Localization and trafficking of Kv channels in cardiac myocytes through the use of novel transfection methods

Voltage-gated K⁺ channels (Kv channels) play important roles in the repolarization and the termination of electrical excitation in cardiomyocytes, but very little is known about how their surface expression and localization are regulated. In this thesis, I report the characterization of Kv1.5 localization as well as the trafficking pathways utilized by Kv4.2 in ventricular myocytes. We have developed a new gene gun transfection method that, for the first time, allows the ready and convenient transfection of acutely isolated adult rat cardiac myocytes. Using this system combined with electrophysiology and confocal imaging experiments, we unequivocally demonstrate that tagged Kv1.5 is efficiently localized to the intercalated disk in ventricular myocytes. Furthermore, Kv1.5 deletion mutations known to reduce the surface expression of the channel in heterologous cells do not affect the channel localization to this structure. The ventricular myocyte transfection system combined with electrophysiological and imaging techniques has also been used identify the small GTPases that regulate the trafficking of cardiac Kv4.2. We demonstrate that the small GTPases Sar1, Rab1, Rab5, Rab4, Rab11and Rab7 are involved in specific steps in the forward and retrograde trafficking of Kv4.2 in rat ventricular myocytes. This work has provided critical insights into the trafficking of cardiac potassium channels and allows us to better understand the modulation of their function in the heart.Medicine, Faculty ofAnesthesiology, Pharmacology and Therapeutics, Department ofGraduat