EPITHELIAL SODIUM CHANNEL/CYTOSKELETON INTERACTIONS

上皮钠通道/细胞骨架相互作用

• 批准号: 2016679
• 负责人: PETER R. SMITH
• 金额: $ 12.04万
• 依托单位: ALLEGHENY UNIVERSITY OF HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 1998-11-10
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2016679
• 关键词: Xenopus; Xenopus oocyte; animal tissue; ankyrins; apical membrane; cellular polarity; cytoskeleton; epithelium; fluorescent dye /probe; human tissue; immunocytochemistry; intermolecular interaction; intracellular transport; laboratory mouse; laboratory rabbit; membrane structure; renal tubular transport; sodium channel; sodium potassium exchanging ATPase; spectrin; tissue /cell culture

Na+ reabsorbing renal epithelia contain Na+ specific channels situated within their apical membranes. These epithelial Na+ channels mediate entry of Na+ from the luminal fluid into the cell during the first stage of electrogenic transepithelial Na+ transport. Restriction of the Na+ channels to the apical membrane domain is essential for the vectorial transport of Na+ across these epithelia. However, the mechanisms responsible for determining and maintaining the distribution of the Na+ channels to the apical membrane domains of renal Na+ reabsorbing epithelia are presently not understood. Preliminary experiments suggest that epithelial Na+ channels are linked to the membrane cytoskeletal proteins ankyrin and spectrin in renal epithelia. It is hypothesized that this interaction between Na+ channels and the membrane cytoskeleton is involved in the maintenance of the polarized distribution of Na+ channels to the apical membrane. Thus, the primary goal of this proposed study is to investigate the role of the membrane cytoskeletal proteins ankyrin and spectrin in determining and maintaining the apical cell surface distribution of epithelial Na+ channels in Na+ reabsorbing renal epithelia. A6 renal epithelial cells, a cell line derived from the distal tubule of Xenopus laevis will be used as the model system. There are three specific aims:(1) To test the hypothesis that there is a direct interaction between epithelial Na+ channels and the membrane cytoskeletal proteins ankyrin and spectrin, and that this interaction restrict the Na+ channels to the microvillar domain of the apical membrane in Na+reabsorbing renal epithelia using biochemical, immunocytochemical and fluorescence photobleach recovery (FPR) techniques (2) To test the hypothesis that Na+ channel-membrane cytoskeleton interactions are involved in determining the distribution of epithelial Na+ channels to the apical membrane of Na+ reabsorbing renal epithelia by examining differentiation from unpolarized precursors to polarized epithelia cells in A6 cells. (3) To further test the hypotheses that the Na+ channel- membrane cytoskeleton complex identified in Specific Aim 1 is involved in determining and maintaining the distribution of Na+ channels within specific membrane domains, the Xenopus oocyte expression system will be used as an in vitro model. This will be achieved by injecting oocytes with Poly (A)+RNA coding for the epithelial Na+ channel or coinfection of Na+ channel Poly (A)+RNA with either sense or antisense oligonucleotides encoding ankyrin followed by immunochemical and FPR analyses of expressed channels. In addition to elucidating how the apical distribution of this essential channel protein is determined and maintained in renal epithelia, the studies proposed in this application will increase our understanding of how other epithelial ion channels are established and maintained within their respective membrane domains. Furthermore, in light of the recent evidence suggesting that ischemic injury disrupts the membrane cytoskeleton of the renal tubules and their ability to maintain distinct apical and basolateral membrane domains, our studies will increase our understanding of ischemia induced renal failure.

Na+重吸收的肾上皮细胞含有位于 在它们的根尖膜内。这些上皮性Na+通道介导 在第一阶段，Na+从腔液进入细胞 电生跨上皮钠离子转运。钠离子的限制 通向根尖膜域的通道对于 Na+在这些上皮细胞中的运输。然而，这些机制 负责确定和维护Na+的分布 肾脏钠离子重吸收的顶膜区通道 上皮细胞目前还不清楚。初步实验表明 上皮性Na+通道与膜细胞骨架相连 肾上皮细胞中的锚蛋白和血影蛋白。这是假设的 钠离子通道和膜细胞骨架之间的这种相互作用 参与维持Na+的极化分布 通向顶膜的通道。因此，这项提议的主要目标是 研究目的是探讨膜上细胞骨架蛋白的作用 锚蛋白和血影蛋白在决定和维持顶端细胞中的作用 Na+重吸收肾上皮细胞Na+通道的表面分布 上皮细胞。A6肾上皮细胞，一种来源于 以非洲爪哇的远端小管为模型系统。那里 有三个具体目标：(1)检验假设，即存在一个直接的 上皮细胞Na+通道与膜细胞骨架的相互作用 这种相互作用限制了Na+ 根尖膜微绒毛域的通道 肾上皮细胞Na+重吸收的生化、免疫细胞化学和免疫组织化学研究 荧光光漂白回收技术(2)，以测试 钠离子通道-膜细胞骨架相互作用的假说 参与决定上皮细胞Na+通道的分布 肾上皮细胞Na~+重吸收顶膜的检测 非极化前体细胞向极化上皮细胞的分化 在A6细胞中。(3)进一步检验钠离子通道-- 特异靶蛋白1中鉴定的膜细胞骨架复合体参与 在确定和维持体内Na+通道的分布中 特定的膜结构域，非洲爪哇卵母细胞表达系统将 用作体外模型。这将通过注射卵母细胞来实现。 用Poly(A)+RNA编码上皮性Na+通道或合并感染 正反义Na+通道Poly(A)+RNA的研究 编码锚蛋白的寡核苷酸，然后是免疫化学和FPR 对表达渠道的分析。除了阐明 这一基本通道蛋白的顶端分布被确定并且 维持在肾上皮细胞中，在本申请中提出的研究 将增加我们对其他上皮离子通道是如何 在它们各自的膜域内建立和维护。 此外，鉴于最近的证据表明，缺血性 损伤破坏肾小管和肾小管的膜细胞骨架 能够保持不同的顶端和基底侧膜结构域，我们的 研究将增加我们对缺血所致肾脏的了解 失败了。