INDUCED GATING OF MEMBRANE CHANNELS WITH HUMAN MINIMAL POTASSIUM EXPRESSION

心脏钾离子通道是一个重要的组成部分，在兴奋- 传播过程 最近，已经证明， 蛋白质（被称为最小K+，或minK），存在于各种 哺乳动物组织，导致表达钾电流（IsK）， 非洲爪蟾卵母细胞;类似的电流也可以记录从人类 用minK cDNA转染的胚胎肾细胞（HEK 293）。 水貂 该蛋白与延迟整流蛋白的表达相关， 豚鼠心室肌细胞的钾电流， 假设貂皮可能是一种基本的（如果不是一种基本的） 人体心脏钾离子通道的一部分 初步 我们实验室的结果表明， minK表达卵母细胞的细胞外间隙导致IsK关闭; 在pH值高于观察值时发生闭合（由其他作者） 当只有细胞外室被酸化时;因此，我们的数据 表明IsK通道也可能对质子浓度敏感 存在于细胞质中。 以前的研究也表明， IsK的生物物理特性和磷酸化调节是 至少部分地由貂蛋白的一级序列决定。 该项目的总体目标是表征 酸化诱导的延迟整流钾电流关闭 与人类minK同种型表达相关的IsK 蛋白 我们将使用电生理学、光学和 分子生物学技术在水貂表达的光滑异种卵母细胞中， 以及在转染貂cDNA的HEK 293细胞中，以研究 酸化对IsK生物物理特性的影响，并确定 minK一级序列之间可能的结构-功能关系， 以及酸化诱导的IsK通道关闭。 具体 目的是：1）表征幅度，电压依赖性和时间 水貂相关通道的pH门控过程中的内部酸化 和/或细胞外空间。 2)通过非- 平稳波动分析，细胞内和/或细胞外的影响 酸化对单元电导和开放概率的影响， 在卵母细胞中表达的附着的IsK通道。 3)表征 细胞质中可溶性成分对pH值的可能作用 IsK的门控，和4）研究功能表达，和pH 在卵母细胞和HEK 292中表达的minK突变形式的敏感性 细胞 这些实验的成功完成将提供一个 更好地理解氢离子抑制IsK的机制， 并可能使我们了解pH诱导的心脏电生理变化， 活动

Cardiac potassium channels are an important component in the excitation- propagation process. Recently, it has been demonstrated that a small protein (dubbed minimal K+, or minK) which is present in a variety of mammalian tissues, leads to the expression of a potassium current (IsK) in Xenopus laevis oocytes; a similar current can also be recorded from human embryonic kidney cells (HEK293) transfected with minK cDNA. The minK protein has been associated with the expression of the delayed rectifier potassium current in guinea pig cardiac ventricular myocytes, and it has been hypothesized that mink may be an essential (if not a constitutive) part of a functional potassium channels in the human heart. Preliminary results from our laboratory show that acidification of both the intra- and extracellular spaces of minK-expressing oocytes lead to IsK closure; the closure occurs at pH values higher than those observed (by other authors) when only the extracellular compartment is acidified; thus, our data suggest that IsK channels may also be sensitive to the proton concentration present in the cytoplasm. Previous studies have also shown that the biophysical properties and regulation by phosphorylation of IsK are determined, at least in part, by the primary sequence of the mink protein. The overall goal of this project is to characterize the molecular bases for acidification-induced closure of the delayed rectifier potassium current IsK that is associated with the expression of the human isoform of the minK protein. We will use a combination of electrophysiological , optical and molecular biological techniques in mink-expressing Xenpous laevis oocytes, as well as in HEK293 cells transfected with mink cDNA, to study the effect of acidification on the biophysical properties of IsK, and to determine a possible structure-function relation between the primary sequence of minK, and the acidification-induced closure of the IsK channels. The Specific Aims are; 1) To characterize the magnitude, voltage dependence and time course of pH gating of minK-related channels upon acidification of the intr and/or the extracellular space. 2) To determine, by means of non- stationary fluctuation analysis, the effects of intra- and/or extracellular acidification on the unitary conductance and open probability of cell- attached IsK channels expressed in oocytes. 3) To characterize the possible role that soluble components of the cytoplasm may have on pH gating of IsK, and 4) to study the functional expression, and pH sensitivity of mutant forms of minK expressed in oocytes and in HEK292 cells. Successful accomplishment of these experiments should provide a better understanding of the mechanisms by which hydrogen ions inhibit IsK, and may give insight into pH-induced changes on cardiac electrical activity.