Ernandez, L. R. | bioRxiv Science

bioRxiv2026

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

The human ether-a-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 IKr, 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 SignificanceSubunit 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.

biophysics↗

bioRxiv2025

A privileged ER compartment for post-translational heteromeric assembly of an ion channel

Mechanisms underlying heterotypic subunit assembly of ion channels and other oligomeric complexes are poorly understood. In the human heart, heteromeric assembly of two isoforms encoded by the human ether-a-go-go related gene (hERG) is essential for the normal function of cardiac IKr in ventricular repolarization, with loss of hERG1b contributing to arrhythmias associated with long QT-syndrome (LQTS). While hERG1a homomers traffic efficiently to the plasma membrane, hERG1b homomers are retained in the endoplasmic reticulum (ER). When expressed together, the two subunits avidly associate during biogenesis. Seeking rules specifying heteromeric association, we characterized the fate of hERG1b proteins using confocal and superresolution imaging in fixed and live HeLa cells. We found hERG1b sequestered in punctate intracellular structures when expressed alone in HeLa cells. These puncta, which depend on the presence of an N-terminal "RXR" ER retention signal, represent a privileged ER sub-compartment distinct from that containing ER-retained, type 2 (hERG-based) LQTS mutant proteins, which were rapidly degraded by the proteasome. Introducing hERG1a to cells with preformed hERG1b puncta dissolved these puncta by rescuing extant hERG1b. Rescue occurred by association of fully translated hERG1b with 1a, a surprising finding given previous studies demonstrating cotranslational heteromeric association. We propose that sequestration limits potentially deleterious surface expression of hERG1b homomeric channels while preserving hERG1b for an alternative mode of heteromeric hERG1a/1b channel assembly post-translationally. These findings reveal a surprising versatility of biosynthetic pathways promoting heteromeric assembly. Significance StatementhERG potassium channels are essential for repolarizing the ventricular action potential. Heteromeric hERG channels, which conduct cardiac IKr, are formed by association of hERG1a and 1b subunits. However, mechanisms governing the assembly of hERG and other heteromeric protein complexes are poorly understood. We identify a noncanonical ER compartment that sequesters hERG1b, distinct from previously described ER quality control pathways that degrade misfolded proteins, including LQTS-associated hERG variants. Instead, this compartment preserves hERG1b and facilitates its post-translational assembly with hERG1a, challenging the current view of simultaneous, cotranslational heteromeric assembly. Our findings redefine ER retention as an active regulatory step in protein complex formation that may broadly govern hetero-oligomeric assembly in many systems and facilitate therapeutic approaches to arrhythmia and other diseases.

cell biology↗