Regulation of ion channel function plays a pivotal role in controlling heart rate and contractility via changes in cardiac myocyte excitability. Humoral mediators and receptors are involved in determining channel responses to changing cardiovascular demands. The dynamic beat-to-beat regulation of ion channels is precisely controlled by autonomic stimulation through complex interplay of second messengers, kinases, G-proteins, and protein-protein interactions. The rapidly activating delayed rectifier current (IKr) produced by the HERG gene is uniquely qualified for an integrative transduction of cellular/hormonal signals due to its unusual biophysical properties and the growing number of second-messenger and protein interactions ascribed to the channel. We have begun mapping alpha- and beta-adrenergic signaling pathways to modulation of HERG/IKr. Beta-adrenergic stimulation leads to a complex series of events that includes; direct binding of cAMP to HERG, interactions with AKAPs, PKA-mediated phosphorylation of the channel, and binding of 14-3-3 to HERG. Thus, an evolving picture of adrenergic regulation of HERG is emerging. It will be essential to understand the mechanisms by which acute and chronic stress adaptation leads to arrhythmia and sudden death in both acquired and hereditary cardiac disease. Accordingly, we propose to explore the effects and mechanisms of each of these interactions in a multidisciplinary approach combining protein biochemistry, immunochemistry, and patch clamp electrophysiology on heterologously expressed proteins and endogenous cardiac tissue. Specifically, we aim to: 1. Determine the significance of 14-3-3e and PKA regulation of HERG 2. Examine PKA targeting to HERG channels by AKAPs. 3. Examine PKA-mediated control of trafficking of HERG protein.
