Ca-induced K transport in human red blood cell ghosts containing arsenazo III: Transmembrane Interactions of Na, K, and Ca and the Relationship to the Functioning Na-K Pump

Abstract

Increasing free intracellular Ca (Cai) from ≤0.1 µM to 10 µM by means of A23187 activated Ca-stimulated K transport and inhibited the Na-K pump in resealed human red cell ghosts. These ghosts contained 2 mM ATP, which was maintained by a regenerating system, and arsenazo III to measure Cai. Ca-stimulated K transport was activated 50% at 2-3 FM free Cai and the Na-K pump was inhibited 50% by 5-10 µM free Cai. Free Cai from 1 to 8 µM stimulated K efflux before it inhibited the Na-K pump, dissociating the effect ofCa on the two systems. 3 µM trifluoperazine inhibited Ca-stimulated Kefflux and 0.5 mM quinidine reduced Na-K pumping by 50%. In other studies, incubating fresh intact cells in solutions containing Ca and 0.5 µM A23187 caused the cells to lose K heterogeneously. Under the same conditions, increasing A23187 to 10 µM initiated a homogeneous loss of Ko In ATP-deficient ghosts containing Cai equilibrated with A23187, K transport was activated at the same free Cai as in the ghosts containing 2 mM ATP. Neither Cao nor the presence of an inward Ca gradient altered the effect of free Cai on the permeability to Ko In these ghosts, transmembrane interactions of Na and K influenced the rate of Ca-stimulated K efflux independent of Na- and K-induced changes in free Cai or sensitivity to Cai. At constant free Cai, increasing Ko from 0.1 to 3 mM stimulated K efflux, whereas further increasing Ko inhibited it. Increasing Nai at constant Ki and free Cai markedly decreased the rate ofefflux at 2 mM Ko, but had no effect when Ko was ≥20 mM. These transmembrane interactions indicate that the mechanism underlying Ca-stimulated K transport is mediated. Since these interactions from either side of the membrane are independent of free Cai, activation of the transport mechanism by Cai must be at a site that is independent of those responsible for the interaction of Na and Ko In the presence of A23187, this activating site is half-maximally stimulated by ~2 µM free Ca and is not influenced by the concentration of ATP. The partial inhibition ofCa-stimulatedK efflux by trifluoperazine in ghosts containing ATP suggests that calmodulin could be involved in the activation of K transport by Cai. Part of the Na-K pump apparatus could be the mechanism that underlies Ca-stimulated K transport because both processes are inhibited by the same drugs, including quinidine, and the pump could account for the transmembrane interactions of Na and K described above. Nevertheless, functioning Na-K pumps are not altered by Cai to transport K selectively, because K transport is activated by Cai before the Na-K pump becomes inhibited. © 1984, Rockefeller University Press., All rights reserved.