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

Autor: Salvage SC; Deparment of Biochemistry, University of Cambridge, Cambridge, UK., Rees JS; Deparment of Biochemistry, University of Cambridge, Cambridge, UK., McStea A; Central Laser Facility, Research Complex at Harwell, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Oxford, UK., Hirsch M; Central Laser Facility, Research Complex at Harwell, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Oxford, UK., Wang L; Central Laser Facility, Research Complex at Harwell, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Oxford, UK., Tynan CJ; Central Laser Facility, Research Complex at Harwell, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Oxford, UK., Reed MW; Department of Nuclear Physics, Research School of Physics and Engineering, Australian National University, Canberrra, ACT, Australia., Irons JR; Deparment of Biochemistry, University of Cambridge, Cambridge, UK., Butler R; Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK., Thompson AJ; Department of Pharmacology, University of Cambridge, Cambridge, UK., Martin-Fernandez ML; Central Laser Facility, Research Complex at Harwell, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Oxford, UK., Huang CL; Deparment of Biochemistry, University of Cambridge, Cambridge, UK.; Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK., Jackson AP; Deparment of Biochemistry, University of Cambridge, Cambridge, UK.
Jazyk: angličtina
Zdroj: FASEB journal : official publication of the Federation of American Societies for Experimental Biology [FASEB J] 2020 Mar; Vol. 34 (3), pp. 3537-3553. Date of Electronic Publication: 2020 Jan 16.
DOI: 10.1096/fj.201701473RR
Abstrakt: 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.
(© 2020 The Authors. The FASEB Journal published by Wiley Periodicals, Inc. on behalf of Federation of American Societies for Experimental Biology.)
Databáze: MEDLINE
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