VOLUME REGULATION OF RENAL CELLS

肾细胞中钙通道的存在和功能是 人们对此知之甚少。然而，已知钙通道阻滞剂 (二氢吡啶(DHP)、苯基己基胺等)可能会影响到众多 肾细胞功能，特别是在某些病理生理状态下 (高血压、糖尿病、缺血等)。最近，我们确定了 兔近端肾小管上皮细胞对DHP敏感的钙通道 肿胀的被肿胀激活的本提案的目标是将 这种肾脏、肿胀激活、DHP敏感的钙通道。四个具体的 建议实现的目标是：1)表征功能 DHP敏感、肿胀激活的钙通道的特性。vbl.使用 肿胀诱导，DHP敏感的钙内流(Fura-2荧光)和单一 通道分析(膜片钳)，钙通道的特征将是 确定通道行为、激活的细胞体积依赖关系和DHP 特异性(激动剂与拮抗剂)。2)确定 肿胀潜在的信号/第二信使通路 激活DHP敏感的钙通道。研究将集中在 阐明可能的转导途径(膜)的作用 去极化、cAMP/蛋白激酶A和二酰甘油/蛋白激酶C) 在肿胀期间激活通道。3)分离和测序 肾DHP敏感钙通道(α1亚基)编码基因的克隆 并确定DHP敏感的钙通道的其他亚单位是否 在肾脏细胞中表达。近端小管一小段(408碱基)片段 与心肌DHP同源的通道已被克隆 敏感，L型钙通道。克隆将被用来筛选近端的 获得全长的cDNA文库。这个 肾细胞中其他钙通道亚单位的表达将是 经Northern印迹分析鉴定。4)确定功能 DHP敏感的克隆钙通道的特性及测定 它与肿胀激活的DHP敏感的钙通道有关。基于 克隆的cDNA1-亚基的全长cdna将是 表达(或与上面确定的其他亚基的cDNA共表达) 非洲爪哇卵母细胞及其电生理特性的比较 肿胀诱导的近曲小管细胞钙通道的改变。反- 将利用正义/正义RNA方法来调节内源性钙通道 在培养的近端肾小管上皮细胞中的表达为 克隆的通道是否为肿胀激活通道。在……里面 综上所述，该项目将提供所需的 酶的功能性质、分子结构及活化机理 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.