Probing the Trafficking Routes of KCNQ1 and KCNE1 After Their ER Exit

Background: KCNQ1 (Q1, pore-forming channel subunit) and KCNE1 (E1, regulatory subunit) associate to form the slow delayed rectifier (IKs) channel, important for cardiac action potential repolarization. Although Q1/E1 are obligatory partners in cardiomyocytes, in adult ventricular myocytes the two are not well colocalized: E1 is on the lateral cell surface while Q1 is mainly in the intracellular junctional SR. This appears to contradict data from heterologous expression or non-cardiac cell types, which suggest that Q1/E1 assemble into IKs when they are still in ER or soon thereafter. The current study is designed to distinguish between 2 scenarios. First, Q1 and E1 traffic from ER to the plasma membrane together, followed by Q1 internalization to the ER/SR compartment. Alternatively, Q1 and E1 traffic on different routes from ER to their separate destinations.Methods: COS-7 cells transfected with Q-GFP/E1-dsR are cultured in presence of brefeldin A (BFA) for 12 – 16 hr, to allow protein translation without ER exit. Cells are imaged in absence of BFA but presence of cycloheximide+dynasore (blocking protein translation and endocytosis). Time-lapse images (Zeiss 710, 37oC) of 4-um optic slice (to maximize capture of trafficking events in thin cytosol of COS-7 cells) are recorded till the Q1-GFP and E1-dsR distribution reaches quasi steady-state (> 2 hr).Results: BFA removal allows ER exit and Golgi reconstruction. E1-dsR exits ER in distinct vesicles, more frequently at the cell periphery. These vesicles gradually cluster to the peri-nuclear region, entering the post-BFA Golgi. Q1-QFP stays in the ER compartment for > 2 hr. Its ER location is confirmed by FRAP experiments.Conclusion: Our data support the second scenario. Experiments are in progress to test whether E1-dsR and Q1-GFP behave the same when expressed in a cardiac myocyte environment.