Volume-sensitive K+/Cl- cotransport in rabbit erythrocytes - Analysis of the rate-limiting activation and inactivation events

The kinetics of activation and inactivation of K+/Cl- cotransport (KCC) hav e been measured in rabbit red blood cells for the purpose of determining th e individual rate constants for the rate-limiting activation and inactivati on events. Four different interventions (cell swelling, N-ethylmaleimide [N EM], low intracellular pH, and low intracellular Mg2+) all activate KCC wit h a single exponential time course; the kinetics are consistent with the id ea that there is a single rate-limiting event in the activation of transpor t by all four interventions. In contrast to LK sheep red cells, the KCC flu x in Mg2+-depleted rabbit red cells is not affected by cell volume. KCC act ivation kinetics were examined in cells pretreated with NEM at 0 degrees C, washed, and then incubated at higher temperatures. The forward rate consta nt for activation has a very high temperature dependence (E-a similar to 32 kCal/mol), but is not affected measurably by cell volume. Inactivation kin etics were examined by swelling cells at 37 degrees C to activate KCC, and then resuspending at various osmolalities and temperatures to inactivate mo st of the transporters. The rate of transport inactivation increases steepl y as cell volume decreases, even in a range of volumes where nearly all the transporters are inactive in the steady state. This finding indicates that die rate-limiting inactivation event is strongly affected by cell volume o ver the entire range of cell volumes studied, including normal cell volume. The rate-limiting inactivation event may be mediated by a protein kinase t hat is inhibited, either directly or indirectly, by cell swelling, low Mg2, acid pH, and NEM.