Structure and function of potassium channels using yeast

利用酵母研究钾通道的结构和功能

• 批准号: 6768590
• 负责人: Steve A N Goldstein
• 金额: $ 5.75万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6768590
• 关键词: CHO cells; Escherichia coli; Saccharomyces cerevisiae; Xenopus; action potentials; cytotoxicity; electrophysiology; fungal genetics; genetic manipulation; immunity; membrane potentials; molecular cloning; phosphorylation; potassium channel; protein structure function; site directed mutagenesis; voltage /patch clamp; voltage gated channel

Structure and function of potassium channels using yeast Potassium channels are important because they set resting membrane potential, repolarize cells after excitatory depolarization, regulate action potential duration and frequency, and, in some cases, mediate bulk transfer of solute. Their function allows for normal physiology, their dysfunction leads to disease. For these reasons, characterizing the structural basis for potassium channel function is the major focus of our research. The past work of this grant was directed toward two large goals: first, cloning and describing the attributes of the newest superfamily of potassium channels - those with 2 P domains. Second, developing and applying genetic methods to expose potassium channel structure and function. In this proposal, we continue toward these goals. This application focuses on three 2 P domain potassium-selective channels we identified in the last period (TOK1 of S. cerevisiae, KCNKO of D. melanogaster and KCNK2 of H. sapiens) and a prokaryotic hyperpolarization-activated potassium channel we describe here for the first time. A key attribute of the proposal is combined utilization of classical approaches and genetic strategies available when channels are studied in yeast cells (even if of animal origin). The three Aims are based on our conviction that potassium channels merit intense scrutiny because of their role in physiology and that yeast genetics can continue to offer unique insights. The 3 Specific Aims are: 1. The basis for K1 killer toxicity and immunity. Four sub-aims consider: (a) residues on K1 and TOK1 that mediate killing; (b) the molecular basis for immunity; (c) residues on K1 and TOK1 that mediate immunity; and, (d) another toxin target. 2. The basis for gating of KCNKO and KCNK2. Four sub-aims seek to assess: (a) KCNKO gating movements and their similarity to C-type inactivation; (b) residues of the KCNKO gating apparatus; (c) how phosphorylation converts KCNK2 from a leak to a voltage-dependent channel; and, (d) KCNK2 residues that mediate ion selectivity and open probability. 3. The function of MVP, a prokaryotic voltage-gated potassium channel. Four sub- aims consider: (a) functional attributes of MVP in yeast; (b) the role of the S4 in activation by hyperpolarization; and (c) residues that couple voltage-sensing and movement of channel gates; and, (d) the function and structure of MVP in E. coli.

使用酵母钾通道的钾通道的结构和功能很重要，因为它们设置了静止的膜电位，在兴奋性去极化后重新极化细胞，调节动作电位持续时间和频率，在某些情况下，介导了溶质的大量溶质转移。 它们的功能允许正常的生理，其功能障碍会导致疾病。 由于这些原因，表征钾通道功能的结构基础是我们研究的主要重点。 这笔赠款的过去工作针对了两个大目标：首先，克隆并描述了钾渠道最新的超家族的属性 - 具有2个P域的钾。 其次，开发和应用遗传方法来揭示钾通道的结构和功能。 在此提案中，我们将继续朝着这些目标迈进。该应用的重点是我们在最后一个时期鉴定的三个2个P结构钾选择性通道（酿酒酵母的Tok1，D。Melanogaster的Kcnko和H. Sapiens的KCNK2）和原核性超极化超极化激活的钾通道，我们在这里首次描述。 该提案的一个关键属性是在酵母细胞中研究通道（即使是动物起源）时可用的经典方法和遗传策略的联合利用。 这三个目标是基于我们坚信钾通道，因为它们在生理学中的作用而值得严格审查，并且酵母遗传学可以继续提供独特的见解。 3个具体目标是：1。K1杀手毒性和免疫力的基础。 四个子iams考虑：（a）介导杀戮的K1和TOK1的残基； （b）免疫的分子基础； （c）介导免疫力的K1和TOK1上的残基；以及（d）另一个毒素靶标。 2。kcnko和kcnk2的门控的基础。 四个子ims试图评估：（a）KCNKO门控运动及其与C型失活的相似性； （b）Kcnko门控设备的残基； （c）磷酸化如何将KCNK2从泄漏转换为电压依赖性通道； （d）介导离子选择性和开放概率的KCNK2残基。 3。MVP的功能，一种原核力电压门控钾通道。 四个子目标考虑：（a）酵母中MVP的功能属性； （b）S4通过超极化在激活中的作用； （c）残基，这些残基伴随着通道大门的电压感应和运动； （d）MVP在大肠杆菌中的功能和结构。