STRUCTURAL STUDIES OF A VOLTAGE-GATED K CHANNEL, KV13

这是香农奖，为这项研究提供部分支持 项目低于指定研究所的资助范围，但 都在最优秀的边缘。香农奖旨在提供 支持测试该方法的可行性；开发进一步的测试 改进研究方法；对可用的数据进行二次分析 数据集；或执行可演示PI的离散项目 研究能力或导致额外的重量已经有价值的 申请。以下应用程序取自原始文档 由首席调查员提交。 电压门控K(Kv)通道调节不同细胞的行为 类型。一个由19个基因组成的大家庭编码这些重要的蛋白质。 这些渠道中任何一个的三级结构的划定将 为理解离子渗透提供了概念框架， 激活、停用和停用，并作为 相关离子通道的发展模型。这样的结构性信息 也可以指导新型离子通道调制的合理设计 可用于治疗各种疾病的药物 包括糖尿病，心律失常，中风，自身免疫性疾病， 高血压、尿失禁和男性型秃顶。 我们建议对Kv1.3进行详细的结构分析，这是一个关键的 T淋巴细胞功能调节剂。Kv1.3频道是最好的频道之一 表征了Kv通道和数百微克的四聚体， 功能纯化的Kv1.3蛋白现在可用于结构 学习。结合互补的定点突变， 电生理学、计算机建模、蛋白质化学和电子 结晶学分为两个互补的策略，我们将探索 Kv1.3的结构。第一个将扩大我们对蝎子毒素的使用 鉴定新毒素的分子卡尺：通道相互作用和提纯 我们的Kv1.3孔和周围区域的模型。将此模型用作 作为指南，我们将确定Kv1.3的S与新一届高中生的互动 Kv1.3的亲和有机阻滞剂，然后可以作为模板 用于设计更具选择性和更有效的通道阻滞剂。 其次，我们将在脂质中产生相干的二维Kv1.3晶体 用电子学方法确定Kv1.3的结构 结晶学。将在电子的基础上生成密度图 以及在多个倾角下收集的光学衍射数据。Kv1.3 将多肽适配到这个图谱上，就可以得到毒素图谱信息 在解释中有价值的。我们的目标将是重建一个 从2D信息中获取通道的三维图像。

THIS IS A SHANNON AWARD PROVIDING PARTIAL SUPPORT FOR THE RESEARCH PROJECTS THAT FALL SHORT OF THE ASSIGNED INSTITUTE'S FUNDING RANGE BUT ARE IN THE MARGIN OF EXCELLENCE. THE SHANNON AWARD IS INTENDED TO PROVIDE SUPPORT TO TEST THE FEASIBILITY OF THE APPROACH; DEVELOP FURTHER TESTS AND REFINE RESEARCH TECHNIQUES; PERFORM SECONDARY ANALYSIS OF AVAILABLE DATA SETS; OR CONDUCT DISCRETE PROJECTS THAT CAN DEMONSTRATE THE PI'S RESEARCH CAPABILITIES OR LEAD ADDITIONAL WEIGHT TO AN ALREADY MERITORIOUS APPLICATION. THE APPLICATION BELOW IS TAKEN FROM THE ORIGINAL DOCUMENT SUBMITTED BY THE PRINCIPAL INVESTIGATOR. Voltage-gated K (Kv) channels regulate the behavior of diverse cell types. An extended family of 19 genes encode these important proteins. Delineation of the tertiary structure of any one of these channels will provide the conceptual framework for understanding ion permeation, activation, inactivation and deactivation, and be the basis for developing models of related ion channels. Such structural information could also guide the rational design of novel ion channel modulating drugs that could be used for the therapy for a wide range of conditions including diabetes, cardiac arrhythmias, stroke, autoimmune disorders, hypertension, urinary incontinence and male pattern baldness. We propose to perform a detailed structural analysis of Kv1.3, a critical modulator of T-lymphocyte function. The Kv 1.3 channel is one of the best characterized Kv channels and hundreds of micrograms of tetrameric, functional purified Kv1.3 protein are now available for structural studies. Combining complementary site-specific mutagenesis, electrophysiology, computer modeling, protein chemistry and electron crystallography into two complementary strategies, we will probe the structure of Kv1.3. The first will extend our use of scorpion toxins as molecular calipers to identify new toxin:channel interactions and refine our model of the Kv1.3 pore and surrounding regions. Using this model as a guide, we will determine Kv1.3's interactions with a new class of high- affinity organic blockers of Kv1.3, which could then serve as a template for the design of more selective and potent blockers of the channel. Second, we will generate coherent two-dimensional Kv1.3 crystals in lipid membranes and determine the structure of Kv1.3 using electron crystallography. A density map will be generated on the basis of electron and optical diffraction data collected at multiple tilt angles. The Kv1.3 polypeptide will be fitted to this map, the toxin-mapping information being valuable in its interpretation. Our goal will be to reconstruct a three-dimensional image of the channel from the 2D information.