Na-Ca exchange: evidence against a ping-pong mechanism and against a Ca pool in ferret red blood cells

Abstract

To determine the mechanism of Na-Ca exchange, we estimated the ratio of maximum velocity to Michaelis constant for extra-cellular Ca by measuring the rate of Ca uptake at very low extracellular Ca. In a Ping-Pong mechanism, one set of sites alternatively transports Ca and Na. In a sequential mechanism, Ca and Na sites are both filled during part of the transport cycle. In each set of experiments, two intracellular Na concentrations were studied. The Ca uptake rate (at low Ca) increased as Na increased; this is consistent with a sequential model, as has been found in other cells. We also examined the alternative hypothesis that the exchanger followed Ping-Pong kinetics and that the red blood cells had a submembrane pool for Ca that limited mixing with the cytosol. In these experiments Ca pump activity was monitored by measuring ATP hydrolysis. This model was disproven by experiments that indicated that greater than 80% of the Ca that entered the cell became bound to EGTA and less than 20% resulted in Ca efflux by the Ca pump.