Traffic Control of Cardiac Kv Channels



 DESCRIPTION (provided by applicant): The long-term objectives of our project are to understand (a) the traffic control of Kv channel subunits in cardiac myocytes under basal conditions and in response to stresses, and (b) how these trafficking events impact on Kv channel function and cardiac electrical activity. The current proposal is focused on the slow delayed rectifier (IKs) channel. IKs is composed of KCNQ1 (pore-forming) and KCNE1 (regulatory) subunits. Due to its small amplitude and slow rate of activation, IKs is not a major repolarizing force in cardiac myocyte under basal conditions. In fact, too much IKs can increase the risk for arrhythmia. However, under stressful conditions when more repolarizing current is needed to shorten the action potential duration (high β-adrenergic tone), the IKs amplitude is increased and its activation becomes faster to provide extra repolarizing current needed for the task. Therefore, IKs is a 'repolarization reserve': it helps the heart cope with stresses. This proposal is based on our recent findings in adult ventricular myocytes: under basal conditions KCNQ1 and KCNE1 are largely segregated from each other. KCNE1 is on the cell surface, while KCNQ1 is in a cytosolic striation compartment. Unpublished experiments further suggest that KCNQ1 and KCNE1 are translated in different rough ER domains, and travel by different routes to their respective destinations. To explore the pathways and targeting/anchoring mechanisms that control the distribution patterns of KCNQ1 and KCNE1 in adult ventricular myocytes, we propose 3 Specific Aims: (1) To delineate the spatiotemporal relationships among the subcellular compartments traversed by KCNQ1 and KCNE1 during their biogenesis, (2) To determine the nature of KCNQ1 striation compartment in adult ventricular myocytes, (3) To determine how functional IKs channels are formed in adult ventricular myocytes under physiological conditions. To accomplish these Aims, we will express fluorescently tagged KCNQ1 and KCNE1 in adult guinea pig ventricular myocytes, and use an 'optical pulse-chase' strategy to follow their movements through subcellular compartments under varying conditions. These confocal experiments are supplemented by patch clamp recording (to assay IKs channel function), and protein biochemistry (to quantify protein levels in different subcellular compartments). We believe IKs channel is not unique among membrane channels: other multiple-component channels may have their components translated separately and assembled at a late stage of biogenesis, and several channels have been shown to have intracellular reservoirs that can quickly deliver new channels to the cell surface in times of need. Knowledge gained here will pave the way for rethinking how membrane channel subunits are translated, stored, and assembled in cardiac and other cell types.