Fluorescence Lifetime Imaging Microscopy Reveals Sodium Pump Dimers in Live Cells. Journal Article

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Authors: Seflova, J; Habibi, NR; Yap, JQ; Cleary, SR; Fang, X; Kekenes-Huskey, PM; Espinoza-Fonseca, LM; Bossuyt, JB; Robia, SL
Article Title: Fluorescence Lifetime Imaging Microscopy Reveals Sodium Pump Dimers in Live Cells.
Abstract: The sodium-potassium ATPase (Na/K-ATPase, NKA) establishes ion gradients that facilitate many physiological functions including action potentials and secondary transport processes. NKA comprises a catalytic subunit (alpha) that interacts closely with an essential subunit (beta) and regulatory transmembrane micropeptides called FXYD proteins. In the heart, a key modulatory partner is the FXYD protein phospholemman (PLM, FXYD1), but the stoichiometry of the alpha-beta-PLM regulatory complex is unknown. Here we used fluorescence lifetime imaging and spectroscopy to investigate the structure, stoichiometry, and affinity of the NKA regulatory complex. We observed a concentration-dependent binding of the subunits of NKA-PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit as well as lower affinity alpha-alpha and alpha-PLM interactions. These data provide the first evidence that, in intact live cells, the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits. Docking and molecular dynamics simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha-alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with the stoichiometry (alpha-beta-PLM).
Journal Title: The Journal of biological chemistry
ISSN: 1083-351X; 0021-9258
Publisher: Unknown  
Date Published: 2022