the linkage of the Na+ channels to the membrane cytoskeleton is a site for modulation of Na+ channel activity in filter grown A6 cells by vasopressin and aldosterone. We hypothesized that if the linkage of the Na+ channels to the membrane cytoskeleton is a site for regulation of Na+ channel activity by vasopressin and aldosterone, then hormone-mediated changes in either the membrane cytoskeleton or the affinity of the Na+ channel for the membrane cytoskeleton, should be reflected in changes in the lateral mobility and/or mobile fraction of Na+ channels on the cell surface. FPR revealed that although the rates of lateral mobility were not affected, there was a twofold increase in mobility fraction (f) of apical Na+ channels in aldosterone-treated (16 hr) monolayers (f = 32.31 +/- 5.42%) when compared to control (unstimulated) (f = 14.2 +/- 0.77%) and vasopressin-treated (20 min) (f = 12.7 +/- 2.4%) monolayers. The twofold increase in mobile fraction of Na+ channels corresponds to the average increase in Na+ transport in response to aldosterone in A6 cells. The aldosterone-induced increase in Na+ transport and mobile fraction can be inhibited by the methylation inhibitor, 3-deazaadenosine, consistent with the hypothesis that a methylation event is involved in aldosterone induced upregulation of Na+ transport. We propose that the membrane cytoskeleton is involved in the aldosterone-mediated activation of epithelial Na+ channels.
我们之前已经证明，A6肾上皮细胞中的顶端钠离子通道与基于血影蛋白的膜细胞骨架蛋白相关联，并且通过荧光光漂白恢复（FPR）分析确定，这些通道的侧向移动性受到这种关联的严重限制（Smith等人，1991年。美国国家科学院院刊88:6971 - 6975）。最近的数据表明，细胞骨架的肌动蛋白成分可能在调节钠离子通道活性中起作用（Cantiello等人，1991年。美国生理学杂志261:C882 - C888）；然而，尚不清楚钠离子通道与基于血影蛋白的膜细胞骨架的连接是否也参与调节通道活性。在本研究中，我们使用FPR来检查在滤膜生长的A6细胞中，钠离子通道与膜细胞骨架的连接是否是血管加压素和醛固酮调节钠离子通道活性的位点。我们假设，如果钠离子通道与膜细胞骨架的连接是血管加压素和醛固酮调节钠离子通道活性的位点，那么激素介导的膜细胞骨架变化或钠离子通道对膜细胞骨架亲和力的变化，应该反映在细胞表面钠离子通道侧向移动性和/或可移动部分的变化中。FPR显示，虽然侧向移动速率不受影响，但与对照（未刺激）（f = 14.2 +/- 0.77%）和血管加压素处理（20分钟）（f = 12.7 +/- 2.4%）单层相比，醛固酮处理（16小时）单层中顶端钠离子通道的移动部分（f）增加了两倍（f = 32.31 +/- 5.42%）。钠离子通道可移动部分增加两倍与A6细胞中醛固酮诱导的钠离子转运平均增加相对应。醛固酮诱导的钠离子转运和可移动部分增加可被甲基化抑制剂3 - 脱氮腺苷抑制，这与甲基化事件参与醛固酮诱导的钠离子转运上调的假设一致。我们提出膜细胞骨架参与醛固酮介导的上皮钠离子通道激活。
