Isoform specificity of human Na(+), K(+)-ATPase localization and aldosterone regulation in mouse kidney cells.
Short-term aldosterone coordinately regulates the cell-surface expression of luminal epithelial sodium channels (ENaC) and of basolateral Na(+) pumps (Na(+), K(+)-ATPase alpha1-beta1) in aldosterone-sensitive distal nephron (ASDN) cells. To address the question of whether the subcellular localization of the Na(+), K(+)-ATPase and its regulation by aldosterone depend on subunit isoform-specific structures, we expressed the cardiotonic steroid-sensitive human alpha isoforms 1-3 by retroviral transduction in mouse collecting duct mpkCCD(c14) cells. Each of the three exogenous human isoforms could be detected by Western blotting. Immunofluorescence indicated that the exogenous alpha1 subunit to a large extent localizes to the basolateral membrane or close to it, whereas much of the alpha2 subunit remains intracellular. An ouabain-sensitive current carried by exogenous pumps could be detected in apically amphotericin B-permeabilized epithelia expressing human alpha1 and alpha2 subunits, but not the alpha3 subunit. This current displayed a higher apparent Na(+) affinity in pumps containing human alpha2 subunits (10 mM) than in pumps containing human alpha1 (33.2 mM) or endogenous (cardiotonic steroid-resistant) mouse alpha1 subunits (mean: 16.3 mM). A very low mRNA level of the Na(+), K(+)-ATPase gamma subunit (FXYD2) in mpkCCD(c14) cells suggested that this ancillary gene product is not responsible for the relatively low apparent Na(+) affinity measured for a1 subunit-containing pumps. Aldosterone increased the pump current carried by endogenous pumps and by pumps containing the human alpha1 subunit. In contrast, the current carried by pumps with a human alpha2 subunit was not stimulated by the same treatment. In summary, quantitative basolateral localization of the Na(+), K(+)-ATPase and its responsiveness to aldosterone require alpha1 subunit-specific sequences that differentiate this isoform from the alpha2 and alpha3 subunit isoforms.
Summa
Vanessa
V
Institute of Physiology, University of Zurich, CH-8057 Zurich, Switzerland.
Camargo
Simone M R
SM
Bauch
Christian
C
Zecevic
Marija
M
Verrey
François
F
D000450:Aldosterone / Q000502:physiology*; D000818:Animals; D002460:Cell Line; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004594:Electrophysiology; D004847:Epithelial Cells / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000502:physiology; D007668:Kidney / Q000166:cytology / Q000502:physiology*; D007685:Kidney Tubules, Collecting / Q000201:enzymology / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D018408:Patch-Clamp Techniques; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000254:Sodium-Potassium-Exchanging ATPase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D013268:Stimulation, Chemical; D013379:Substrate Specificity; D014161:Transduction, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D000450:Aldosterone; D000254:Sodium-Potassium-Exchanging ATPase