Supramolecular clustering of the cardiac sodium channel Nav1.5 in HEK293F cells, with and without the auxiliary ?3-subunit.

ABSTRACT: Voltage-gated sodium channels comprise an ion-selective ?-subunit and one or more associated ?-subunits. The ?3-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 ?3-subunit forms trimers in the plasma membrane. We extend this result with biochemical assays and use the proximity ligation assay (PLA) to identify oligomeric ?3-subunits, not just at the plasma membrane, but throughout the secretory pathway. We then investigate the corresponding clustering properties of the ?-subunit and the effects upon these of the ?3-subunits. The oligomeric status of the Nav1.5 ?-subunit in vivo, with or without the ?3-subunit, has not been previously investigated. Using super-resolution fluorescence imaging, we show that under conditions typically used in electrophysiological studies, the Nav1.5 ?-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 ?3-subunit is not required for this clustering but ?3 does significantly change the distribution of cluster sizes and nearest-neighbor distances between Nav1.5 ?-subunits. However, when assayed by PLA, the ?3-subunit increases the number of PLA-positive signals generated by anti-(Nav1.5 ?-subunit) antibodies, mainly at the plasma membrane. Since PLA can be sensitive to the orientation of proteins within a cluster, we suggest that the ?3-subunit introduces a significant change in the relative alignment of individual Nav1.5 ?-subunits, but the clustering itself depends on other factors. We also show that these structural and higher-order changes induced by the ?3-subunit do not alter the degree of electrophysiological gating cooperativity between Nav1.5 ?-subunits. Our data provide new insights into the role of the ?3-subunit and the supramolecular organization of sodium channels, in an important model cell system that is widely used to study Nav channel behavior.

SUBMITTER: Salvage SC 

PROVIDER: S-EPMC7079131 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature