Supramolecular clustering of the cardiac sodium channel Nav1.5 in HEK293F cells, with and without the auxiliary β3‐subunit

Voltage-gated sodium channels comprise an ion-selective alpha-subunit and one or more associated beta-subunits. The beta3-subunit (encoded by the SCN3B gene) is an important physiological regulator of the heart-specific sodium channel, Nav1.5. We have previously shown that when expressed alone in HEK293F cells, the full-length beta3-subunit forms trimers in the plasma membrane. We extend this result with biochemical assays and use the proximity ligation assay (PLA) to identify oligomeric beta3-subunits, not just at the plasma membrane, but throughout the secretory pathway. We then investigate the corresponding clustering properties of the alpha-subunit and the effects upon these of the beta3-subunits. The oligomeric status of the Nav1.5 alpha-subunit in vivo, with or without the beta3-subunit, has not been previously investigated. Using super-resolution fluorescence imaging, we show that under conditions typically used in electrophysiological studies, the Nav1.5 alpha-subunit assembles on the plasma membrane of HEK293F cells into spatially localized clusters rather than individual and randomly dispersed molecules. Quantitative analysis indicates that the beta3-subunit is not required for this clustering but beta3 does significantly change the distribution of cluster sizes and nearest-neighbor distances between Nav1.5 alpha-subunits. However, when assayed by PLA, the beta3-subunit increases the number of PLA-positive signals generated by anti-(Nav1.5 alpha-subunit) antibodies, mainly at the plasma membrane. Since PLA can be sensitive to the orientation of proteins within a cluster, we suggest that the beta3-subunit introduces a significant change in the relative alignment of individual Nav1.5 alpha-subunits, but the clustering itself depends on other factors. We also show that these structural and higher-order changes induced by the beta3-subunit do not alter the degree of electrophysiological gating cooperativity between Nav1.5 alpha-subunits. Our data provide new insights into the role of the beta3-subunit and the supramolecular organization of sodium channels, in an important model cell system that is widely used to study Nav channel behavior.