The proposed study will examine how the ATP-sensitive potassium channel (KATP) in heart muscle is controlled. The work will investigate the interaction of physiological and pharmacological agents in short-and long- term regulation. The specific planned experiments are intended to elucidate the sub-cellular and molecular mechanisms underlying the modulation of KATP channel activity in mammalian heart cells. Whole-cell and patch-clamped preparations from rate and guinea-pig hearts will be used to investigate the mechanisms by which nucleotides inhibit channel activity and by which specific drugs (cromakalim, pinacidil, RP 49356) open the channel. The hypothesis that very long-term modulation of channel activity in response to environmental stimuli will be examined by experiments using tissue from different parts of the heart, and hearts exposed to different workloads. The planned experiments will address the following specific questions: (1) Does channel phosphorylation modulate ATP-sensitivity and is this an in vivo mechanism of channel regulation? The answers to this question will help understanding of the regulation of channel activity over a time-scale of minutes. (2) What kinetic scheme describes the interaction of ATP and other nucleotides with the channel? This investigation is designed to elucidate the short-term (millisecond) regulation of channel behavior. (3) What is the interaction between the KATP channel and the potassium channel opening drugs and sulfonylureas? This investigation will determine the interactions between potassium channel opening drugs and nucleotides in regulating channel behavior. (4) Is there a preferential source of ATP for channel inhibition? The route by which ATP diffuses to the channel is important to our understanding of how the channel is controlled in the intact cell. (5) Do environmental factors (such as mechanical load, hypertrophy, age) influence the numer, or ATP-sensitivity of KATP channels? The results of the proposed experiments, and answers to the above questions, will provide insight into the regulation of ion transport processes at the molecular level. The work will therefore provide information on the interactions between electrical and chemical features of the heart cell, relevant to normal and pathological function. The work should therefore lay the foundations for more rational therapies for myocardial diseases involving metabolic compromise.
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