REGULATORY MECHANISMS OF CARDIAC REPOLARIZATION

描述(改编自申请人的摘要)：电压门控K+ 通道，负责动作电位复极和设置 静息膜电位，是蛋白质编码的最复杂的 在心血管系统中发现的一组离子通道基因。一小块 血管平滑肌膜K+通透性的变化，导致 膜电位和钙通道活性发生明显变化。 因此，K+通道功能的改变可以对 血管张力。拟议的工作将审查三个具体目标。 1.分析大鼠K+通道亚基的定位和组装 心肌细胞和血管平滑肌组织。将有两个假设 检查：(I)在心脏中，Kva1.5要么是同分异构体，要么是 在插入盘处与Kva1.2组装。在脑室(但不是 中庭)含有Kva1.5的复合体与 失活相关的KVB亚基。(Ii)在血管平滑肌，Kva 1.5 以与Kva1.4亚基结合的异构体结构存在 Kvb1.2β亚基。2.钾离子通道的机制 将确定a/b亚基的相互作用。将有两个假设 检查过了。(I)AB装配涉及将新生的航道与 促进亚单位组装的伴侣样蛋白。(Ii)。特定的 B亚基上的氨基酸决定物理ab相互作用。测试版 对电压敏感性、失活和失活的影响可以追溯到 不同的氨基酸。此外，所有的ab交互作用都发生在 α和β亚基的N-末端结构域。3.蜂窝 和IKR钾通道蛋白H-ERG的亚细胞分布， 将在正常人和患病的人心肌中被检测到。一个假设 将被调查：IKR表达不是静态的，本地化是 在病变心肌中发生改变。AIMS 1和AIMS 3的免疫组织化学 将主要在大鼠、犬和人类组织切片上进行 目前存在的抗体和正在生产的抗血清。阿尔法-贝塔 相互作用将通过电压钳位技术和 身体上通过免疫净化。亚基组装将通过以下方式确定 免疫纯化方法，其中两个不同的亚基用 仅对一种抗体具有特异性的抗体是组装的操作定义。 参与这种相互作用的氨基酸将通过各种不同的方式进行鉴定 诱变的方法。

DESCRIPTION (adapted from the applicant's abstract): Voltage-gated K+ channels, responsible for action potential repolarization and setting the resting membrane potential, are proteins encoded by one of the most complex group of ion channel genes found in the cardiovascular system. A small change in K+ permeability in vascular smooth muscle membranes, results in a significant change in membrane potential and in Ca2+ channel activity. Thus, changes in K+ channel function can have an important effect on vascular tone. Three specific aims will be examined in the proposed work. 1. K+ channel subunit localization and assembly will be analyzed in rat cardiac myocytes and vascular smooth muscle tissue. Two hypotheses will be examined: (i) In heart Kva1.5 is either a homomeric alpha structure or assembled with Kva1.2 at the intercalated disk. In the ventricle (but not the atrium) the Kva1.5-containing complex is assembled with an inactivation-conferring Kvb subunit. (ii) In vascular smooth muscle, Kva1.5 exists as a heteromeric structure in association with the Kva1.4 subunit and the Kvb1.2 beta subunit. 2. The mechanisms underlying potassium channel a/b subunit interactions will be determined. Two hypotheses will be examined. (i) ab assembly involves the association of nascent channels with chaperon-like proteins that facilitate subunit assembly. (ii). Specific amino acids on b subunits determine physical ab interactions. The beta effects on voltage-sensitivity, deactivation, and inactivation can be traced to different amino acids. Furthermore all ab interactions occur at the N-terminal domains of both the alpha and beta subunits. 3. The cellular and subcellular distribution of the IKr potassium channel protein, h-erg, will be determined in normal and diseased human myocardium. One hypothesis will be investigated: IKr expression is not static, with localization being altered in diseased myocardium. The immunohistochemistry in aims 1 and 3 will be performed primarily on rat, canine and human tissue sections with antibodies that currently exist and antisera under production. Alpha-beta interactions will be monitored functionally by voltage-clamp techniques and physically by immunopurification. Subunit assembly will be determined by immunopurification methods where purification of two distinct subunits with an antibody specific for only one is the operational definition of assembly. The amino acids involved in this interaction will be identified by a variety of mutagenesis approaches.