EFFECT OF INTRACELLULAR MAGNESIUM ON CALCIUM EXTRUSION BY THE PLASMA-MEMBRANE CALCIUM-PUMP OF INTACT HUMAN RED-CELLS

1. The effect of varying the concentration of intracellular magnesium on the Ca2+-saturated Ca2+-extrusion rate through the Ca2+ pump (phi(max)) was investigated in human red blood cells with the aid of the divalent cation ionophore A23187. The aim was to characterize the [Mg2+](i) dependence of the Ca2+ pump in the intact cell. 2. The initial experimental protocol consisted of applying a high ionophore concentration to obtain rapid sequential Mg2+ and [Ca-45]CaCl2 equilibration, prior to measuring phi(max) at constant internal [Mg-T](i) by either the Co2+ block method or by ionophore removal. With this protocol, competition between Ca2+ and Mg2+ through the ionophore prevented Ca2+ equilibration at high [Mg2+](0). To provide rapid and comparable Ca2+ loads and maintain intracellular ATP within normal levels it was necessary to separate the Mg2+ and the Ca2+ loading-extrusion stages by an intermediate ionophore and external Mg2+ removal step, and to use different metabolic substrates during Mg2+ loading (glucose) and Ca2+ loading-extrusion (inosine) periods. 3. Intracellular Co2+ was found to sustain Ca2+ extrusion by the pump at subphysiological [Mg2+](i). Ionophore removal was therefore used to estimate the [Mg2+](i) dependence of the pump at levels below [Mg-T](i)(similar to 2 mmol (340 g Hb)(-1)), whereas both ionophore removal and Co2+ block were used for higher [Mg-T](i) levels. 4. [Mg2+](i) was computed from measured [Mg-T](i) using known cytoplasmic Mg2+-buffering data. The phi(max) of the Ca2+ pump increased hyperbolically with [Mg2+](i). The Michaelis parameter (K-1/2) of activation was 0.12 +/- 0.04 mmol (1 cell water)(-1) (mean +/- S.E.M.). Increasing [Mg-T](i) and [Mg2+](i) to 9 mmol (340 g Hb)(-1) and 2.6 mmol (1 cell water)(-1), respectively, failed to cause significant inhibition of the phi(max) of the Ca2+ pump. 5. The results suggest that within the physiological and pathophysiological range of [Mg2+](i), from 0.3 mmol (1 cell water)(-1) in the oxygenated state to 1.2 mmol (1 cell water)(-1) in the deoxygenated state, the Ca2+-saturated Ca2+ pump remains unaffected by [Mg2+](i) at normal ATP levels.