IONIC MOVEMENTS ACROSS NERVE CELL BODY MEMBRANES

长期目标是提供离子的分子理解 离子渗透和电压依赖性构象变化（门控） 钾离子通道和钾离子通道。 内向整流 钾通道（IRK）被用作实现这种功能的靶点 认识 IRK很重要，因为它们调节静息电位 和动作电位的形状。 反过来，它们可能受到一个 许多重要的生理配体，如G蛋白，ATP和 磷酸化激酶 IRK具有结构简单的优点 比大多数依赖电压的离子通道都要多。 具体目标是：1）定位IRK的孔和门控结构域 并分析其机制; 2）识别可能作为 IRK的生理阻断剂;和3）确定生化结构 的IRK。 该研究设计使用了两个克隆的IRK，其中一个纠正了 一个强，另一个弱。 负责这些问题的结构 使用组合来定位和分析整流中的差异 诱变、异源表达和电生理学的研究。 整流涉及细胞质分子如Mg2+，并且还可以 涉及天然存在的多胺如精胺或亚精胺。 多胺的作用是否是生理性的，将由 生物化学和电生理学方法。 IRK的更直接的结构数据可以通过分析 纯化蛋白 没有丰富的IRK蛋白质来源，所以我们将使用 过表达，然后进行免疫纯化，以获得足够的 是介于 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， 在IRK中的门控，我们可以获得对这些机制的更一般的理解 在电压依赖性离子通道中。 离子通道是人体内 兴奋性分子是神经系统的基本特性 系统和活细胞的基本性质。 电压依赖离子 通道可能参与并成为治疗疾病的靶点， 神经系统疾病，如阿尔茨海默病和癫痫。 实现我们 目标将有助于为治疗提供更合理的基础， 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.