Tissue-specific distribution and modulatory role of the γ subunit of the Na,K-ATPase

Abstract

The Na,K-ATPase comprises a catalytic α subunit and a glycosylated β subunit. Another membrane polypeptide, γ, first described by Forbush et al. (Forbush, B., III, Kaplan, J. H., and Hoffman, J. F. (1978) Biochemistry 17, 3667-3676) associates with α and β in purified kidney enzyme preparations. In this study, we have used a polyclonal anti-γ antiserum to define the tissue specificity and topology of γ and to address the question of whether γ has a functional role. The trypsin sensitivity of the amino terminus of the γ subunit in intact right-side-out pig kidney microsomes has confirmed that it is a type I membrane protein with an extracellular amino terminus. Western blot analysis shows that γ subunit protein is present only in membranes from kidney tubules (rat, dog, pig) and not those from axolemma, heart, red blood cells, kidney glomeruli, cultured glomerular cells, α1- transfected HeLa cells, all derived from the same (rat) species, nor from three cultured cell lines derived from tubules of the kidney, namely NRK-52E (rat), LLC-PK (pig), or MDCK (dog). To gain insight into γ function, the effects of the anti-γ serum on the kinetic behavior of rat kidney sodium pumps was examined. The following evidence suggests that γ stabilizes E1 conformation(s) of the enzyme and that anti-γ counteracts this effect: (i) anti-γ inhibits Na,K-ATPase, and the inhibition increases at acidic pH under which condition the E2(K) → E1 phase of the reaction sequence becomes more rate-limiting, (ii) the oligomycin-stimulated increase in the level of phosphoenzyme was greater in the presence of anti-γ indicating that the antibody shifts the E1 mutually implies mutually implies E2P equilibria toward E2P, and (iii) when the Na+-ATPase reaction is assayed with the Na+ concentration reduced to levels (≤2 mM) which limit the rate of the E1 → → E2P transition, anti-γ is stimulatory. These observations taken together with evidence that the pig γ subunit, which migrates as a doublet on polyacrylamide gels, is sensitive to digestion by trypsin, and that Rb+ ions partially protect it against this effect, indicate that the γ subunit is a tissue-specific regulator which shifts the steady-state equilibria toward E1. Accordingly, binding of anti-γ disrupts αβ-γ interactions and counteracts these modulatory effects of the γ subunit.