How do KCNQ1 and KCNE1 Assemble to form the Slow- Delayed-Rectifier (IKs) Channels in Adult Ventricular Mocytes (AVMs)?

Abstract

I Ks plays a key role in ventricular-repolarization during high b-adrenergic tone, and is composed of KCNQ1 (channel component) and KCNE1 (regulatory subunit). Although KCNQ1 and KCNE1 are obligate partners, we showed that they are largely segregated in AVMs: KCNQ1 mostly in cytosolic compartment while KCNE1 on lateral cell surface (LCS). Only a small portion of KCNQ1 appears to overlap with KCNE1 on LCS. To address the above question, we conduct proof-of-principle experiments by expressing fluorescent protein (FP)-tagged KCNQ1 and KCNE1 in COS-7 cells, and track their movements using confocal, TIRF and structured-illumination microscopies. We use a strategy called 'retention-using-selective-hooks' to differentiate how KCNQ1 and KCNE1 traffic after they exit the endoplasmic reticulum (ER). We use proximity-ligation-amplification (PLA) to locate KCNQ1/KCNE1 assemblies in cells. We conduct physiological experiments by in vitro or in vivo expression of FP-tagged KCNQ1 and KCNE1 in AVMs, using adenovirus or AAV9 respectively. We observe bright spots of KCNQ1-GFP moves within the ER network. When reaching cell edge, KCNQ1-GFP appears to bud off ER in vesicles and disappears presumably by fusing with the plasma membrane (PM). Vesicles of KCNE1-dsRed move in the cytoplasm, and often appear to drag KCNQ1-GFP/ER along tracks or carry bright spot of KCNQ1-GFP transiently. When expressed in AVMs, KCNQ1-GFP clusters to the nuclear envelope and along the z-lines and KCNE1-dsRed is detected in vesicles and LCS. KCNQ1-GFP/KCNE1-dsRed assemblies are present on the LCS and in the sub-sarcolemma compartment beneath LCS. We conclude that KCNQ1 and KCNE1 traffic by different routes to PM and assemble once there: KCNQ1 via an ER/SR route while KCNE1 via a vesicular route. KCNE1 appears capable of guiding KCNQ1 to exit ER/SR, facilitating the formation of I Ks channels on cell surface.

Gating and Regulation of KCNQ1 and KCNQ1 + KCNE1 Channel Complexes

“…How KCNQ1 and its accessory subunit KCNE1 traffic and assemble to form complexes with unique physiological roles is still a subject of some debate. Overall, two main theories for trafficking and assembly exist, one involves KCNE1 and KCNQ1 trafficking separately, KCNE1 by a vesicular route and KCNQ1 trafficking by an endoplasmic reticulum or sarcoplasmic reticulum route (Poulsen and Klaerke, 2007;Jiang et al, 2017;Kaur et al, 2020). Once at the plasma membrane the two subunits then assemble.…”