Abstract 1070: An Unconventional Vesicular Transport Pathway Regulates the Cell Surface Expression of hERG K + Channels

Abstract

Approximately 35– 45% of patients that are genotype positive for congenital Long QT Syndrome (LQT) have mutations in the human Ether-a-go-go Related Gene ( hERG ). The purpose of this study was to elucidate the mechanisms that regulate ER export and cell surface expression of hERG channel protein, because these steps are impaired for ~90% of LQT-linked hERG missense mutations. The small GTPases Sar1 and Arf1 regulate the conventional vesicular transport (trafficking) for the ER export of proteins to the Golgi apparatus (Golgi). We generated dominant negative (DN) mutations for Sar1 and Arf1, and co-expressed these DN GTPases with hERG in HEK 293 cells. The trafficking of hERG through the Golgi can be visualized biochemically using Western blot analysis, because additional glycosylation of hERG in the Golgi (Golgi processing) increases the MW of hERG protein from 135kDa to 155kDa. Co-expression of hERG and DN-Sar1 inhibited Golgi processing, decreased hERG current (I hERG ) by 85% compared to control (n≥8 cells per group, p<0.05), and decreased the staining of hERG protein at the cell surface, while co-expression of hERG and DN-Arf1 showed no significant effect on Golgi processing or I hERG . This lack of an effect by DN-Arf1 was selective for hERG as it efficiently blocked the transport of previously reported proteins. Rab11 GTPases regulate the trafficking of proteins from endosomal compartments to the cell surface membrane and/or the Golgi. Rab11a is ubiquitously expressed, whereas Rab11b is expressed primarily in brain and heart. Co-expression of DN-Rab11a did not alter Golgi processing of hERG but reduced I hERG by 51% compared to control (n≥8 cells per group, p<0.05), whereas co-expression of DN-Rab11b inhibited Golgi processing of hERG and reduced I hERG by 79% compared to control (n=8 cells per group, p<0.05). Thus, Rab11a appears to regulate the trafficking of hERG to the cell surface after processing in the Golgi, whereas Rab11b regulates the trafficking of hERG prior to processing in the Golgi. These data suggest that hERG does not traffic via the conventional pathway from the ER to the Golgi, but rather in an unconventional pathway from the ER to endosomal compartments prior to Rab11b-mediated transport to the Golgi and subsequent delivery to the cell membrane.