K-Cl cotransport, pH, and role of Mg in volume-clamped low-K sheep erythrocytes: three equilibrium states.

Ouabain-resistant K efflux and Rb influx in Cl and NO3 media were studied in volume-clamped low-K (LK) sheep red blood cells (SRBC) with normal and experimentally reduced cytoplasmic Mg (Mgi) levels as function of pH and at 37 degrees C. Sucrose was added to solutions with constant ionic strength and variable pH to maintain normal cell volume. Cl-dependent ouabain-resistant K(Rb) fluxes (K-Cl cotransport) at unity relative cell volume exhibited a maximum at pH approximately 7 in normal-Mgi LK cells consistent with the apparent acid pH activation reported for human erythrocytes. However, in LK SRBC with Mgi lowered by A-23187 and an external Mg chelator, K(Rb)-Cl cotransport was reversibly activated as the pH was raised from 6.5 to 9. The alkaline pH effect on Cl-dependent Rb influx in low-Mgi LK SRBC was due to a 10-fold rise in the maximum velocity values without a major change in the Km values. The pH dependence of the experimental flux reversal point, i.e., the extracellular Rb concentration at which no net K-Cl cotransport occurs, approximately paralleled that of the flux reversal point predicted from the ratio of the ion products, in both control and low-Mgi LK cells, albeit with a small displacement to higher extracellular Rb concentration at all pH values. The kinetic data can be explained by a general minimum three-state equilibrium in which deprotonation recruits transporters from a resting R state into the active A state modified by Mgi to an inactive I state.(ABSTRACT TRUNCATED AT 250 WORDS)