Asymmetric affinity of Na+-H+ antiporter for Na+ at the cytoplasmic versus external transport site.
The affinity for Na+ of the cytoplasmic vs. external transport site of the amiloride-sensitive Na+-H+ antiporter was studied in confluent cultures of MDCK cells. Na+-H+ antiport activity was fluorometrically determined by monitoring changes in intracellular pH (pHi) using the pH-sensitive fluorescent probe, BCECF. Na+-dependent H+ fluxes were studied both in the functionally operative (H+ efflux/Na+ influx) and reverse (H+ influx/Na+ efflux) mode of antiport activity, under pH equilibrium, but Na+-gradient conditions. Thus the driving force for antiport activity was solely dependent on the transmembrane Na+ gradient. Independent experiments established that pHi and intracellular Na+ [Na+i] had been set at the desired values before the initiation of a particular experiment. Under conditions of pHi = pHo = 7.0, [Na+i] = 0 mM and varying extracellular Na+ concentration [Na+o], the apparent affinity for Na+ (KtNa) for the external transport site was 24 +/- 3 mM. When antiport activity was measured in the reverse mode of operation, but under identical pH conditions, KtNa at the internal site was 7 +/- 1 mM. When ambient pH was elevated to 7.5, KtNa at the internal site was 14 +/- 1 mM. Maximum H+ flux (JmaxH+) for the antiporter under all three conditions was not significantly different. In summary, the Na+-H+ antiporter displays asymmetric affinity for Na+ at the internal vs. external transport site. Under pH equilibrium conditions, the affinity of the Na+-H+ antiporter for Na+ is three- to four-fold greater at the internal vs. external locus, and the affinity for Na+ at the internal site is enhanced by lower pHi. The close similarity between values for KtNa (inside) and reported values for intracellular Na+ concentration suggests that regulation of the Na+-H+ antiporter may be affected by changes in intracellular Na+ concentration.
Goldfarb
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Department of Medicine, University of California, School of Medicine, Los Angeles 90024.
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D000818:Animals; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism*; D004285:Dogs; D005452:Fluoresceins / Q000378:metabolism; D005470:Fluorometry; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000166:cytology; D012964:Sodium / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D005452:Fluoresceins; D017923:Sodium-Hydrogen Exchangers; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium