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

描述(申请人提供)：本研究旨在从分子水平了解原核生物电压门控钠离子通道的离子选择性基础。电压门控钠离子通道是一种完整的膜蛋白，根据膜电位的不同，钠离子优先通过细胞膜。它们在整个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。一些过度兴奋的疾病与这些通道的缺陷有关。 目前的研究集中在原核生物的电压门控钠离子通道，以深入了解钠通道选择性机制的分子基础，也将有助于揭示电压门控钙通道的功能特性，其离子传导孔被认为与钠通道相似。原核钠通道与真核钠钙通道有很高的序列相似性，但与真核钠通道相比，它更容易克隆到合适的表达载体上并大量表达，因此具有一些实用的优势。 将采用结构和功能相结合的办法。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射线结晶学确定其三维结构。重点放在孔洞区域，因为电压传感器组件被认为是高度灵活的，这给结晶带来了技术困难。同时，系统诱变和电生理方法将被用来从另一个方向来解决这个问题。为了实现这些目标，已经从六个不同的细菌物种中克隆了该基因。对表达良好的克隆进行了鉴定和条件优化。 这项研究与公共卫生的相关性。许多美国人和世界各地的更多人患有肌肉、心脏或神经系统的几种疾病，这些疾病是由钠通道蛋白突变引起的。其中一些疾病是癫痫、长QT综合征、周期性瘫痪、肌强直等。如果达到预期的研究目标，将为系统的药物设计和治疗策略提供结构和分子框架。

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.