REGULATION OF G PROTEIN COUPLED K+ CHANNEL FUNCTION

描述(改编自申请者的摘要)：心脏毒鼠强 K+(Kach)电流在副交感神经调节中起重要作用 心率的变化。目前，人们普遍认为， Kach电流由G蛋白的刺激程度决定 乙酰胆碱，导致通道开放频率的改变。 然而，申请人最近在切除的膜片中的研究 表明有两种胞内分子(ATP和一种不明原因的胞浆 因数)可以产生多达10倍的开盘概率变化 由G蛋白激活的KACH通道，由于其显著影响 通道打开状态的持续时间。三磷酸腺苷延长与胞浆因子 缩短打开时间。这些发现有力地表明 生理条件下ACh对心房Kach电流的激活作用 也可能涉及由这两个胞质分子介导的这种效应。这个 本申请中提出的研究试图了解这些角色 ATP和未知胞浆因子在Kach电流激活和FAST中的作用 脱敏，以及这些过程中涉及的机制。目前， 关于三磷酸腺苷是否对血管活性有影响还存在争议。 G蛋白激活的Kach通道。因此，第一个具体目标是 就是清楚地定义ATP在Kach电流激活中的作用， 尤其是在更生理的情况下，使用快速和短时间 (毫秒)应用ACH。第二个具体目标是提纯 对心房细胞溶解因子进行同质性鉴定。这将有助于 揭示胞浆因子的细胞作用机制以及 卡赫海峡上的ATP。第三个具体目标是检查 KACH通道(GIRK1/CIR)在缺乏KACH通道的卵母细胞中的表达 心房细胞溶解因子的活性。使用这个表达系统， 申请者将测试GIRK1/CIR的动力学行为是否一致 与三磷酸腺苷和心房性胞浆因子在心力衰竭中的作用 Kach电流(GIRK1/CIR)的激活和快速脱敏。

DESCRIPTION (adapted from the applicant's abstract): The cardiac muscarinic K+ (KACh) current plays an important role in the parasympathetic regulation of heart rate. At present, it is generally believed that the magnitude of the KACh current is determined by the degree of G protein stimulation by acetylcholine, resulting in changes in the frequency of channel opening. However, recent studies by the applicant in excised membrane patches indicate that two intracellular molecules (ATP and an unidentified cytosolic factor) can produce as much as a 10 fold change on the open probability of the KACh channels activated by G protein, due to their marked effects on the duration of the channel open state. ATP prolongs and the cytosolic factor shortens the open time duration. These findings strongly suggest that activation of the atrial KACh current by ACh under physiological conditions may also involve such effects mediated by the two cytosolic molecules. The studies proposed in this application seek to gain understanding of the roles of ATP and the unknown cytosolic factor in KACh current activation and fast desensitization, and the mechanisms involved in these process. At present, there is a controversy as to whether ATP has any effect on the activity of the KACh channel activated by G protein. Therefore, the first specific aim is to clearly define the role of ATP in KACh current activation, particularly under more physiological conditions using rapid and short (millisecond) applications of ACh. The second specific aim is to purify the atrial cytosolic factor to homogeneity and identify it. This will help to reveal the cellular mechanism of action of the cytosolic factor as well as that of ATP on the KACh channel. The third specific aim is to examine the behavior of the KACh channel (GIRK1/CIR) expressed in oocytes which lack the activity of the atrial cytosolic factor. Using this expression system, the applicant will test whether the kinetic behavior of GIRK1/CIR is consistent with the hypothesized roles of ATP and the atrial cytosolic factor in the activation and fast desensitization of the KACh current (GIRK1/CIR).