Tissue-specific Distribution and Modulatory Role of the γ Subunit of the Na,K-ATPase

Abstract

The Na,K-ATPase comprises a catalytic ␣ subunit and a glycosylated ␤ subunit. Another membrane polypeptide, ␥, first described by Forbush et al. (Forbush, B., III, Kaplan, J. H., and Hoffman, J. F. (1978) Biochemistry 17, 3667-3676) associates with ␣ and ␤ in purified kidney enzyme preparations. In this study, we have used a polyclonal anti-␥ antiserum to define the tissue specificity and topology of ␥ and to address the question of whether ␥ has a functional role. The trypsin sensitivity of the amino terminus of the ␥ 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 ␥ 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, ␣ 1 -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 ␥ function, the effects of the anti-␥ serum on the kinetic behavior of rat kidney sodium pumps was examined. The following evidence suggests that ␥ stabilizes E 1 conformation(s) of the enzyme and that anti-␥ counteracts this effect: (i) anti-␥ inhibits Na,K-ATPase, and the inhibition increases at acidic pH under which condition the E 2 (K) 3 E 1 phase of the reaction sequence becomes more ratelimiting, (ii) the oligomycin-stimulated increase in the level of phosphoenzyme was greater in the presence of anti-␥ indicating that the antibody shifts the E 1 7 7 E 2 P equilibria toward E 2 P, and (iii) when the Na ؉ -ATPase reaction is assayed with the Na ؉ concentration reduced to levels (<2 mM) which limit the rate of the E 1 3 3 E 2 P transition, anti-␥ is stimulatory. These observations taken together with evidence that the pig ␥ 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 ␥ subunit is a tissue-specific regulator which shifts the steady-state equilibria toward E 1 . Accordingly, binding of anti-␥ disrupts ␣␤-␥ interactions and counteracts these modulatory effects of the ␥ subunit.

Phospholemman (FXYD1) associates with Na,K-ATPase and regulates its transport properties

“…In the present paper, we provide evidence that PLM may be a tissue-specific regulator of NKA similar to other members of the FXYD protein family such as the ␥ subunit of NKA (14,35,36), CHIF (12), and FXYD7 (44). Indeed, our results show that PLM is associated with NKA in heart and skeletal muscles and that it Fig.…”

Na pump isoforms in human erythroid progenitor cells and mature erythrocytes

“…3 and 5 show definitively that the ␥ modulator is a constituent component in the progenitor cells, and more importantly in mature human erythrocytes. By using the same antibody (B-Ab) in a much higher titer than used previously (13) in a study where the ␥ modulator was not detected, it was now clearly found (Fig. 5).…”

“…The basis for this difference in isoform expression between sheep and humans is unknown. The ␥ modulator of the Na pump (2,6,12) has not been found in rat red cells (13), but the evidence presented below clearly documents, by both Northern and Western blot analyses, that the ␥ modulator is a normal constituent of precursor as well as mature human red blood cells. We have changed the notation to refer to the ␥ component as the ␥ modulator or FXYD2 rather than the ␥ subunit; a change that is consistent with its evident function as a member of the FXYD family (14).…”

Stress-induced Expression of the γ Subunit (FXYD2) Modulates Na,K-ATPase Activity and Cell Growth

“…1) Stable transfection of ␥ in NRK-52E cells, which do not express ␥ under normal conditions, results in a significant delay of cell growth compared with non-transfected cells (8,20). 2) While being exclusively expressed in kidney, expression of ␥ is normally absent in renal cells in culture (20,21) but can be induced with genotoxic stress (hypertonicity (22,25), heat shock, oxidative stress, and treatment with heavy metals). As we showed here, induction of endogenous ␥a in NRK-52E treated with hypertonicity correlated with significant reduction in the V max of the pump and was accompanied by a reduction in the rate of cell division.…”