Regulation of Na+ pump function by aldosterone is alpha-subunit isoform specific.
1. 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. 2. To test whether this action also applies to pumps containing mammalian alpha-subunits of different isoforms, we generated A6 cell lines expressing the naturally ouabain-resistant rat alpha1 subunit or the rat alpha2* and alpha3* subunits made ouabain resistant by site-directed mutagenesis. 3. Cell lines were obtained which expressed the exogenous alpha-subunits in active, basolateral Na+ pumps, such that ouabain-resistant pump current (Ip) could be measured following apical permeabilization with amphotericin B. 4. The inhibition constants (Ki) for ouabain of the current carried by the pumps containing exogenous rat alpha-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 alpha1 than for those containing the alpha2* subunit (34.9 +/- 1.9 versus 26.3 +/- 2.6 mM). 5. 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 alpha1 subunit (by 1.8- to 2.2-fold). In contrast, the current carried by pumps containing the exogenous rat alpha2* subunit remained unchanged. 6. The fact that this early transcriptionally mediated activation of Na+ pumps by aldosterone is specific for pumps containing an alpha1 subunit should permit the identification in this subunit of structures involved in its regulation.
Pfeiffer
R
R
Institute of Physiology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Beron
J
J
Verrey
F
F
D000450:Aldosterone / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004351:Drug Resistance; D004594:Electrophysiology; D007527:Isoenzymes / Q000187:drug effects; D007700:Kinetics; D010042:Ouabain / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000187:drug effects*; D014982:Xenopus laevis
D007527:Isoenzymes; D000450:Aldosterone; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase