G PROTEIN-MEDIATED K+ CHANNEL ACTIVATION IN HEART

本研究旨在探讨毒蕈碱受体介导的 酶促分离的单个细胞中钾通道的激活 牛蛙心房细胞 乙酰胆碱诱导的，向内纠正的 钾电流也在受体非依赖性 在抗水解GTP类似物的存在下， 因此，激活机制必须包括对GTP的理解 结合蛋白（G蛋白）-通道相互作用，以及 毒蕈碱受体刺激对G蛋白功能的影响。 几个假设将被测试，在实验中， 利用全细胞和单通道膜片钳 技术. 一个关键问题是， 动力学特性归因于钾通道本身是 G蛋白）-通道相互作用的结果。 这将被处理 实验上有两种方法，第一，用抗水解的GTP 类似物（消除G蛋白周转，并产生持久的 激活），第二，与试剂，扰乱流动性 肌膜 另一个需要解决的问题是， 钾离子通道激活在ACh的情况下，尽管 G蛋白周转率显著（0.3 min-1）。 我们将测试 这是否是因为活化的G蛋白对 频道低。 第三个调查领域是 通过毒蕈碱受体调节G蛋白功能， 特别是关于国内生产总值的增长 释放速率取决于受体类型。 最后一个假设 要测试的是G蛋白水解GTP的速率 可能会受到与渠道交互的影响，因此 通道自我限制其自身的激活。 这也将涉及一个 对脱敏现象的研究。 这些研究旨在深入了解 受体G蛋白介导的信号转导机制。 一个定量模型的组成部分之间的相互作用， 系统对于理解神经递质介导的 控制心脏兴奋性和收缩性，两个关键 正常心脏功能的决定因素。 这些研究还将 为研究其他G蛋白转导的 系统.

This proposal studies the mechanism of muscarinic receptor-mediated activation of potassium channels in enzymatically isolated, single bullfrog atrial cells. The ACh-induced, inwardly rectifying potassium current is also activated in a receptor-independent manner in the presence of hydrolysis-resistant GTP analogs, and thus the activation mechanism must include an understanding of GTP binding protein (G protein)-channel interactions, as well as the effect of muscarinic receptor stimulation on G protein function. Several hypotheses will be tested, in experiments which will utilize both the whole cell and single channel patch clamp techniques. A key question is whether any of the characteristic kinetic properties ascribed to the potassium channel itself are the result of G protein)-channel interactions. This will be approached experimentally in two ways, first, with hydrolysis-resistant GTP analogs (which eliminate G protein turnover, and produce persistent activation), and second, with agents that perturb the fluidity of the sarcolemma. Another issue which will be addressed is the lack of potassium channel activation in the absence of ACh, despite significant rates of G protein turnover (0.3 min-1). We will test whether this is because the affinity of the activated G protein for the channel is low. The third area of investigation is the modulation of G protein function by muscarinic receptor, specifically addressing the question of whether the increase in GDP release rate is dependent on receptor type. The final hypothesis to be tested is that the rate of GTP hydrolysis by the G protein may be influenced by interaction with the channel, and thus the channel self-limits its own activation. This will also involve an investigation of the phenomenon of desensitization. These studies are designed to produce insight into the kinetic mechanism of receptor-G protein - mediated signal transduction. A quantitative model of the interactions among components of the system is crucial to an understanding of neurotransmitter-mediated control of cardiac exitability and contractility, two key determinants of normal cardiac function. These studies will also provide a useful model for the study of other G protein-transduced systems.