G PROTEIN-MEDIATED K+ CHANNEL ACTIVATION IN HEART

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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.