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Activation Gating in Human Heart Na+ Channels

人类心脏 Na 通道的激活门控

基本信息

批准号：
6763240
负责人：
SHO-YA Y WANG
金额：
$ 25.75万
依托单位：
STATE UNIVERSITY OF NEW YORK AT ALBANY
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-09-01 至 2007-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6763240
关键词：
cell line cysteine drug receptors heart human genetic material tag neurotoxins protein engineering protein structure function receptor binding receptor expression sodium channel voltage gated channel

项目摘要

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand better how the activation gate of voltage-gated Na+ channels works during state transitions. As a first step, we plan to delimit the whereabouts of an inner obstruction site critical for activation gating. We hypothesize that the cytoplasmic portions of Na+ channel S6 segments form such a constricted site. Our rationale is based on two S6-situated receptors and their "gated" access for local anesthetics and batrachotoxin. Our specific aims are (1) to create, express, and characterize a series of cysteine-substituted mutants at positions 15-28 of all four homologous S6 segments (D1-S6 to D4-S6), (2) to determine the accessibility of these cysteine-mutants with charged cysteine-modifying reagents, and (3) to create, express, and characterize additional mutants with residues of different size, hydrophobicity, and polarity at this putative constricted site. Mutants of the human heart a-subunit Na+ channel (hH1) clone wifi be expressed in human embryonic kidney cells by transient transfection. Mutant Na+ channels and their gating properties will be first characterized under whole-cell configuration. Cysteine-mutants will be then assessed after internal application of charged cysteine-modifying reagents with and without repetitive pulses to evaluate their "gated" accessibility during state transitions. If needed, in-side-out patches will be used for direct measurements of chemical reactivity rate. Gated and ungated profiles of various cysteine-mutants will allow us to infer the clustered pore-lining residues along the S6 a-helical structures. In addition, UV irradiation of a tethered photo-activatable linker attached to cysteine-mutants may further reveal the S6 movement during channel opening. Subsequent characterizations of the junction between gated- and ungated-accessible region with additional single or double mutations may unravel how such a constricted site opens upon depolarization at the molecular level. This pore-lining site also governs the access of a variety of clinical drugs such as local anesthetics, antiarrhythmics, and anticonvulsants to their receptor(s) within the Na+ channel inner vestibule. Detailed mapping of the cytoplasmic S6 regions along with their linkage with the Na+ channel activation gating may provide insights for the design of new therapeutic drugs that target this important region.

描述（由申请人提供）：本项目的长期目标是为了更好地了解电压门控Na+通道的激活门在状态转换期间工作。作为第一步，我们计划划定内梗阻部位的位置对激活门控至关重要。我们假设Na+通道S6片段的细胞质部分形成这种狭窄的地方我们的理论基础是两个位于S6的受体，他们的“门控”进入局部麻醉剂和蟾毒。我们的具体目标是（1）创建，表达和表征一系列半胱氨酸取代的所有四个同源S6片段（D1-S6至D4-S6）的15-28位突变体， (2)为了确定这些半胱氨酸突变体与带电的半胱氨酸修饰试剂，以及（3）产生、表达和表征具有不同大小、疏水性和疏水性的残基的其它突变体，在这个假定的收缩部位的极性。人类心脏的突变体人胚肾α亚单位钠离子通道（hH 1）克隆及其表达细胞瞬时转染。突变型Na+通道及其门控特性将首先在全细胞配置下表征。半胱氨酸突变体然后将在内部应用带电的具有和不具有重复脉冲的半胱氨酸修饰试剂以评估它们在状态转换期间的“门控”可访问性。如果需要，由内而外贴片将用于直接测量化学反应率。门控各种半胱氨酸突变体的非门控图谱将使我们能够推断出沿着S6 α-螺旋结构聚集的孔衬残基。此外，本发明还提供了一种方法，连接至光致抗蚀剂的拴系的可光活化的连接体的UV照射半胱氨酸突变体可以进一步揭示通道开放过程中的S6运动。随后对门控和具有另外的单突变或双突变的未设门的可接近区域可解开这样一个收缩的网站如何打开后，去极化的分子水平这个孔衬部位也控制着各种临床应用的进入。药物如局部麻醉药、抗癫痫药和抗惊厥药， Na+通道内前庭内的受体。详细的地图细胞质S6区沿着与Na+通道激活的连接门控可以为设计靶向治疗的新药物提供见解，这一重要区域。