β-Subunit of cardiac Na+-K+-ATPase dictates the concentration of the functional enzyme in caveolae

Abstract

Previous studies showed the presence of a significant fraction of Na⁺-K⁺-ATPase α-subunits in cardiac myocyte caveolae, suggesting the caveolar interactions of Na⁺-K⁺-ATPase with its signaling partners. Because both α- and β-subunits are required for ATPase activity, to clarify the status of the pumping function of caveolar Na⁺-K⁺-ATPase, we have examined the relative distribution of two major subunit isoforms (α₁and β₁) in caveolar and noncaveolar membranes of adult rat cardiac myocytes. When cell lysates treated with high salt (Na₂CO₃or KCl) concentrations were fractionated by a standard density gradient procedure, the resulting light caveolar membranes contained 30–40% of α₁-subunits and 80–90% of β₁-subunits. Use of Na₂CO₃was shown to inactivate Na⁺-K⁺-ATPase; however, caveolar membranes obtained by the KCl procedure were not denatured and contained ∼75% of total myocyte Na⁺-K⁺-ATPase activity. Sealed isolated caveolae exhibited active Na⁺transport. Confocal microscopy supported the presence of α,β-subunits in caveolae, and immunoprecipitation showed the association of the subunits with caveolin oligomers. The findings indicate that cardiac caveolar inpocketings are the primary portals for active Na⁺-K⁺fluxes, and the sites where the pumping and signaling functions of Na⁺-K⁺-ATPase are integrated. Preferential concentration of β₁-subunit in caveolae was cell specific; it was also noted in neonatal cardiac myocytes but not in fibroblasts and A7r5 cells. Uneven distributions of α₁and β₁in early and late endosomes of myocytes suggested different internalization routes of two subunits as a source of selective localization of active Na⁺-K⁺-ATPase in cardiac caveolae.