STRUCTURAL DETERMINANTS OF K(CA) CHANNEL FUNCTION

这项建议的长远目标是： “maxim Kca”的钙和电压激活的决定因素和机制 通道，并确定负责其蛋白质结构域 通过磷酸化、G蛋白和β亚基进行调节。我们有 最近克隆并功能性表达了人最大KCa通道，hkB， 和剪接变体（hslo-A）。初步数据显示：（一） 表达成孔α亚单位hepatocyte及其β亚单位 显著增加其Ca 2+敏感性;和2）可以调节 GS蛋白质因此，我们将通过诱变，嵌合体构建， 药理学和定点抗体之间的功能耦合 阻碍和这些调节蛋白，以及结构决定因素， 他们的互动。我们要解决的主要问题是：1）如何 我们能解释天然组织中KCA通道功能的多样性吗？2)可以 我们确定了负责钙离子和电压依赖性的结构域， 激活？3)磷酸化和G蛋白是如何调节 形成一个亚基，涉及哪些结构域？（4）什么是 β亚单位对histamine功能的作用机制和作用域？到 为了回答这些问题，研究将主要通过使用histamine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-A和hslo-A）已经可用，并分离其他hslo-A剪接 β亚基的变体和人同源物。具体目标是 1）克隆人源化的人源化 剪接变体。我们将研究不同的剪接变体 行为相同，以及hepatocyte是否可以与不同的β亚型偶联， 亚单位。我们还将研究α和β亚基的mRNA分布 在不同的激素条件下; 2）研究的机制（S） 电压和Ca 2+依赖性激活。研究钙离子的耦合作用， 结合，电荷移动和孔开放，我们将测量钙， 在hqs，hqs中离子和门控电流的电压灵敏度 同源物，以及羧基末端和S4区突变体; 3）检查 通过磷酸化（PKA，cGMP-1）调节成孔α亚基， PK）和推定的G蛋白作用（Gs-α、β-γ亚基），以及 通过磷酸化建立调节位点。我们将分析 在电压和钙依赖性的变化诱导这些 调节机制;和4）研究的结构决定因素， KCa通道复合物的α和β亚基之间的相互作用， 和功能性后果。人Kca的结构与功能研究 通道可用于治疗装置的设计 冠心病或早产时肌肉痉挛。

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.