REGULATION OF G PROTEIN COUPLED K+ CHANNEL FUNCTION

描述（改编自申请人的摘要）： K+（KACh）电流在副交感神经调节中起重要作用 心率 目前，人们普遍认为， KACh电流由G蛋白刺激的程度决定， 乙酰胆碱，导致通道开放频率的变化。 然而，申请人最近在切除的膜贴片中的研究 表明两种细胞内分子（ATP和一种未鉴定的胞质 因素）可以产生高达10倍的变化，对开放的概率， G蛋白激活的KACh通道，由于其对细胞膜的显著影响， 通道开放状态的持续时间。 ATP酶和胞浆因子 缩短了开放时间。 这些发现有力地表明， 生理条件下ACh激活心房KACh电流 也可能涉及由两种胞质分子介导的这种效应。 的 本申请中提出的研究试图了解 ATP和未知的胞质因子在KACh电流激活和快速 脱敏，以及这些过程中涉及的机制。 目前， 关于ATP是否对细胞的活性有任何影响， G蛋白激活的KACh通道。 因此，第一个具体目标 是为了明确ATP在KACh电流激活中的作用， 特别是在更多的生理条件下，使用快速和短的 （毫秒）ACh的应用。 第二个具体目标是净化 心房胞浆因子的同质性，并确定它。这将有助于 揭示了细胞溶质因子的细胞作用机制， KACh通道上的ATP。 第三个具体目标是审查 KACh通道（GIRK 1/CIR）在缺乏GIRK 1/CIR的卵母细胞中表达的行为。 心房胞浆因子的活性。 使用该表达系统， 申请人将测试GIRK 1/CIR的动力学行为是否一致 假设ATP和心房胞浆因子在 KACh电流的激活和快速脱敏（GIRK 1/CIR）。

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).