An ER retention motif controls the heteromeric stoichiometry of hERG1a/1b channels

Abstract The human ether-à-go-go related gene ( hERG ) encodes a potassium channel essential for cardiac repolarization and neuronal excitability. In the heart, heteromeric assemblies of hERG1a and hERG1b subunits produce cardiac I Kr , and mutations in either subunit are associated with long QT syndrome. Although hERG1a and 1b contain identical transmembrane and C-terminal cytosolic domains, they differ in N-terminal cytosolic domains, with hERG1b harboring an arginine-based endoplasmic reticulum (ER) retention/retrieval motif that limits its surface expression in the absence of hERG1a. While the association of hERG1a and 1b subunits is known to influence channel function, the stoichiometry of heteromeric hERG channels and mechanisms that regulate it have remained unresolved. Here, using single molecule photobleaching step analysis in HeLa cells, we show that heteromeric hERG1a/1b channels assemble predominantly with a fixed 2:2 stoichiometry. Mutation of the hERG1b ER retention motif disrupts this bias, resulting in a broader, near-random distribution of subunit compositions. Independent functional assays using dominant-negative pore mutants in Xenopus oocytes yielded quantitative current suppression consistent with a 2:2 assembly and similarly revealed loss of stoichiometric bias upon RXR mutation. Together, these results establish the oligomeric composition of hERG1a/1b channels and identify ER retention as a previously unrecognized determinant of heteromeric stoichiometry. Statement of Significance Subunit stoichiometry is a key determinant of ion channel function, yet how defined stoichiometries are established during biogenesis remains poorly understood. Here we demonstrate that heteromeric hERG1a/1b channels assemble with a fixed 2:2 stoichiometry and that an arginine-based ER retention motif in hERG1b is required to maintain this assembly bias. Using single-molecule photobleaching and independent functional assays, we show that disrupting this motif leads to a broadened, near-random distribution of tetrameric subunit compositions. These findings reveal that ER retention contributes not only to quality control and trafficking but also specifies subunit stoichiometry during ion channel assembly.