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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 is among the simplest mechanisms containing more than one event in the activation–inactivation process. However, many other relatively simple mechanisms involving multiple steps are possible. For example, a cascade mechanism of the kind described recently by Lytle 1998 for duck red cell Na-K-2Cl cotransport could, in principle, describe the data shown here. In this kind of mechanism, the rates of forward and reverse transitions between active and inactive states are not directly affected by cell volume, but rather are modulated by an enzyme (e.g., kinase) whose activity is sensitive to cell volume. For example, the reverse rate constant k21 could represent a kinase activity that is, in turn, controlled by a separate phosphorylation/dephosphorylation cycle (shown in Fig. 2). This kind of modulation of rate constants by a separate phosphorylation/dephosphorylation cycle could of course be applied to either a two- or three-state mechanism, but, for simplicity, we consider only the two-state case. In this model, cell volume could in principle affect k34 and/or k43, and thereby affect k21. That is, k21 could be inhibited by cell swelling, but the parameter directly affected by cell volume could actually be k34 or k43. Our data cannot rule out a model of this type. However, it should be pointed out that the inactivation rate constant k21 increases without a detectable lag time when cells are suspended in media of increasing osmolality (Fig. 10). The measurements do not have the time resolution to make quantitative estimates of the rate of change of k21, but the data can be fit very easily by assuming that, when the cells first shrink to the new volume, k21 changes much more rapidly than the rate of change of transport. The rapid change in k21 implies that, if the inactivation rate constant is regulated by a volume-dependent phosphorylation/dephosphorylation cycle, then that cycle must be able to reach a new steady state in much less than 1 min.


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 is among the simplest mechanisms containing more than one event in the activation–inactivation process. However, many other relatively simple mechanisms involving multiple steps are possible. For example, a cascade mechanism of the kind described recently by Lytle 1998 for duck red cell Na-K-2Cl cotransport could, in principle, describe the data shown here. In this kind of mechanism, the rates of forward and reverse transitions between active and inactive states are not directly affected by cell volume, but rather are modulated by an enzyme (e.g., kinase) whose activity is sensitive to cell volume. For example, the reverse rate constant k21 could represent a kinase activity that is, in turn, controlled by a separate phosphorylation/dephosphorylation cycle (shown in Fig. 2). This kind of modulation of rate constants by a separate phosphorylation/dephosphorylation cycle could of course be applied to either a two- or three-state mechanism, but, for simplicity, we consider only the two-state case. In this model, cell volume could in principle affect k34 and/or k43, and thereby affect k21. That is, k21 could be inhibited by cell swelling, but the parameter directly affected by cell volume could actually be k34 or k43. Our data cannot rule out a model of this type. However, it should be pointed out that the inactivation rate constant k21 increases without a detectable lag time when cells are suspended in media of increasing osmolality (Fig. 10). The measurements do not have the time resolution to make quantitative estimates of the rate of change of k21, but the data can be fit very easily by assuming that, when the cells first shrink to the new volume, k21 changes much more rapidly than the rate of change of transport. The rapid change in k21 implies that, if the inactivation rate constant is regulated by a volume-dependent phosphorylation/dephosphorylation cycle, then that cycle must be able to reach a new steady state in much less than 1 min.

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