IONIC MOVEMENTS ACROSS NERVE CELL BODY MEMBRANES

长期目标是提供对离子的分子理解。 离子的渗透和电压相关构象变化(GATING) 一般是通道，特别是钾通道。内向整流器 钾通道(Irk)被用作实现这一目标的靶点。 理解。IRK很重要，因为它们调节静息电位 以及动作电位的形状。反过来，它们可能会受到 重要的生理配体的数量，如G蛋白、ATP和 磷酸化的激酶。IRKS的优点是结构更简单 比大多数电压依赖的离子通道更好。 其具体目的是：1)定位irks的气孔和浇注区域 并分析其机制；2)识别可能起作用的分子 IRKs的生理阻滞剂；3)决定生化结构 讨厌的人。 研究设计利用了两个克隆的irk，其中一个整形 一个强，另一个弱。负责该项目的结构 使用组合来定位和分析整改中的差异 突变、异源表达和电生理学。 整流涉及到像镁离子这样的细胞质分子，也可能 涉及自然产生的多胺，如精胺或亚精胺。 多胺的影响是否是生理的，将通过以下方式进行测试 生化和电生理方法。 更直接的结构数据可以通过分析 纯化的蛋白质。没有丰富的irk蛋白来源，所以我们将使用 过量表达，然后进行免疫纯化，以获得足够的 数量。蛋白质折叠的拓扑模型将使用 糖基化位点插入。 通过确定离子渗透和电压依赖的机制 我们可以对这些机制有一个更全面的了解。 在电压依赖的离子通道中。离子通道是必不可少的 兴奋性分子，这是神经的基本属性 系统和活细胞的一个基本属性。电压相关离子 经络可能参与并以此为靶点的疾病治疗 神经系统，如阿尔茨海默病和癫痫。实现我们的 目的有助于为该病的治疗提供更合理的依据。 这些疾病。

The long term objectives are to provide a molecular understanding of ion permeation and voltage-dependent conformational changes (gating) for ion channels in general and potassium channels in particular. Inward rectifier potassium channels (IRKs) are used as the targets for achieving such understanding. IRKs are important because they regulate resting potential and the shape of action potentials. In turn, they may be regulated by a number of important physiological ligands such as G proteins, ATP and phosphorylating kinases. IRKs have the advantage of a simpler structure than most voltage dependent ion channels. The specific aims are to: 1) locate the pore and gating domains of IRKs and analyze their mechanisms; 2) identify molecules that may act as physiological blockers of IRKs; and 3) determine the biochemical structure of IRKs. The research design makes use of two cloned IRKs, one of which rectifies strongly and the other weakly. The structures responsible for the differences in rectification are localized and analyzed using a combination of mutagenesis, heterologous expression, and electrophysiology. Rectification involves cytoplasmic molecules such as Mg2+_ and may also involve naturally occurring polyamines such as spermine or spermidine. Whether the polyamine effects are physiological will be tested by biochemical and electrophysiological methods. More direct structural data on IRKs may be obtained from analysis of purified protein. There is no rich source of IRK protein so we will use overexpression followed by immunopurification to obtain sufficient quantities. Topological models of protein folding will be tested using glycosylation site insertion. By determining the mechanisms of ion permeation and voltage-dependent gating in IRKs we may gain a more general understanding of these mechanisms in voltage-dependent ion channels. Ion channels are the essential molecules of excitability which is the fundamental property of the nervous system and a basic property of living cells. Voltage-dependent ion channels may be involved in and are targets for treatment of diseases of the nervous system such as Alzheimer's disease and epilepsy. Achieving our objectives will help to provide a more rational basis for the therapy of these diseases.