The Trafficking of Mutant HERG K+ Channels Linked to Long QT Syndrome are Regulated by a Subdomain in the Endoplasmic Reticulum

Abstract

Type 2 Long QT syndrome (LQT2) is caused by human Ether-a-go-go Related Gene (hERG) mutations, and studies suggest that most LQT2 missense mutations are retained in pre-Golgi compartments. We tested the hypothesis that trafficking-deficient LQT2 (tdLQT2) channels are regulated by quality control mechanisms in specialized subdomains of the Endoplasmic Reticulum (ER) and/or the ER Golgi Intermediate Compartment (ERGIC). An inherent problem with studying the localization of proteins to ER subdomains and the ERGIC is that they are in close proximity and it is difficult to distinguish them from one another. We found that acutely treating cells with nocodazole (a microtubule depolymerizing agent) and brefeldin (bfa, an inhibitor of Coat Protein I vesicular transport) allowed us to delineate specialized ER subdomains and the ERGIC using confocal microscopy. We assessed whether the tdLQT2 mutation G601S-hERG colocalized with the different ER subdomains and/or the ERGIC in stably expressing HEK293 cells. Imaging data show that G601S-hERG accumulated in the peripheral ER subdomains and did not colocalize with markers for perinucler ER subdomains, transitional ER subdomains, or the ERGIC. Treating cells in E-4031, a drug that increases G601S-hERG trafficking in the secretory pathway, prevented G601S-hERG from accumulating in the peripheral ER subdomains and promoted the movement of G601S-hERG into the ERGIC. The data suggest that cellular quality control mechanisms within specialized ER subdomains are primarily responsible for the ER retention of G601S-hERG, and that E-4031 facilitates the movement of G601S-hERG out of the specialized ER subdomains and into the ERGIC.