BPS2026 – Orthogonal approaches for determining the stoichiometry of heteromeric hERG1a/1b channels

The human ether-à-go-go related gene (hERG) encodes a potassium channel important for repolarizing the cardiac action potential and modulating neuronal firing. Heteromeric assemblies of hERG1a and hERG1b subunits produce cardiac I Kr , and dysfunction or depletion of hERG1b is associated with arrhythmia. hERG1a and 1b contain identical transmembrane and C-terminal cytosolic domains but differ in N-terminal cytosolic domains. hERG1b N-terminal domain contains an ER retention motif that prevents aberrant trafficking of homomeric hERG1b and promotes heteromeric assembly. Although the balance between hERG1a and 1b subunits is important in normal function, the stoichiometry of heteromeric hERG channels and mechanisms that regulate the proper stoichiometry was previously unknown. Here, we expressed hERG1a and 1b in HeLa cells and used TIRF microscopy and single-molecule photobleaching analysis to count the number of fluorescently tagged subunits. Photobleaching step distributions showed that the stoichiometry of the heteromeric hERG1a/1b channel is predominantly 2:2. When the hERG1b ER retention signal was mutated, the stoichiometry was shifted from fixed 2:2 to a random distribution. To determine stoichiometry using an independent method, we used dominant-negative poison subunits in Xenopus oocytes, where current suppression matched the predictions for a 2:2 arrangement and shifted to random assembly upon RXR mutation. This suggests a role for ER sequestration or the biochemistry of the RXR motif in establishing proper stoichiometry. In conclusion, we have resolved the oligomeric composition of hERG1 channels and uncovered a new mechanism regulating their stoichiometry.