Mass spectrometric studies on potassium channels

说明(申请人提供)：电压依赖离子通道有 有助于产生膜电位、受体电位和 动作电势。它们是分子构建块， 神经科学中许多细胞的基本特征：兴奋性。这些 蛋白质与所有兴奋性疾病的生理学和病理生理学有关 组织，是许多疾病过程的基础，包括癫痫和心律失常， 是临床应用基本药物的主要靶点。尽管 这些蛋白质明显的生物学和临床重要性，我们仍然知道 对它们的结构了解相对较少。尽管已经取得了重要的进展 在理解钾离子通道的离子渗透方面取得的进展，大部分 这些必需的蛋白质在结构上仍然没有被分解。 这项提议探索了一种解决结构性问题的新技术。 涉及离子通道和其他膜蛋白。我们将使用组合 半胱氨酸扫描诱变、烷基化和质谱学研究 两个钾通道，KCSA和Shaker的结构方面。我们指的是这个 新技术，如现场定向质量标记。这样做的长期目标是 建议提供有关功能的低分辨率结构信息 离子通道。从我们还不知道的意义上说，这项提议风险很高 这项技术是否会发挥其潜力并被证明是一种有用的 结构工具。然而，如果成功，现场定向质量标记可能能够 为许多稀有膜的大体结构提供重要的洞察力 蛋白质。预计这项研究的结果将提供新的 深入了解Shaker的总体结构布局。

DESCRIPTION (provided by applicant): Voltage-dependent ion channels are instrumental in the generation of membrane potential, receptor potential, and action potential. They are the molecular building blocks for what is an essential characteristic of many cells in neuroscience: excitability. These proteins are implicated in the physiology and pathophysiology of all excitable tissues, underlie many disease processes including epilepsies and arrhythmias, and are major targets of essential drugs used in clinical medicine. Despite the obvious biological and clinical importance of these proteins, we still know relatively little about their structure. Although important progress has been made in understanding ion permeation in potassium channels, large parts of these essential proteins remain structurally unresolved. This proposal explores a novel technique for addressing structural questions involving ion channels and other membrane proteins. We will use a combination of cysteine scanning mutagenesis, alkylation, and mass spectrometry to study structural aspects of two potassium channels, KcsA and Shaker. We refer to this novel technique as site-directed mass tagging. The long-term goal of this proposal is to provide low-resolution structural information about functional ion channels. The proposal is high risk in the sense that we do not yet know whether the technique will fulfill its potential and prove to be a useful structural tool. If successful, however, site-directed mass tagging may be able to provide important insight into the gross structure of many rare membrane proteins. It is anticipated that results from this study will provide new insight into the general structural layout of Shaker.