Pk. Lauf et al., K-CL COTRANSPORT, PH, AND ROLE OF MG IN VOLUME-CLAMPED LOW-K SHEEP ERYTHROCYTES - 3 EQUILIBRIUM STATES, The American journal of physiology, 266(1), 1994, pp. 30000095
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.