Structural Studies of Voltage Gating in Voltage-Gated Sodium Channels

描述（由申请人提供）：动作电位通常由钠通道（Navs）引发。Navs在许多组织中共享这种作用，Navs的突变或其他功能障碍导致组织特异性疾病，包括心律失常、癫痫和慢性神经性疼痛。所有通道病的一个关键相似之处是，对通道基本结构的损伤会导致功能的改变。确定原子分辨率的基本结构和选择性、门控和抗高血压药物结合的立体化学细节是本提案的目的。虽然K+通道的结构研究相当先进，但目前没有类似的研究，也没有对Na+和Ca2+选择性或药物结合的立体化学理解。这个建议的主要目的是确定一个电压门控离子通道家族中钠和钙选择性和抗高血压药物结合的机制。第一个具体目标将完成我们目前的结构，第二个具体目标将确定Na+和Ca2+的离子选择性机制，第三个目标将开发和利用稳定固有柔性分子的方法。多个抗体和工程化二硫键将用于稳定特定的门控状态。这些状态将通过结构研究来确认，并且它们的结构将通过X射线晶体学来确定。这项工作的长期益处将是数百种致病突变的统一模型，导致改善治疗的药物结合的立体化学观点，更好地理解离子选择性以及Navs和Cavs之间的关系，以及通道门控的物理模型。另一个好处是开发了固定固有柔性分子以帮助结晶的方法。该项目将使用X射线晶体学来确定第一个Na+通道结构。公共卫生受益将是改善抗心律失常药物导致的钠离子通道功能障碍的治疗方法。

DESCRIPTION (provided by applicant): Action potentials are typically initiated by sodium channels (Navs). This role is shared by Navs in many tissues, and mutations or other dysfunction of Navs lead to tissue specific diseases, including arrythmias, epilepsy, and chronic neuropathic pain. A key similarity in all channelopathies is that an insult to the basic architecture of the channel leads to a change in function. Determining this basic architecture at atomic resolution and the stereo-chemical details of selectivity, gating, and of anti-hypertension drug binding are the purposes of this proposal. While structural studies of K+ channels are quite advanced, there are currently no analogous studies and no stereo-chemical understanding of Na+ and Ca2+ selectivity or of drug binding. The broad aim of this proposal is to determine the mechanisms of sodium and calcium selectivity and anti-hypertension drug binding in a family of voltage-gated ion channels. The first specific aim will complete our current structure, the second specific aim will determine the mechanism of ion selectivity for both Na+ and Ca2+, and the third aim will develop and utilize methods to stabilize inherently flexible molecules. Multiple antibodies and engineered disulfide bonds will be used to stabilize specific gating states. These states will be confirmed by structural studies, and their structures will be determined by x-ray crystallography. The long-term benefit of this work will be a unifying model for the hundreds of disease causing mutations, a stereo-chemical view of drug binding leading to improved therapeutics, a better understanding of ion selectivity and the relationship between Navs and Cavs, and a physical model for channel gating. An additional benefit will be the development of methods for immobilizing inherently flexible molecules to aid in crystallization. This project will use x-ray crystallography to determine the first Na+ channel structure. The public health benefit will be improved treatment methods for Na+ channel dysfunction resulting from improved anti- arrhythmias drugs.