Control of the sodium-proton antiporter in human placental microvillous membranes by transport substrates

Abstract

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 K m determined for Na + was 8.2 ± 4.0 mM, while the external p K was 7.29 ± 0.02. The V max 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.