In human heart, pore-forming KCNQ1 (KV7.1; Q1) subunits assemble with auxiliary KCNE1 (E1) subunits to form the slowly activating, delayed rectifier potassium current, IKs, which is essential for normal cardiac action potential (AP) repolarization. Decreased cardiac IKs prolongs the ventricular action potential duration (APD), resulting in long QT syndrome (LQTS), a disorder that predisposes to exertion-triggered fatal arrhythmias and sudden cardiac death (SCD). LQTS accounts for a significant portion of ~400,000 cases of SCD in the United States each year affecting all age groups from infants to the elderly. Current treatment options for LQTS (?- blocker therapy, implantable defibrillators, left cardiac sympathetic denervation) do not correct the underlying repolarization abnormality and all have significant limitations. Pathological decreases in cardiac IKs can arise due to inherited mutations in channel subunits (Q1? LQT1; E1? LQT5), or can be acquired in the failing heart, potentially as a consequence of the adverse neuro-hormonal milieu in this condition. Mechanistically, reduced cardiac IKs may be due to: (i) improper assembly of channel subunits; (ii) diminished trafficking of Q1 and/or E1 subunits to the heart cell surface; (iii) abnormal biophysical properties of surface channels (including diminished Po and rightward shifts in voltage-dependence of channel activation); and (iv) impaired sympathetic regulation. The precise molecular mechanisms underlying reductions in cardiac IKs density and/or functional regulation in most cases of inherited and acquired LQTS is unknown. This lack of clarity is a critical barrier to rational development of new therapies for this dangerous condition Factors contributing to the lack of progress are: (1) IKs is absent and does not contribute to action potential repolarization in popularly used mouse models; (2) lack of tools to quantitatively monitor dynamic IKs channel trafficking in heart; and (3) paucity of studies investigating functional impact of LQT1 mutations directly in adult cardiomyocytes. Our long term objective is to elucidate the molecular mechanisms controlling the surface density and functional regulation of Q1/E1 channels in heart under both physiological and pathological conditions, and to bridge the mechanistic insights to advance personalized therapy for LQTS and life-threatening cardiac arrhythmias. We have made several innovations to advance these objectives including developing optical tools to measure Q1/E1 assembly, surface density, and dynamic trafficking in live cells, and establishing two complementary adult cultured cardiomyocyte model systems to elucidate LQT1 mechanisms directly in heart cells. We propose three specific Aims: (1) Utilize optical approaches to illuminate mechanisms controlling surface density of Q1/E1 channel complexes in heart. (2) Elucidate mechanisms by which distinct LQT1 mutations in Q1 C-terminus impair IKs function in adult ventricular cardiomyocytes. (3) Determine the impact of protein kinases that are chronically elevated in heart failure on Q1/E1 surface density, trafficking, function, and regulation in heart.