Molecular Cloning of Epithelial K Channels

上皮 K 通道的分子克隆

• 批准号: 7192404
• 负责人: HENRY SACKIN
• 金额: $ 34.76万
• 依托单位: ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7192404
• 关键词: Affect; Ally; Cations; Characteristics; Chimera organism; Closure; Communication; Condition; Coupled; Crystallization; Crystallography; Cysteine; Data; Electrodes; Elevation; Engineering; Epithelial; Equilibrium; Goals; Hand; Helix (Snails); Ions; KCNJ1 gene; Kidney; Kinetics; Ligands; Mechanics; Modeling; Molecular Cloning; Numbers; Oocytes; Patch-Clamp Techniques; Perfusion; Physiological; Potassium Channel; Preparation; Principal Investigator; Protons; Series; Specific qualifier value; System; Testing; Tweens; base; ear helix; large-conductance calcium-activated potassium channels; mutant; particle; programs; protonation; research study; sensor; voltage; voltage clamp

DESCRIPTION (provided by applicant): The proposed project continues the original specific goal of understanding K permeation and gating through the renal, inward rectifying, K channel: ROMK (Kir1.1). However, the project now encompasses a more general theme of understanding the general mechanics of K channel gating. This is particularly timely in view of recent crystallographic studies on bacterial KcsA and Ca-activated BK channels suggesting that K channel gating is associated with specific structural changes. In this model, the K selectivity filter not only selects among cations but also functions as an outer gate for the channel. A second, inner gate, in series with the outer gate, is believed to consist of a hinged section of the inner transmembrane helix. Particular sensors (voltage, ligand, pH) that are coupled to this inner gate would define the functional characteristics of a particular channel. Electrophysiological experiments would be conducted to test this two-gate hypothesis, using ROMK and its mutants and chimeras, expressed in Zenopus oocytes. Oocytes would be studied with: the two electrode voltage clamp (TEVC), the cut-open oocyte technique and patch-clamp recording of single channels. This proposal is a unique opportunity to combine available crystallographic information with a functional electrophysiological study to elucidate what may be a general paradigm for K channel gating, as well as a specific mechanism for regulating renal K secretion.

描述（由申请人提供）：拟议的项目延续了最初的具体目标，即了解钾通过肾脏的渗透和门控，向内整流，K通道：ROMK （Kir1.1）。然而，该项目现在包含了一个更普遍的主题，即理解K通道门控的一般机制。鉴于最近对细菌KcsA和ca激活的BK通道的晶体学研究表明，K通道门控与特定的结构变化有关，这是特别及时的。在该模型中，K选择性滤波器不仅在阳离子之间进行选择，而且还作为通道的外门。第二个，内门，与外门串联，据信由内跨膜螺旋的铰链部分组成。特定的传感器（电压，配体，pH值）耦合到这个内门将定义一个特定通道的功能特征。电生理实验将利用在顶蟾卵母细胞中表达的ROMK及其突变体和嵌合体来验证这一双门假说。卵母细胞的研究方法有：双电极电压钳（TEVC）、切开卵母细胞技术和单通道膜片钳记录技术。这一建议是一个独特的机会，将现有的晶体学信息与功能性电生理学研究结合起来，阐明钾通道门控的一般模式，以及调节肾脏钾分泌的特定机制。