Activation Gating in Human Heart Na+ Channels

描述（申请人提供）：该项目的长期目标是 为了更好地理解电压门控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。

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.