Activation Gating in Human Heart Na+ Channels

人类心脏 Na 通道的激活门控

• 批准号: 6638713
• 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
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6638713
• 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

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）：该项目的长期目标是 更好地了解电压门控Na+通道的激活门如何 在状态转换期间工作。作为第一步，我们计划划定 对激活门控至关重要的内部障碍位点的位置。我们 假设 Na+ 通道 S6 片段的细胞质部分形成这样的 一个狭窄的场地。我们的基本原理基于两个 S6 受体 他们对局部麻醉剂和箭毒毒素的“门禁”访问。我们的具体目标 (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）的位置 15-28 处的突变体， (2) 确定这些带电荷的半胱氨酸突变体的可及性 半胱氨酸修饰试剂，以及（3）创建、表达和表征 具有不同大小、疏水性和残基的其他突变体 这个假定的收缩位点的极性。人类心脏的突变体 a亚基Na+通道(hH1)克隆wifi在人胚肾中表达 通过瞬时转染细胞。突变的 Na+ 通道及其门控 首先将在全细胞配置下表征特性。 半胱氨酸突变体将在内部应用带电后进行评估 使用或不使用重复脉冲进行评估的半胱氨酸修饰试剂 它们在状态转换期间的“门控”可访问性。如果需要，可从内向外 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特征将使我们能够推断出 沿 S6 a 螺旋结构聚集的孔内衬残基。此外， 附着在光激活连接体上的紫外线照射 半胱氨酸突变体可能进一步揭示通道开放期间 S6 的运动。 门控和门控之间连接的后续特征 具有额外单突变或双突变的非门控可及区域可能 揭示这样一个狭窄的位点如何在分子去极化时打开 等级。该孔衬位点还控制着各种临床药物的进入。 局部麻醉药、抗心律失常药、抗惊厥药等药物 Na+ 通道内前庭内的受体。详细测绘 细胞质 S6 区域及其与 Na+ 通道激活的联系 门控可能为设计针对目标的新治疗药物提供见解 这个重要的地区。