Activation Gating in Human Heart Na+ Channels

描述(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钠通道的激活门是如何 在状态转换期间工作。作为第一步，我们计划将 对激活门控至关重要的内部障碍物的位置。我们 假设钠通道S6片段的细胞质部分形成这样的 狭窄的场地。我们的理论基础是两个位于S6的受体和 他们获得局部麻醉剂和巴曲霉毒素的“大门”。我们的具体目标 是(1)创造、表达和表征一系列半胱氨酸取代的 位于所有四个同源S6片段(D1-S6至D4-S6)第15-28位的突变体， (2)确定这些半胱氨酸突变体带电的可及性 半胱氨酸修饰试剂，以及(3)创建、表达和表征 具有不同大小、疏水性和残基的附加突变体 在这个假定的狭窄部位的两极。人类心脏的变种人 A亚单位钠通道(HH1)克隆在人胚胎肾脏中的表达 通过瞬时转染法获得细胞。突变型钠通道及其门控 属性将首先在全单元配置下进行表征。 半胱氨酸突变体将在内部应用带电后进行评估 评价有无重复脉冲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。如有需要，可由内而外 补丁将用于直接测量化学反应率。门控 而各种半胱氨酸突变体的未锁定轮廓将允许我们推断 沿S6α螺旋结构成簇的孔衬残留物。此外, 紫外光照射连接到的可系留光激活连接体 半胱氨酸突变体可能进一步揭示S6在通道开放期间的运动。 GATED-和之间的结点的后续刻画 具有额外单突变或双突变的非门控可访问区域可能 解开在分子去极化时这样一个狭窄的位置是如何打开的 水平。这个毛孔衬里部位也控制着各种临床 局麻药、抗心律失常药和抗惊厥药等药物 钠通道内前庭内的受体(S)。详细的地图绘制 细胞质S6区及其与钠通道激活的连锁 门控可能为设计新的靶向治疗药物提供见解 这个重要的地区。

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.