Isoform specificity of human Na+,K+-ATPase localization and aldosterone regulation in mouse kidney cells
| Autor: | Summa, Vanessa, Camargo, Simone M R, Bauch, Christian, Zecevic, Marija, Verrey, François |
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| Jazyk: | angličtina |
| Rok vydání: | 2003 |
| Předmět: |
DNA
Complementary Patch-Clamp Techniques Reverse Transcriptase Polymerase Chain Reaction Immunoblotting Fluorescent Antibody Technique Epithelial Cells Kidney Research Papers Gene Expression Regulation Enzymologic Stimulation Chemical Cell Line Membrane Potentials Substrate Specificity Electrophysiology Isoenzymes Mice Transduction Genetic Animals Humans Kidney Tubules Collecting Sodium-Potassium-Exchanging ATPase Aldosterone DNA Primers |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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