Cytosolic pH sensitivity of an expressed human NHE-1 Na(+)-H+ exchanger.
These studies examined the effects of protein kinase C activation and calmodulin inhibition on the amiloride-sensitive NHE-1 isoform of the Na(+)-H+ exchanger in defined host cells. Our objective was to define differences in the cellular regulatory responses using a specified isoform of the Na(+)-H+ exchanger. Suspended cells were loaded with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and preacidified to a cytosolic pH of 6.2. Wild-type mouse Ltk- cells, human A-431 cells, and mutant mouse fibroblasts stably transfected with the human NHE-1 isoform (LAP+ cells) were examined to define the maximal rate of transport (Vmax) in response to 140 mM external Na+, the Hill stoichiometric coefficient, and the cytosolic pH at which the NHE-1 isoform was half-maximally stimulated (pH50). The mouse NHE-1 isoform had a greater affinity for cytosolic H+ than the human NHE-1 isoforms. Calmodulin antagonism with N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide reduced the Vmax and shifted the pH50 in the acidic direction, especially in the A-431 cells. Protein kinase C stimulation had a similar effect in A-431 cells and little effect in the wild-type (Ltk-) and transfected (LAP+) mouse cells. While the NHE-1 isoform contains several potential phosphorylation sites, the cellular milieu in which the isoform is expressed has an important effect on the modulation of NHE-1 activity.
Takaichi
K
K
Department of Medicine, University of Alabama, Birmingham 35294.
Balkovetz
D F
DF
Van Meir
E
E
Warnock
D G
DG
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007739:L Cells; D051379:Mice; D010957:Plasmids; D015183:Restriction Mapping; D017923:Sodium-Hydrogen Exchangers; D013449:Sulfonamides / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured
D002147:Calmodulin; D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; D013449:Sulfonamides; C069139:growth factor-activatable Na-H exchanger NHE-1; C017967:W 7; D000584:Amiloride; D013755:Tetradecanoylphorbol Acetate