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

Jennings ML - J. Gen. Physiol. (1999)

Bottom Line: The forward rate constant for activation has a very high temperature dependence (E(a) approximately 32 kCal/mol), but is not affected measurably by cell volume.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA. jenningsmichaell@exchange.uams.edu

ABSTRACT
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 Mg(2+)) 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 Mg(2+)-depleted rabbit red cells is not affected by cell volume. KCC activation kinetics were examined in cells pretreated with NEM at 0 degrees C, washed, and then incubated at higher temperatures. The forward rate constant for activation has a very high temperature dependence (E(a) approximately 32 kCal/mol), but is not affected measurably by cell volume. Inactivation kinetics were examined by swelling cells at 37 degrees 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 Mg(2+), acid pH, and NEM.

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Mentions: The three-state model of Dunham et al. 1993 proposes that the activation process involves a rate-limiting step (A to B) followed by a fast step (B to C) (Fig. 1). To discuss relaxation rates, no assumptions are necessary about the detailed kinetic properties of the three states, other than that the flux is much higher in the C than in the A state. In this model, the relaxation rate for approach to any new steady state is (see ): 2\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}{\mathrm{{\tau}}}^{-1}=k_{12}+{k_{21}}/{ \left \left(1+K_{{\mathrm{BC}}}\right) \right }{\mathrm{,}}\end{equation*}\end{document} where KBC is the equilibrium constant k23/k32 for the rapid second step.


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

Jennings ML - J. Gen. Physiol. (1999)

© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230653&req=5

Mentions: The three-state model of Dunham et al. 1993 proposes that the activation process involves a rate-limiting step (A to B) followed by a fast step (B to C) (Fig. 1). To discuss relaxation rates, no assumptions are necessary about the detailed kinetic properties of the three states, other than that the flux is much higher in the C than in the A state. In this model, the relaxation rate for approach to any new steady state is (see ): 2\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}{\mathrm{{\tau}}}^{-1}=k_{12}+{k_{21}}/{ \left \left(1+K_{{\mathrm{BC}}}\right) \right }{\mathrm{,}}\end{equation*}\end{document} where KBC is the equilibrium constant k23/k32 for the rapid second step.

Bottom Line: The forward rate constant for activation has a very high temperature dependence (E(a) approximately 32 kCal/mol), but is not affected measurably by cell volume.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA. jenningsmichaell@exchange.uams.edu

ABSTRACT
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 Mg(2+)) 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 Mg(2+)-depleted rabbit red cells is not affected by cell volume. KCC activation kinetics were examined in cells pretreated with NEM at 0 degrees C, washed, and then incubated at higher temperatures. The forward rate constant for activation has a very high temperature dependence (E(a) approximately 32 kCal/mol), but is not affected measurably by cell volume. Inactivation kinetics were examined by swelling cells at 37 degrees 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 Mg(2+), acid pH, and NEM.

Show MeSH
Related in: MedlinePlus