PMID- 14694143 OWN - NLM STAT- MEDLINE DCOM- 20041005 LR - 20240315 IS - 0022-3751 (Print) IS - 1469-7793 (Electronic) IS - 0022-3751 (Linking) VI - 555 IP - Pt 2 DP - 2004 Mar 1 TI - Isoform specificity of human Na(+), K(+)-ATPase localization and aldosterone regulation in mouse kidney cells. PG - 355-64 AB - 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. FAU - Summa, Vanessa AU - Summa V AD - Institute of Physiology, University of Zurich, CH-8057 Zurich, Switzerland. FAU - Camargo, Simone M R AU - Camargo SM FAU - Bauch, Christian AU - Bauch C FAU - Zecevic, Marija AU - Zecevic M FAU - Verrey, Francois AU - Verrey F LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20031223 PL - England TA - J Physiol JT - The Journal of physiology JID - 0266262 RN - 0 (DNA Primers) RN - 0 (DNA, Complementary) RN - 0 (Isoenzymes) RN - 4964P6T9RB (Aldosterone) RN - EC 7.2.2.13 (Sodium-Potassium-Exchanging ATPase) SB - IM MH - Aldosterone/*physiology MH - Animals MH - Cell Line MH - DNA Primers MH - DNA, Complementary/biosynthesis/genetics MH - Electrophysiology MH - Epithelial Cells/enzymology MH - Fluorescent Antibody Technique MH - Gene Expression Regulation, Enzymologic/drug effects MH - Humans MH - Immunoblotting MH - Isoenzymes/physiology MH - Kidney/cytology/*physiology MH - Kidney Tubules, Collecting/enzymology/metabolism MH - Membrane Potentials/physiology MH - Mice MH - Patch-Clamp Techniques MH - Reverse Transcriptase Polymerase Chain Reaction MH - Sodium-Potassium-Exchanging ATPase/biosynthesis/genetics/*metabolism MH - Stimulation, Chemical MH - Substrate Specificity MH - Transduction, Genetic PMC - PMC1664841 EDAT- 2003/12/25 05:00 MHDA- 2004/10/06 09:00 PMCR- 2005/03/01 CRDT- 2003/12/25 05:00 PHST- 2003/12/25 05:00 [pubmed] PHST- 2004/10/06 09:00 [medline] PHST- 2003/12/25 05:00 [entrez] PHST- 2005/03/01 00:00 [pmc-release] AID - jphysiol.2003.054270 [pii] AID - 10.1113/jphysiol.2003.054270 [doi] PST - ppublish SO - J Physiol. 2004 Mar 1;555(Pt 2):355-64. doi: 10.1113/jphysiol.2003.054270. Epub 2003 Dec 23.