We hypothesize that targeting of channels to particular membrane microdomains and their organization in macromolecular complexes allow cells to increase the efficiency of their response to extracellular signals. We plan to study the molecular mechanisms underlying the trafficking and turnover of Kv1.5 and KV2.1 potassium channels in the heart. We will characterize the macromolecular complex (channelosome) that modulates the function of a delayed rectifier potassium channel, Kv1.5. To this end, we will use a combination of biochemical and molecular tools, patch-clamp analyses, and in vivo mouse models to determine the mechanisms that regulate trafficking of Kv1.5 polypeptide to caveolae, specifically to characterize the interactions between Kv1.5 and caveolin-3 (Cav-3) and SAP97. In addition, we plan to examine the role of posttranslational modifications on the trafficking, assembly, and stability on Kv1.5/Cav-3/SAP97 macromolecular complex. We will map the site of interactions between SAP97 and Cav-3 and prove that this interaction contributes to the targeting of Kv1.5 to caveolae. We also plan to study a second voltage-gated potassium channel (Kv2.1). We plan to characterize the role of N-terminal cysteines of Kv2.1 and their posttranslational modifications in regulating the assembly, trafficking, and gating of Kv2.1. The work outlined in this application should greatly increase our understanding of the regulation of expression of Kv1.5 and Kv2.1.
Long QT syndrome (LQTS) is associated with delayed cardiac repolarization, prolonged QT intervals, recurrent syncope, ventricular arrhythmias, and sudden death. Since mouse models for both LQT1 and LQT2 have failed to mimic the arrhythmias in human LQT1 and LQT2, we created transgenic rabbit models deficient in voltage gated potassium channels (IKs and IKr) by overexpressing either KvLQT1-Y315S in the heart in order to attenuate IKs, or HERG-G628S to attenuate IKr. We next plan to analyze and compare the phenotype of these models with surface ECG; monitoring of alert, free-moving rabbits; and programmed electrical stimulation (PES) of the right ventricle of anesthetized rabbits. In addition, we plan to characterize the biochemical and electrophysiological phenotype of rabbit cardiomyocytes derived from the epicardial, mid-myocardial, and endocardial layers of the left ventricle of KvLQT1-Dn and ERG-DN rabbits. Specifically, to characterize and compare the expression of native potassium channel polypeptides, the APDs, the inward and outward potassium currents, and the inward calcium currents expressed in these cardiomyocytes. We will evaluate the effect of the adrenergic agonist isoproterenol and transmural distribution of transmembrane action potential characteristics across the ventricular wall in KvLQT1-DN and ERG-DN rabbit models using a wedge preparation. We will assess the contribution of transmural electrical heterogeneity induced by isoproterenol to the development of long QT intervals, broad-based T waves, transmural dispersion of repolarization, and the development of spontaneous as well as programmed electrical stimulation (PES)-induced TdP in the two models. These rabbits will likely provide new insights into the mechanisms that underlie the ventricular arrhythmias and the electrical remodeling detected in LQT1 and LQT2. These models may also enable drug screening for acquired LQTS, and the development of gene therapy for congenital LQTS.