VOLUME REGULATION OF RENAL CELLS

肾细胞中钙（Ca）通道的存在和功能是 不太了解。然而，已知Ca通道阻断剂 （二氢吡啶（DHP）、苯基烷基胺等]可以影响许多 肾细胞功能，特别是在某些病理生理状态下 （高血压、糖尿病、局部缺血等）。最近，我们发现 兔近端小管中的一个DHP敏感的Ca通道， 通过肿胀激活。本提案的目标是， 这个肾脏的，肿胀激活的，对DEHP敏感的钙离子通道。四个具体 提出了实现的目标：1）表征功能 DHP敏感的、膨胀激活的Ca通道的特性。使用 肿胀诱导的，DHP敏感的Ca内流（fura-2荧光）和单个 通道分析（膜片钳），Ca通道将被表征为 确定通道行为、细胞体积依赖性激活和DHP 特异性（激动剂vs拮抗剂）。 2)确定 信号/第二信使途径的基础肿胀诱导 激活对DHP敏感的Ca通道。研究将集中在 阐明可能的转导途径（膜 去极化、cAMP/蛋白激酶A和二酰基甘油/蛋白激酶C） 在膨胀过程中激活通道。3)为了分离和排序 编码肾脏DHP敏感性Ca通道（α 1亚基）的cDNA克隆 并确定DHP敏感性Ca通道的其他亚基是否 在肾细胞中表达。近端小管Ca的一个短片段（408 bp） 通道已被克隆，这是同源的心肌DHP- 敏感的L型钙通道。克隆将用于筛选近端 小管cDNA文库，获得全长cDNA序列。 的 肾细胞中其它Ca通道亚单位的表达将被抑制。 通过北方印迹分析评估。4)为了确定函数 克隆的，DHP敏感的Ca通道的特性，并确定 其与肿胀激活的DHP敏感性Ca通道的关系。基于 克隆的cDNA序列，即α 1-亚基的全长cDNA， 表达（或与上文鉴定的其它亚基的cDNA共表达）于 非洲爪蟾卵母细胞的电生理特性 近曲小管细胞肿胀诱导的钙通道。反 正义/正义RNA方法将用于调节内源性Ca通道 在培养的近曲小管细胞中表达，以提供直接证据， 克隆的通道是否是膨胀激活通道。在 总之，该项目将提供一个必要的表征， 功能特性、分子结构和活化机制 一个重要的肾钙通道

The presence and function of calcium (Ca) channels in kidney cells is poorly understood. It is known, however, that Ca channel blockers (dihydropyridines (DHP), phenylklkylamines, etc.] can affect numerous kidney cell functions, particularly in some pathophysiological states (hypertension, diabetes mellitus, ischemia, etc.). Recently, we identified a DHP-sensitive Ca channel in proximal tubules of rabbit which could be activated by swelling. The goal of the present proposal is to characterize this renal, swelling-activated, DHP-sensitive Ca channel. Four specific aims are proposed to be achieved: 1) To characterize the functional properties of the DHP-sensitive, swelling-activated Ca channel. Using swelling-induced, DHP-sensitive Ca influx (fura-2 fluorescence) and single channel analysis (patch clamp), the Ca channel will be characterized to determine channel behavior, cell volume-dependency of activation, and DHP specificity (agonist vs antagonists). 2) To determine the signalling/second messenger pathways underlying swelling-induced activation of the DHP-sensitive Ca channel. Studies will focus on elucidating the role of likely transduction pathways (membrane depolariztion, cAMP/protein kinase A, and diacylglycerol/protein kinase C) in activating the channel during swelling. 3) To isolate and sequence a cDNA clone encoding the renal, DHP-sensitive Ca channel (alpha1-subunit) and to determine whether other subunits of DHP-sensitive Ca channels are expressed in renal cells. A short segment (408 bp) of a proximal tubule Ca channel has been cloned which is homologous to cardiac muscle DHP- sensitive, L-type Ca channels. The clone will be used to screen a proximal tubule cDNA library to obtain the full-length cDNA sequence. The expression of other Ca channel subunits in the renal cells will be assessed by Northern blot analysis. 4) To determine the functional characteristics of the cloned, DHP-sensitive Ca channel and to determine its relation to the swelling-activated, DHP-sensitive Ca channel. Based on the cloned cDNA sequence, full-length cDNA of the alpha1-subunit will be expressed (or coexpressed with cDNA of other subunits identified above) in Xenopus oocytes and the electrophysiological properties compared to those of the swelling-induced Ca channels of proximal tubule cells. Anti- sense/sense RNA methods will be utilized to modulate endogenous Ca channel expression in cultured proximal tubule cells to provide direct evidence as to whether the cloned channel is the swelling-activate channel. In summary, the project will provide a needed characterization of the functional properties, molecular structure, and mechanism of activation of an important, renal Ca channel.