Modulation of K-Cl cotransport in volume-clamped low-K sheep erythrocytes by pH, magnesium, and ATP

Cellular pH, ionized Mg (Mg-i(2+)), and MgATP concentration of red blood cells, concomitantly with cell volume, change transiently during circulation. The action of these three effecters on Cl-dependent K efflux was examined in low-K sheep red blood cells with constant cell volume. Activation of K-Cl efflux by Mg-i(2+) extraction required ATP, suggesting that phosphorylation of a putative component occurred before Mg-i(2+) extraction. Conversely, Mg and ATP were synergistic inhibitors of K-Cl cotransport, since maximal inhibition was observed only in cells containing both ATP and >300 mu M Mg-i(2+). Both findings suggest dual roles for Mg and ATP. At 300-600 mu M Mg-i(2+), lowering the pH from similar to 7.4 to similar to 6.5 stimulated K-Cl efflux only in fed cells, suggesting that protons oppose or release the inhibition by Mg2+ and ATP. A direct effect of both protons and Mg-i(2+) on the cotransporter is suggested by their inhibition of K-CI efflux in ATP-depleted cells. These findings are discussed in light of the current phosphorylation/dephosphorylation hypothesis.