Abstract 18298: Trafficking-Deficient hERG K+ Channels Linked to Long QT Syndrome are Regulated by a Quality Control Compartment in the ER

Abstract

The human Ether-a-go-go Related Gene (hERG) encodes the pore-forming voltage-gated K+ channel (Kv) α-subunit that underlies the rapidly-activating delayed-rectifier current in cardiomyocytes. hERG is synthesized in the Endoplasmic Reticulum (ER) as “immature” 135 kDa asparagine-linked (N-linked) glycoprotein & is terminally glycosylated in the Golgi apparatus (Golgi) to form a “mature” 155 kDa glycoprotein. Most hERG missense mutations linked to Long QT syndrome Type 2 (LQT2) reduce terminal glycosylation & functional expression. E-4031 is a class III anti-arrhythmic that binds to hERG & blocks current (IhERG), & can increase terminal glycosylation & functional expression for many “trafficking-deficient” LQT2 mutations (pharmacological correction). Treating HEK293 cells expressing the trafficking-deficient LQT2 mutation G601S-hERG in 10 uM E-4031 (E4) for 6 or 12 hours showed a time-dependent increase in maximal IhERG after drug washout, Western blot analyses showed similar increase in the fractional density (FD) of terminally glycosylated G601S-hERG. Treating cells in 0.2mg/ml cycloheximide (cyclo), that inhibits protein translation, did not alter the E4 induced increase in terminally glycosylated G601S-hERG, suggesting pharmacological correction occurs post-translationally. Immunocytochemistry & colocalization analyses showed that in control conditions G601S-hERG primarily colocalized w/ BAP31, an itinerant ER protein that moves between the peripheral ER and perinuclear ER. G601S-hERG weakly colocalized w/ other ER proteins, like lecithin chaperone calnexin, ERAD complex protein Derlin, or transitional ER marker Sec31. Treating cells in E4 decreased G601S-hERG and BAP31 colocalization after 6 hrs & 12 hr. Treatment with microtubular depolymerizing agent nocodazole (10 uM) for 2 hrs increased G601S-hERG colocalization with BAP31 & caused them to accumulate into peripheral ER compartments. We conclude that in control conditions, G601S-hERG is an itinerant ER protein that moves between the peripheral and perinuclear ER via a microtubule-dependent mechanism, & pharmacological correction with E-4031 prevents this by allowing transport to the Golgi & cell surface membrane.