STRUCTURE AND FUNCTION OF THE TOK1 POTASSIUM CHANNEL

TOK1是新的k+通道家族的第一个成员[1]。这个海峡， 我们从萌芽中的酿酒酵母中克隆的，有两个 新颖的特征。首先，它在结构上是不同的。与之形成鲜明对比的是 以前确定的K=通道α亚基，它只带有一个孔- TOK1有两个结构域，形成P结构域。其次，TOK1是第一个新的 功能型K+选择性通道，是一种“外向整流”。 内向整流器超家族通道，TOK1‘激活’耦合到 钾的平衡电位(EK)，但TOK1通过K+电流在 向外方向，一种以前未描述的通道表型。我们的整体 这项工作的目标是详细说明TOK1通道和 在可能的情况下，阐明它们的结构基础。我们的6个具体目标是 接下来的5年是：(1)详细说明TOK1的基本单通道特性 (2)研究TOK1的分子机制 外向纠偏；(3)评价两个P结构域在脑内的作用。 完整通道和每个P结构域分别表达时；(4) 研究航道的膜状拓扑结构，重点研究与之相关的 其2 P结构域的排列；(5)克隆通道的同源物 以及，(6)将酵母分子遗传学的工具应用于 TOK1钾通道结构与功能的研究初步 研究结果支持上述每个目标的可行性。 使用所表达的TOK1通道的宏观和单通道记录 在非洲爪哇的卵母细胞和标准分子遗传工具。

TOK1 is the first member of a new family of k+ channels [1]. The channel, which we cloned from the budding yeast Saccharomyces cerevisiae, has two novel features. First, it is structurally distinct. In contrast to previously identified K= channel alpha-subunits which bear only one pore- forming P domain, TOK1 has two. Second, TOK1 is the first example of a new functional type of K+-selective channel, an "outward rectifier'. Like inward rectifier superfamily channels, TOK1 'activation' is coupled to the equilibrium potential for potassium (EK), but TOK1 passes K+ current in the outward direction, a previously undescribed channel phenotype. Our overall goal in this work is to detail the basic attributes of TOK1 channels and where possible, elucidate their structural basis,. Our 6 specific aims for the next 5 years are: (1) to detail basic single channel properties of TOK1 channels; (2) to investigate the molecular mechanism underlying TOK1 outward rectification; (3) to evaluate function of the 2 P domains in the intact channel and when each of the P domains is expressed separately; (4) to study the membrane topology of the channel with emphasis on the relative arrangement of its 2 P domains; (5) to clone homologues of the channel from higher organism; and, (6) to apply the tools of yeast molecular genetics to study of TOK1 potassium channel structure and function. Preliminary findings are presented to support the feasibility of each of these aims using macroscopic and single channel recording of TOK1 channels expressed in Xenopus laevis oocytes and standard molecular genetic tools.