G PROTEIN-MEDIATED K+ CHANNEL ACTIVATION IN HEART

本方案研究了M受体介导的作用机制。 酶法分离的单细胞钾离子通道的激活 牛蛙的心房细胞。ACh诱导的内向纠偏 钾电流也是在一种非受体依赖的 在存在耐水解的GTP类似物时的方式，以及 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 刺激M受体对G蛋白功能的影响。 几个假设将在实验中得到验证， 利用全细胞和单通道膜片钳 技巧。一个关键的问题是，是否有任何特征 归因于钾通道本身的动力学特性是 G蛋白)-通道相互作用的结果。我们将着手解决这一问题 实验中有两种方式，第一，使用抗水解的GTP 类似物(消除G蛋白周转，并产生持久的 激活)，第二，使用扰乱流动性的试剂 肌膜。另一个要解决的问题是缺乏 在没有ACh的情况下钾通道的激活，尽管 显著的G蛋白周转率(0.3min-1)。我们将测试 这是否是因为被激活的G蛋白对 这条通道很低。第三个调查领域是 M受体对G蛋白功能的调节 具体解决国内生产总值的增长是否 释放率取决于受体类型。最后一个假设 要测试的是G蛋白对GTP的水解率 可能受到与通道的交互的影响，因此 渠道自我限制自己的激活。这还将涉及到一个 对脱敏现象的研究。 这些研究的目的是为了深入了解 受体G蛋白介导的信号转导机制。 组件间相互作用的定量模型 系统对于理解神经递质介导的 控制心脏兴奋性和收缩性是两个关键 正常心功能的决定因素。这些研究还将 为其他G蛋白转导的研究提供了一个有用的模型 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.