STRUCTURAL DETERMINANTS OF K(CA) CHANNEL FUNCTION

这项提案的长期目标是：界定结构性的 钙激活和电压激活的决定因素及机制 并确定负责其功能的蛋白质结构域 通过磷酸化、G蛋白和β亚基进行调节。我们有 最近克隆并功能表达了人最大KCA通道hslo， 和一个剪接变异体(hslo-A)。初步数据显示：i)联合 孔道形成α亚基hslo及其β亚基的表达 显著提高其对钙离子的敏感性；2)hslo可以调节 通过Gs蛋白。因此，我们将通过突变来研究嵌合体构建， 药理学和定点抗体之间的功能偶联 Hslo和这些调节蛋白，以及hslo和这些调控蛋白的结构决定因素 他们的互动。我们要解决的主要问题是：1)如何 我们能解释KCA通道在天然组织中的功能多样性吗？2)可以 我们确定了与钙和电压相关的结构域 激活？3)磷酸化和G蛋白如何调节毛孔- 形成一个亚单位，涉及哪些结构域？和4)什么是 β亚基作用于hslo功能的机制和结构域？至 回答这些问题，研究将主要通过使用hslo进行， 其β亚基、cGMP-蛋白激酶和G蛋白克隆。为了 比较自然发生的突变，我们将研究两个KCA通道 克隆(hslo和hslo-A)已可用，并隔离其他hslo剪接 β亚基的变异体和人类同源物(S)。具体目标是 目的：1)克隆hsloα亚基和其他亚基的人类同源物(S) Hslo的剪接变体。我们将研究不同的剪接变体 行为均等，以及hslo是否可以与不同的β亚型偶联 亚单位。我们还将检查a和β亚基的mrna分布。 在不同激素条件下；2)探讨其作用机制(S)。 电压和钙依赖的激活。探讨钙离子的偶联作用 结合，电荷运动和开孔，我们将测量钙和 离子电流和门控电流的电压灵敏度 同源物，以及在羧基末端和S4区突变体中；3)检查 通过磷酸化(PKA、cGMP-)调节成孔的α亚基 Pk)和可能的G蛋白作用(Gs-α、β-伽马亚基)，以及 确定通过磷酸化进行调控的位点。我们将分析 由这些因素引起的电压和钙依赖性的改变 调节机制；以及4)研究结构决定因素。 KCA通道复合体的α和β亚基之间的相互作用， 以及由此产生的功能后果。人KCA的结构与功能研究 经络可能在设计治疗手段时有用，以防止通畅 冠状动脉疾病或早产时的肌肉痉挛。

The long term objectives of this proposal are: to define the structural determinants and mechanisms of calcium and voltage activation of "maxim Kca channels, and to determine the protein domains responsible for their modulation by phosphorylation, G proteins and beta subunits. We have recently cloned and functionally expressed a human maxi Kca channel, hslo, and a splice variant (hslo-A). Preliminary data shows that: i) co- expression of hslo, the pore-forming alpha subunit, with its beta subunit dramatically increases its Ca2+ sensitivity; and 2) hslo can be modulated by Gs proteins. Thus, we will study, by mutagenesis, chimera construction, pharmacology, and site-directed antibodies, the functional coupling between hslo and these modulatory proteins, and the structural determinants of their interaction. The main questions that we want to address are: 1) how can we explain Kca channel functional diversity in native tissues? 2) can we identify the domains responsible for Ca2+- and voltage-dependent activation? 3) how do phosphorylation and G proteins regulate the pore- forming a subunit and what domains are involved? and 4) what are the mechanisms and the domains for the beta subunit action on hslo function? To answer these questions, studies will be performed primarily by using hslo, its beta subunit, cGMP-protein kinase and G protein clones. In order to compare naturally occurring mutations, we will study the two Kca channel clones (hslo and hslo-A) already available, and isolate other hslo splicing variants and human homologue(s) of the beta subunit. The specific aims are to: 1) clone a human homologue(s) of the hslo alpha subunit and other splice variants of hslo. We will investigate if different splice variants behave equally, and whether hslo can couple with different isoforms of beta subunits. We will also examine the mRNA distribution of a and beta subunits under different hormonal conditions; 2) investigate the mechanism(s) of voltage- and Ca2+-dependent activation. To investigate the coupling of Ca2+ binding, charge movement and pore opening we will measure the calcium and voltage sensitivities of ionic and gating currents in hslo, hslo homologues, and in carboxyl terminus and S4 region mutants; 3) examine the modulation of the pore-forming alpha subunit by phosphorylation (PKA, cGMP- PK) and putative G protein action (Gs-alpha, beta-gamma subunits), and establish the sites of modulation by phosphorylation. We will analyze modifications in the voltage and calcium dependencies induced by these modulatory mechanisms; and 4) study the structural determinants of the interaction between the alpha and beta subunits of the KCa channel complex, and the functional consequences. Structure-function studies of human Kca channels may be useful in the design of therapeutic means to prevent smooth muscle spasm in coronary disease or premature labor.