Arterial membrane potential is an essential regulator of contractility. Smooth muscle cell (myocyte) BKCa channels are a major physiological modulator of arterial membrane potential. In arterial myocytes, BKCa channels are formed from pore-forming alpha subunits and auxiliary beta 1 subunits. BKCa channel activation induces membrane hyperpolarization and vasodilation. Signaling pathways that control the number of functional ion channels on the plasma membrane in arterial myocytes are poorly understood. This application stems from exciting preliminary data indicating for the first time that BKCa channel plasma membrane subunit composition in arterial myocytes is dynamic and rapidly modulated by physiological stimuli to control arterial contractility. Our preliminary data indicate that, 1. BKCa alpha subunits are primarily plasma membrane-located, whereas beta 1 is primarily intracellular, 2. Nitric oxide or membrane depolarization stimulate rapid (minutes) beta 1 subunit plasma membrane trafficking and membrane insertion, 3. Knockdown of rab11A, a recycling vesicle small GTPase, abolishes NO- and depolarization-induced beta 1 trafficking, 4. NO-induced beta 1 subunit trafficking elevates BKCa channel activity, Ca2+ sensitivity and activation by lithocholate, a beta 1 subunit ligand and 5. NO-induced trafficking of beta 1 subunits to the plasma membrane and subsequent BKCa channel activation induces vasodilation. We will test the central hypothesis that vasoregulatory stimuli activate rab11A-dependent plasma membrane insertion of beta 1 subunits in arterial myocytes, leading to an elevation in BKCa channel Ca2+ sensitivity and vasodilation. Techniques to be used include surface biotinylation, immuno-Fluorescence Resonance Energy Transfer (Immuno-FRET) microscopy, western blotting, gene knockdown, patch-clamp electrophysiology, high-speed confocal Ca2+ imaging, membrane potential measurements and pressurized artery diameter measurements. This work aims to identify a novel cellular trafficking pathway that modulates BKCa channel activity and vascular tone.
|Program type||Postdoctoral Fellowship|
|Effective start/end date||07/01/2012 → 06/30/2014|