Assembly and Localization of hERG Channel Cotranslational Complexes

Project Summary In the heart, perturbation in the balance of channels that produce the ventricular action potential (AP) can lead to dangerous arrhythmias and sudden cardiac death. How excitable cells regulate the precise the bal- ance of ion channels mediating electrical signaling is poorly understood. Previous studies in the Robertson lab have shown that ion channels are co-regulated at the translational level, via pairs of interacting mRNA during biosynthesis. My project aims to investigate how and where this cotranslational regulation occurs. Previous studies found that mRNAs of functionally related ion channels – including hERG1a, Cav1.2, Nav1.5, and KCNQ1- critical for the AP co-purify with nascent proteins via antibody pull-down. These associations were also identified using single-molecule fluorescence in situ hybridization (smFISH) combined with immu- nofluorescence. Similarly, alternative transcripts hERG1a and 1b associate during biosynthesis of heter- omeric channels producing the repolarizing current IKr. However, technical obstacles have limited the charac- terization of hERG1b within the regulatory framework of functionally related but distinct ion channels pairs. My project leverages RNAscope, a smFISH technique capable of detecting hERG1b to probe several key questions, and for which I provide preliminary data supporting its implementation. In Aim 1, I will determine whether hERG1a channels co-synthesized with functionally related channels are homotetramers, or whether they are heterotetramers with hERG1b. In Aim 2, I will determine whether these channel pairs are synthesized in the perinuclear ER, or near their functional domains. Using engineered micropatterned iPSC-CMs, and mature human myocardium I will apply RNAscope to test the hypothesis that a subset of translational com- plexes localize near insertion sites on the T-tubule and intercalated disc. This fellowship training plan is intended to prepare me for a career as tenure-track faculty and will include weekly mentoring sessions with Dr. Robertson to discuss progress toward IDP goals, participation in lab meetings, participation and presentation in journal clubs, managing collaboration with Dr. Lee Eckhardt’s lab, the cardiac tissue bank and the Translational Research Initiatives in Pathology lab, and mentoring students in a richly diverse laboratory. I will gain more knowledge in RNA biology, biogenesis, single-molecule micros- copy, advanced analysis techniques, and new human cellular and tissue cardiac models. I plan to broaden my network and present my work at local symposia and international conferences, with the goal of becoming a versatile and collaborative scientist prepared for the next steps in my career.