Regulation of Na+ pump function by aldosterone is α-subunit isoform specific

摘要

class="enumerated" style="list-style-type:decimal">During its early ‘genomic’ phase of action (< 3 h), aldosterone activates pre-existing Na⁺ pumps (Na⁺,K⁺-ATPase) in epithelia formed by Xenopus laevis A6 kidney cells.To test whether this action also applies to pumps containing mammalian α-subunits of different isoforms, we generated A6 cell lines expressing the naturally ouabain-resistant rat α1 subunit or the rat α2* and α3* subunits made ouabain resistant by site-directed mutagenesis.Cell lines were obtained which expressed the exogenous α-subunits in active, basolateral Na⁺ pumps, such that ouabain-resistant pump current (Ip) could be measured following apical permeabilization with amphotericin B.The inhibition constants (Ki) for ouabain of the current carried by the pumps containing exogenous rat α-subunits were similar to those reported previously for ATPase activity inhibition. The apparent Michaelis constant (Km) for Na⁺ (K⁺ replacement) was slightly higher for pumps containing the rat α1 than for those containing the α2* subunit (34.9 ± 1.9 versus 26.3 ± 2.6 mM).At a Na⁺ concentration of 10 mM, aldosterone (2.5 h) increased the pump current carried by endogenous pumps as well as that carried by pumps containing the exogenous rat α1 subunit (by 1.8- to 2.2-fold). In contrast, the current carried by pumps containing the exogenous rat α2* subunit remained unchanged.The fact that this early transcriptionally mediated activation of Na⁺ pumps by aldosterone is specific for pumps containing an α1 subunit should permit the identification in this subunit of structures involved in its regulation.