title: Na(+)-K(+)-ATPase that redistributes to apical membrane during ATP depletion remains functional.
abstract: We have previously demonstrated using immunocytochemical, histochemical, and biochemical techniques that ischemia in vivo and ATP depletion in vitro result in dissociation of Na(+)-K(+)-adenosinetriphosphatase (ATPase) from the actin cytoskeleton and redistribution to the apical domain in renal proximal tubule cells. To directly evaluate whether apical Na(+)-K(+)-ATPase retained Na+ pumping activity, a rapidly reversible model of cellular ATP depletion in confluent LLC-PK1 cells grown on semipermeable membranes was utilized. Tight-junction integrity, monitored by electrical resistance, was lost during ATP depletion and reestablished during 2 h of ATP repletion. Total cellular Na(+)-K(+)-ATPase activity and total surface membrane [3H]ouabain binding remained constant, but specific apical [3H]ouabain binding increased (7 vs. 26 fmol/filter, P < 0.01). Apical [3H]ouabain binding returned to baseline during 5 h of ATP repletion. Apically applied ouabain was then used to selectively inhibit apical Na(+)-K(+)-ATPase. It had no effect on apical-to-basolateral Na+ flux under physiological conditions (1.3 +/- 0.61 vs. 1.27 +/- 0.46 meq.filter-1.30 min-1), but it increased the apical-to-basolateral flux in ATP-depleted and then repleted monolayers (0.39 +/- 0.12 vs. 0.83 +/- 0.27 meq.filter-1.30 min-1, P < 0.01), implying that apical Na(+)-K(+)-ATPase retained Na+ pumping activity. Together, these data imply that ATP depletion induces loss of surface membrane polarity resulting in redistribution of functional proteins to the alternate domain.
authors.affiliation: Department of Medicine, University of Colorado, School of Medicine, Denver.
authors.forename: B A
authors.initials: BA
authors.lastname: Molitoris
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D000968:Antimycin A / Q000494:pharmacology*; D001692:Biological Transport; D002462:Cell Membrane / Q000201:enzymology*; D002999:Clone Cells; D007365:Intercellular Junctions / Q000502:physiology; D007668:Kidney; D007700:Kinetics; D008961:Models, Structural; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
chemical_list: D000968:Antimycin A; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase