Abstract 418: Heteromeric Assembly Of hERG1 Channels Occurs Co-Translationally Via Amino-Terminal Interactions
The human Ether-à-go-go-Related Gene (hERG) encodes channels producing IKr currents in the human heart. hERG subunits are encoded by two alternate transcripts, 1a and 1b, which are identical except for their divergent amino (N) termini. Mutations or drug block of the hERG channel can result in Long QT Syndrome (LQTS) leading to potentially fatal ventricular arrhythmias. Studying the assembly process of the hERG subunits is important in elucidating the varied mechanisms by which hERG mutations, many of which are unique to the 1a N-terminus, can result in trafficking defects and disease. Here, we tested the hypothesis that the preferential assembly of hERG 1b with 1a is mediated by N-terminal interactions. Our results demonstrate that 1a and 1b heteromerize in the endoplasmic reticulum. A previously identified C-terminal tetramerization domain is not required for this interaction. hERG 1a and1b N-termini were sufficient for inter-subunit association and in vitro binding assays demonstrated that this binding is direct and dose-dependent. To determine whether heteromerization takes place after or during the subunit synthesis, we exploited an observation that truncation of the 1b C-terminus (1bΔCT) disrupts Golgi-mediated glycosylation, leaving only an N-linked core glycoform. Core glycosylation of 1bΔCT occurred within 5 minutes of metabolic labeling with 35S-methionine, consistent with other studies indicating core glycosylation occurs during protein synthesis. Core glycosylation was disrupted when 1bΔCT was co-expressed with 1a N-terminal fragments, suggesting that 1a and 1b N-termini associated before the glycosylation event and, therefore, co-translationally. This study suggests that hERG 1a and 1b assembly is mediated by their N-termini as they emerge from the ribosome. Inefficient heteromerization of hERG subunits caused by mutations in the N-termini represents a potentially novel mechanism underlying trafficking defects associated with LQTS.