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

Abstract

Voltage‐gated sodium channels comprise an ion‐selective α‐subunit and one or more associated β‐subunits. The β3‐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 β3‐subunit forms trimers in the plasma membrane. We extend this result with biochemical assays and use the proximity ligation assay (PLA) to identify oligomeric β3‐subunits, not just at the plasma membrane, but throughout the secretory pathway. We then investigate the corresponding clustering properties of the α‐subunit and the effects upon these of the β3‐subunits. The oligomeric status of the Nav1.5 α‐subunit in vivo, with or without the β3‐subunit, has not been previously investigated. Using super‐resolution fluorescence imaging, we show that under conditions typically used in electrophysiological studies, the Nav1.5 α‐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 β3‐subunit is not required for this clustering but β3 does significantly change the distribution of cluster sizes and nearest‐neighbor distances between Nav1.5 α‐subunits. However, when assayed by PLA, the β3‐subunit increases the number of PLA‐positive signals generated by anti‐(Nav1.5 α‐subunit) antibodies, mainly at the plasma membrane. Since PLA can be sensitive to the orientation of proteins within a cluster, we suggest that the β3‐subunit introduces a significant change in the relative alignment of individual Nav1.5 α‐subunits, but the clustering itself depends on other factors. We also show that these structural and higher‐order changes induced by the β3‐subunit do not alter the degree of electrophysiological gating cooperativity between Nav1.5 α‐subunits. Our data provide new insights into the role of the β3‐subunit and the supramolecular organization of sodium channels, in an important model cell system that is widely used to study Nav channel behavior.

Cell-Adhesion Properties of β-Subunits in the Regulation of Cardiomyocyte Sodium Channels

“…The β2-subunit can bind in trans to extracellular laminin [ 32 ]. The β3-subunit can bind homophilically in cis [ 20 , 33 ] and heterophilically in trans with neurofascins [ 34 ]. The β4-subunits can bind homophilically both in cis and trans [ 35 ].…”

“…In particular, they contain a high local density of Nav1.5 channels that form multi-molecular two-dimensional clusters on the membrane surfaces [ 49 ]. This clustering may partly reflect an inherent tendency of Nav1.5 channels to self-associate [ 20 ]. But Nav1.5 channels are additionally stabilised by interactions with connexin-43 hemi-channels and with multi-modular cytosolic scaffolding proteins such as ankyrin-G, ZO-1 and 14-3-3 [ 50 , 51 , 52 ].…”

“…We therefore suggest that unlike β1, the β3-subunit may not play a major role in stabilising the perinexal space by trans -mediated cell-adhesion. On the other hand, when expressed alone in HEK293 cells, the β3-subunits bind homophilically in cis , using their Ig domains [ 19 , 20 , 33 ]. Super-resolution imaging experiments show that when co-expressed with Nav1.5, the β3-subunit affects the relative geometry between Nav1.5 channel α-subunits, possibly by promoting particular orientations of individual Nav1.5 α-subunit dimers within larger clusters [ 20 ].…”

“…They also shift the voltage ranges over which Nav channel steady-state activation and/or inactivation occur, and in some cases enhance the rates of inactivation and recovery from inactivation [ 18 ]. As an illustrative example, the β3-subunit shifts the V ½ for inactivation of Nav1.5 in a depolarising direction: i.e., the voltage at which half the channels are inactivated is displaced to a more positive value compared to the α-subunit alone ( Figure 3 D) [ 19 , 20 , 21 , 22 ]. For a cardiomyocyte with a resting potential of about -90 mV [ 2 ], this would act to increase the fraction of functional Nav1.5 channels available in the membrane [ 17 ].…”

“…Based on biochemical and electrophysiological data, it is probable that the β3-subunit transmembrane alpha-helix also binds to Nav1.5 DIII voltage sensing domain [ 19 , 20 , 21 ]. Yet, there is evidence that it may bind closer to Nav1.5 DIII helix S3 rather than to the binding site for the β1 transmembrane region on the DIII helix S2 [ 19 , 21 ].…”