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Isoform specificity of human Na + ,K + ‐ATPase localization and aldosterone regulation in mouse kidney cells
Author(s) -
Summa Vanessa,
Camargo Simone M. R.,
Bauch Christian,
Zecevic Marija,
Verrey François
Publication year - 2004
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2003.054270
Subject(s) - aldosterone , gene isoform , protein subunit , ouabain , epithelial sodium channel , microbiology and biotechnology , biology , intracellular , chemistry , endocrinology , biochemistry , sodium , gene , organic chemistry
Short‐term aldosterone coordinately regulates the cell‐surface expression of luminal epithelial sodium channels (ENaC) and of basolateral Na + pumps (Na + ,K + ‐ATPase α1–β1) 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 α 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 α1 subunit to a large extent localizes to the basolateral membrane or close to it, whereas much of the α2 subunit remains intracellular. An ouabain‐sensitive current carried by exogenous pumps could be detected in apically amphotericin B‐permeabilized epithelia expressing human α1 and α2 subunits, but not the α3 subunit. This current displayed a higher apparent Na + affinity in pumps containing human α2 subunits (10 m m ) than in pumps containing human α1 (33.2 m m ) or endogenous (cardiotonic steroid‐resistant) mouse α1 subunits (mean: 16.3 m m ). A very low mRNA level of the Na + ,K + ‐ATPase γ 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 α1 subunit. In contrast, the current carried by pumps with a human α2 subunit was not stimulated by the same treatment. In summary, quantitative basolateral localization of the Na + ,K + ‐ATPase and its responsiveness to aldosterone require α1 subunit‐specific sequences that differentiate this isoform from the α2 and α3 subunit isoforms.