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，是由一个最复杂的蛋白质编码 在心血管系统中发现的一组离子通道基因。 一个小 血管平滑肌膜中K+渗透性的变化，导致 膜电位和Ca 2+通道活性的显著变化。 因此，K+通道功能的变化可以对 血管张力 在拟议的工作中将审查三个具体目标。 1. K+通道亚基的定位和组装将在大鼠中进行分析。 心肌细胞和血管平滑肌组织。 两个假设将是 检查：（i）在心脏中，Kva 1.5是同聚体α结构或 与Kva 1.2组装在闰盘上。 在心室（但不是 中庭），含有Kva 1.5的复合物与 失活赋予Kvb亚基。 (ii)在血管平滑肌中，Kva 1.5 作为与Kva 1.4亚基相关的异聚体结构存在， Kvb1.2 β亚基。 2. 钾离子通道的作用机制 将测定a/B亚基相互作用。 两个假设将是 考察 (i)AB组装涉及新生通道与 促进亚基组装的伴侣样蛋白。 （ii）。 具体 B亚基上的氨基酸决定物理AB相互作用。 贝塔 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ab相互作用都发生在 α和β亚基的N端结构域。 3. 蜂窝 和IKr钾通道蛋白，h-erg， 将在正常和患病的人心肌中测定。 一个假设 IKr表达不是静态的，定位是 在病变心肌中改变。 目的1和目的3的免疫组化 将主要在大鼠、犬和人体组织切片上进行， 目前存在的抗体和正在生产的抗血清。 Alpha-beta 将通过电压钳技术功能性地监测相互作用， 通过免疫纯化。 子单元组装将由 免疫纯化方法，其中纯化两种不同的亚基， 仅对一种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多种方法鉴定。 诱变方法。

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.