Abstract 1524: hERG and MiRP1 Do Not Associate Prior to Export Out of the Endoplasmic Reticulum
Congenital Long QT Syndrome (LQTS) has been linked to mutations in the human Ether-a-go-go Related Gene (hERG) and KCNE2 gene. These genes encode the α- and auxiliary subunits that underlie the rapidly activating delayed rectifier K+ current (IKr) in cardiac myocytes. In addition to hERG K+ channels, MiRP1 is also thought to regulate the function of several other voltage-gated K+ channels expressed in the heart. Many hERG K+ channel mutations linked to LQTS disrupt the intracellular transport (trafficking) of hERG K+ channels out of the Endoplasmic Reticulum (ER). The purpose of this study was to determine whether hERG K+ channels and MiRP1 co-assemble prior to ER export. We first determined whether or not trafficking-deficient hERG K+ channels disrupted the trafficking of wild type (WT) MiRP1. We transiently expressed the trafficking-deficient LQTS-linked mutations, N470D hERG or G601S hERG, and WT MiRP1 that contained a poly-histidine tag in HEK 293 cells. We used immunocytochemical techniques and confocal imaging to visualize the localization of these proteins. Images showed that N470D or G601S hERG channel proteins were retained intracellularly, whereas, the transport of WT MiRP1 to the cell surface did not appear disrupted. These data suggest that hERG K+ channels and MiRP1 may associate after their transport out of the ER. To test this, we co-expressed WT hERG and WT MiRP1 with WT or dominant-negative small G-proteins that regulate protein trafficking out of the ER. Immunocytochemical techniques show that the cell surface expression of WT MiRP1, but not WT hERG, was inhibited by the co-expression of the dominant-negative mutation in the small G-protein ADP-Ribosylation Factor 1 (Q71L-ARF1). Together, these data suggest that hERG K+ channels and MiRP1 do not associate with one another in the ER, and their transport out of the ER may be regulated by different small G-proteins. These data are important because they suggest that trafficking-deficient LQTS-linked hERG mutations do not appear to disrupt MiRP1 transport.
This research has received full or partial funding support from the American Heart Association, AHA Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).