Control of the sodium-proton antiporter in human placental microvillous membranes by transport substrates.
The microvillous membrane of the human placental syncytiotrophoblast contains an amiloride-inhibitable, electroneutral, Na+/H+ antiporter. The kinetic characteristics of this antiporter have been investigated to determine its response to alterations in intracellular and extracellular H+ and Na+ concentrations. Antiporter activity was measured using a pH-sensitive fluorescent probe entrapped in placental microvillous vesicles. We report here on the kinetic characterization of the antiporter, a transporter which displays simple, saturable kinetics for the external site but complex kinetics at the internal site. Measurement of the external Na+ and H+ dependences demonstrated that Na+ and H+ compete for binding to a single external binding site which displays saturation kinetics. The external Km determined for Na+ was 8.2 +/- 4.0 mM, while the external pK was 7.29 +/- 0.02. The Vmax calculated from these experiments was 0.57 +/- 0.10 nequiv./s per mg membrane protein. By contrast, the internal dependences for both Na+ and H+ showed significant deviations from simple linear kinetics. Decreasing internal pH to 6.0 stimulated Na+/H+ exchange to a greater degree than predicted for a single-site saturable binding model, in a manner which suggested allosteric activation. At the other extreme, Na+/H+ exchange ceased above an internal pH of 7.1, despite the existence of an inwardly-directed Na+ gradient. Increasing intracellular Na+ caused inhibition of Na+/H+ exchange but the intracellular Na+ dependence showed that the effect is due to a mechanism more complex than simple, competitive inhibition between Na+ and H+. These results show that the microvillous Na+/H+ antiporter is insensitive to changes in extracellular Na+ and H+ concentrations in the physiological range. Changes in intracellular Na+ and H+ however are likely to cause marked changes in antiporter activity. These characteristics suggest that cellular Na+ and H+ concentrations are tightly controlled in the placental syncytiotrophoblast and that the Na+/H+ antiporter may play a significant role in their regulation.
Illsley
N P
NP
Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco 94143-0556.
Jacobs
M M
MM
D000584:Amiloride / Q000494:pharmacology; D001665:Binding Sites; D002352:Carrier Proteins / Q000032:analysis* / Q000502:physiology; D002712:Chlorides / Q000378:metabolism; D005260:Female; D006706:Homeostasis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008871:Microvilli / Q000378:metabolism; D010920:Placenta / Q000378:metabolism* / Q000648:ultrastructure; D011247:Pregnancy; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D002712:Chlorides; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D012964:Sodium