K-CL COTRANSPORT, PH, AND ROLE OF MG IN VOLUME-CLAMPED LOW-K SHEEP ERYTHROCYTES - 3 EQUILIBRIUM STATES

Ouabain-resistant K efflux and Rb influx in Cl and NOB media were stud ied in volume-clamped low-K (LK) sheep red blood cells (SRBC) with nor mal and experimentally reduced cytoplasmic Mg (Mg-i) levels as functio n of pH and at 37 degrees C. Sucrose was added to solutions with const ant ionic strength and variable pH to maintain normal cell volume. Cl- dependent ouabain-resistant K(Rb) fluxes (K-Cl cotransport) at unity r elative cell volume exhibited a maximum at pH similar to 7 in normal-M g LK cells consistent with the apparent acid pH activation reported fo r human erythrocytes. However, in LK SRBC with Mg-i lowered by A-23187 and an external Mg chelator, K(Rb)-Cl cotransport was reversibly acti vated as the pH was raised from 6.5 to 9. The alkaline pH effect on Cl -dependent Rb influx in low-Mg-i LK SRBC was due to a 10-fold rise in the maximum velocity values without a major change in the K-m values. The pH dependence of the experimental flux reversal point, i.e., the e xtracellular Rb concentration at which no net K-Cl cotransport occurs, approximately paralleled that of the flux reversal point predicted fr om the ratio of the ion products, in both control and low-Mg-i LK cell s, albeit with a small displacement to higher extracellular Rb concent ration at all pH values. The kinetic data can be explained by a genera l minimum three-state equilibrium in which deprotonation recruits tran sporters from a resting R state into the active A state modified by Mg -i to an inactive I state [GRAPHICS] Based on this model, Mg-i modifie s in LK SRBC the response of the K-Cl cotransporter to changes in pH, i.e., a chemical signal, to maintain constant cell volume. Thiol reage nts activate K-Cl cotransport by preventing the Mg-i-mediated regulati on, thus maintaining the transporter in a modified active state.