A possible role for phase separation in heteromeric biogenesis of hERG1a/1b channels.

Abstract

The human ether-a-go-go related gene (hERG) encodes a K+ channel important for repolarizing the cardiac action potential. Cardiac IKr is produced by heteromeric assemblies of hERG1a and 1b subunits, and depletion of hERG1b subunits confers proarrhythmic behavior in cellular cardiac models. hERG1a homomers can traffic to the membrane, but hERG1b homomers fail to progress efficiently out of the endoplasmic reticulum (ER). We found that hERG1b protein is sequestered and forms punctate intracellular structures when expressed heterologously in HeLa cells. When co-expressed with hERG1a, previously shown to promote hERG1b maturation, few puncta are observed and both signals are robustly present at the plasma membrane. We show that 1b puncta are primarily localized to the ER space and do not overlap with other membranous compartments, including COPII, lysosome, and autophagosome. Testing the hypothesis that hERG1b puncta are condensates arising from liquid-liquid phase separation, we used live cell imaging and observed that sequestered hERG1b puncta undergo fusion events, although rarely as might be expected from ER-delimited membrane proteins. Using truncation experiments, we found that removing the disordered C-terminus altered the size and distribution of the puncta but removing the structured cyclic nucleotide-binding homology domain (CNBhD) abolished the puncta entirely. These data suggest the puncta are formed by multivalent interactions of ordered and disordered domains of hERG1b and may sequester hERG1b in the absence of hERG1a. In this way sequestration in puncta may limit deleterious homomeric expression of hERG1b and thus promote heteromeric hERG1a/1b channel assembly.