Molecular basis of ion selectivity in the prokaryotic voltage-gated Na+ channel

原核电压门控Na通道离子选择性的分子基础

• 批准号: 7487976
• 负责人: MEHABAW G DEREBE
• 金额: $ 2.77万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7487976
• 关键词: Action Potentials; American; Arrhythmia; Behavior; Calcium Channel; Cell membrane; Cell physiology; Cells; Characteristics; Classification; Condition; Coupled; Crystallization; Data; Defect; Disease; Drug Design; Epilepsy; Eukaryota; Eukaryotic Cell; Evolution; Genes; Goals; Human; Immunoglobulin Fragments; Integral Membrane Protein; Invertebrates; Investigation; Ion Channel; Ions; Light; Long QT Syndrome; Mammalian Cell; Membrane Potentials; Methods; Molecular; Monoclonal Antibodies; Muscle Cells; Mutate; Mutation; Myocardium; Myopathy; Myotonia; Nerve; Nervous system structure; Paralysed; Permeability; Pharmaceutical Preparations; Phase; Physiological; Potassium Channel; Property; Proteins; Public Health; Recombinant Proteins; Refractory; Research; Roentgen Rays; Side; Sodium; Sodium Channel; Structure; Thinking; Toxin; X-Ray Crystallography; base; expression vector; gene cloning; insight; mutant; sensor; sodium channel proteins; three dimensional structure; voltage

DESCRIPTION (provided by applicant): In this investigation it is intended to get molecular level understanding to the basis of ion selectivity in the prokaryotic voltage-gated sodium channel. Voltage-gated sodium channels are integral membrane proteins that preferentially pass Na+ ions across the cell mebrane depending on the membrane potential. They are highly conserved throughout evolution. In humans, they regulate many cellular processes, mainly the excitation of nerve and muscle cells. Several diseases of hyperexcitability have been associated with defects in these channels. The current research focuses on the prokaryotic voltage-gated Na+ channel to get crucial insights into the molecular basis underlying the selectivity mechanism of sodium channels and also will shed light on the functional properties of the voltage-gated calcium channels, whose ion conduction pore are believed to be similar to that of sodium channels. The prokaryotic sodium channel shares high sequence similarity to eukaryotic sodium and calcium channels but has several practical advantages over its eukaryotic counterparts in that it is easier to clone into suitable expression vectors and express in large quantities. A combined structural and functional approach will be employed. X-ray crystallographic structure data can reveal the underlying molecular basis for Na+ selectivity. The first approach is thus to crystallize the pore of the channel and determine its three dimensional structure using X-ray crystallography. The emphasis is on the pore region because the voltage sensor component is believed to be highly flexible, which introduces technical difficulties for crystallization. Concurrently, systematic mtagenesis coupled with electrophysiological methods will be employed to tacke the problem from another direction. To achieve these goals the gene has already been cloned from six different bacterial species. Well-expressing clones have been characterized and conditions optimized. Relevance of the research to public health. Many Americans and many more across the globe surfer from several diseases of the muscle, heart or the nervous system that result from mutations in the sodium channel protein. Some of these diseases are epilepsy, long QT syndrome, periodic paralysis, myotonia, etc. If the goals of the propsed research are achieved it would provide a structural and molecular framework for systematic drug design and treatment strategies.

描述（由申请人提供）：本研究旨在从分子水平了解原核细胞电压门控钠通道中离子选择性的基础。电压门控钠通道是一种膜蛋白，其依赖于膜电位优先通过Na+离子穿过细胞膜。它们在进化过程中高度保守。在人类中，它们调节许多细胞过程，主要是神经和肌肉细胞的兴奋。几种过度兴奋性疾病与这些通道的缺陷有关。 目前的研究主要集中在原核细胞的电压门控Na+通道，以获得重要的见解的分子基础的钠通道的选择性机制，也将揭示电压门控钙通道的功能特性，其离子传导孔被认为是类似的钠通道。原核钠通道与真核钠和钙通道具有高度的序列相似性，但与真核对应物相比具有几个实际优势，因为它更容易克隆到合适的表达载体中并大量表达。 将采用结构和功能相结合的方法。X射线晶体结构数据可以揭示Na+选择性的潜在分子基础。因此，第一种方法是使通道的孔结晶并使用X射线晶体学确定其三维结构。重点是孔隙区域，因为电压传感器组件被认为是高度柔性的，这为结晶引入了技术困难。同时，系统的遗传学结合电生理学的方法将从另一个方向解决这个问题。为了实现这些目标，已经从六种不同的细菌物种中克隆了该基因。表达良好的克隆已进行了表征和条件优化。 研究与公共卫生的相关性。许多美国人和地球仪上的许多冲浪者都患有几种肌肉、心脏或神经系统疾病，这些疾病是由钠通道蛋白质突变引起的。这些疾病中的一些是癫痫，长QT综合征，周期性麻痹，肌强直等，如果所提出的研究目标实现，它将提供一个结构和分子框架，系统的药物设计和治疗策略。