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Original Article

Analysis of the Rate-Limiting Activation and Inactivation Events

Michael L. Jennings^a

^a From the Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, 4301 W. Markham St., Mail Slot 505, Little Rock, AR 72205.501-686-8167

jenningsmichaell{at}exchange.uams.edu

The kinetics of activation and inactivation of K⁺/Cl^– cotransport (KCC) have been measured in rabbit red blood cells for the purpose of determining the individual rate constants for the rate-limiting activation and inactivation events. Four different interventions (cell swelling, N-ethylmaleimide [NEM], low intracellular pH, and low intracellular Mg²⁺) all activate KCC with a single exponential time course; the kinetics are consistent with the idea that there is a single rate-limiting event in the activation of transport by all four interventions. In contrast to LK sheep red cells, the KCC flux in Mg²⁺-depleted rabbit red cells is not affected by cell volume. KCC activation kinetics were examined in cells pretreated with NEM at 0°C, washed, and then incubated at higher temperatures. The forward rate constant for activation has a very high temperature dependence (E_a 32 kCal/mol), but is not affected measurably by cell volume. Inactivation kinetics were examined by swelling cells at 37°C to activate KCC, and then resuspending at various osmolalities and temperatures to inactivate most of the transporters. The rate of transport inactivation increases steeply 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 the rate-limiting inactivation event is strongly affected by cell volume over the entire range of cell volumes studied, including normal cell volume. The rate-limiting inactivation event may be mediated by a protein kinase that is inhibited, either directly or indirectly, by cell swelling, low Mg²⁺, acid pH, and NEM.

Key Words: osmoregulation • red blood cell • phosphatase • kinase • kinetics

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