Abstract
Alternate transcripts of the human ether-à-go-go-related gene (hERG1) encode two subunits, hERG 1a and 1b, which form potassium channels regulating cardiac repolarization, neuronal firing frequency, and neoplastic cell growth. The 1a and 1b subunits are identical except for their unique, cytoplasmic N termini, and they readily co-assemble in heterologous and native systems. We tested the hypothesis that interactions of nascent N termini promote heteromeric assembly of 1a and 1b subunits. We found that 1a and 1b N-terminal fragments bind in a direct and dose-dependent manner. hERG1 hetero-oligomerization occurs in the endoplasmic reticulum where co-expression of N-terminal fragments with hERG1 subunits disrupted oligomerization and core glycosylation. The disruption of core glycosylation, a cotranslational event, allows us to pinpoint these N-terminal interactions to the earliest steps in biogenesis. Thus, N-terminal interactions mediate hERG 1a/1b assembly, a process whose perturbation may represent a new mechanism for disease.
Highlights
RESULTS hERG1 Subunits Assemble in the endoplasmic reticulum (ER)—Core glycosylation of proteins has been shown to be a cotranslaminitab)-supplemented 10 mM Tris-HCl, pH 7.4, 250 mM sucrose, tional event in the ER lumen (29 –31)
The We showed previously that hERG 1b subunits expressed in supernatant was subjected to a final centrifugation at 100,000 ϫ g HEK-293 cells mature from an ϳ85-kDa core-glycosylated spefor 90 min to separate small organelles (e.g. endosomes, lysosomes; cies in the ER to a ϳ90-kDa species in the medial Golgi [24], 100,000 ϫ g pellet) and the cytosol (100,000 ϫ g supernatant)
We investigated the biochemical basis for heteromeric assembly of hERG1 channels during biogenesis
Summary
Plasmids—For mammalian expression, hERG 1a (aa 1–1159) and 1b (aa 1– 819) cDNA were subcloned into pcDNA3.1 vector (Invitrogen). B, lysate from HEK-293 cells transiently expressing 1a and 1b was immunoprecipitated with 1b-specific rabbit antibody, and the Western blot was probed with mouse antibody against the common C terminus of hERG1 isoforms. The We showed previously that hERG 1b subunits expressed in supernatant was subjected to a final centrifugation at 100,000 ϫ g HEK-293 cells mature from an ϳ85-kDa core-glycosylated spefor 90 min to separate small organelles (e.g. endosomes, lysosomes; cies in the ER to a ϳ90-kDa species in the medial Golgi [24], 100,000 ϫ g pellet) and the cytosol (100,000 ϫ g supernatant). Pellet was dissolved in 300 l of lysis buffer (protease-inhibitor Here we show both the mature and immature glycoforms of supplemented 50 mM Tris-HCl, pH 7.2, 1 mM EDTA, 150 mM hERG 1a and 1b can be co-immunoprecipitated from cells transodium chloride, 1% Triton X-100). We conclude that the C terminus, which includes a tetramerization coiled-coil (TCC) domain [34], is not required for hERG 1a/1b heteromeric association
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