Cardiac function is regulated by various receptor-dependent cell signalling pathways, e.g. beta-adrenergic and muscarinic acetylcholine (ACh receptors. GTP-binding proteins play a key role in linking many membrane receptors and effectors, such as adenylate cyclase and phospholipase C. Upon agonist-binding, the heterotrimeric G proteins are functionally dissociated into their subunits (GTP-bound G alpha and G beta gamma). Both subunits serve as potential messengers. A number of cardiac ion channels can be directly regulated by G proteins. In this proposal we will focus on the inwardly-rectifying muscarinic K channel (KAch). ACh and adenosine activate the KAch channel via-pertussis toxin-sensitive G proteins in nodal and atrial myocytes, resulting in deceleration of the heart rate. The channel is consistently activated by intracellular GTP (in the presence of agonists) and purified G protein subunits in isolated patches. The KAch channel, therefore, serves as a sensitive indicator of G protein function and may be used to explore the receptor-G protein-effector interactions, with unprecedented time resolution. We will quantitatively investigate the molecular mechanisms underlying activation, modulation and desensitization of the G protein-gated KAch channel by using the patch clamp technique on isolated cardiac atrial myocytes. Briefly, the specific aims of this project are: (1) To examine GTP- activation of the channel and identify the kinetic states affected by GTP, thereby constructing a quantitative kinetic model for the G protein-channel interaction. (2) To examine activation of the KAch channel by G protein subunits with the aim of identifying the subunit activating the channel in vivo and elucidating the molecular mechanisms of G beta gamma subunit-activation (3) To elucidate the underlying mechanisms and clarify the physiological roles of modulation of the KAch channel by non-G protein substances, e.g. arachidonic acid metabolites. (4) To elucidate the molecular mechanisms underlying desensitization of muscarinic activation of the KAch channel. Our long term goal is to elucidate the functional and molecular mechanisms of receptor-dependent regulation of cardiac ion channel under both physiological and pathophysiological conditions. Alterations in the functions of any of these pathways may lead to cardiac dysfunction. Therefore, an understanding of the mechanisms will improve the therapeutic strategies employed in the treatment of various heart diseases.
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