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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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Effect of cell volume on the rate of inactivation of KCC at 25°C. Cells were preincubated in hyposmotic (200 mosmol/kg) medium for 15 min at 37°C to activate KCC and were pelleted and maintained at 37°C before resuspending at 25°C in 86Rb-containing media of the indicated osmolality. All flux mediate contained 5 mM KCl and 10−4 M ouabain. The solid curves through the data represent single exponential decreases in the flux (), with initial flux equal to the initial slope of the curve in 196 mosmol/kg medium. The lag times for the individual curves (from the top) are 17.5, 11.3, 6.0, 3.7, and 1.8 min.
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Figure 10: Effect of cell volume on the rate of inactivation of KCC at 25°C. Cells were preincubated in hyposmotic (200 mosmol/kg) medium for 15 min at 37°C to activate KCC and were pelleted and maintained at 37°C before resuspending at 25°C in 86Rb-containing media of the indicated osmolality. All flux mediate contained 5 mM KCl and 10−4 M ouabain. The solid curves through the data represent single exponential decreases in the flux (), with initial flux equal to the initial slope of the curve in 196 mosmol/kg medium. The lag times for the individual curves (from the top) are 17.5, 11.3, 6.0, 3.7, and 1.8 min.

Mentions: The influx data were fit (Sigma Plot; Jandel Scientific) to , with two adjustable parameters, τ and J1, for most experiments. In the NEM activation experiments, the steady state flux J1 was determined independently in a parallel suspension that had been incubated 15–20 min at 37°C to allow KCC to activate. In these experiments, the only adjustable parameter in the curve fits was the lag time τ, which can be estimated accurately when all the other parameters are determined independently. In the inactivation experiments (Fig. 10Fig. 11Fig. 12), the initial flux was estimated in swollen cells, and the lag time was determined in a two-parameter fit (τ and J1). The estimate of τ is reasonably accurate in an inactivation experiment (despite the two-parameter fit) because the final steady state flux is small.


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

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

Effect of cell volume on the rate of inactivation of KCC at 25°C. Cells were preincubated in hyposmotic (200 mosmol/kg) medium for 15 min at 37°C to activate KCC and were pelleted and maintained at 37°C before resuspending at 25°C in 86Rb-containing media of the indicated osmolality. All flux mediate contained 5 mM KCl and 10−4 M ouabain. The solid curves through the data represent single exponential decreases in the flux (), with initial flux equal to the initial slope of the curve in 196 mosmol/kg medium. The lag times for the individual curves (from the top) are 17.5, 11.3, 6.0, 3.7, and 1.8 min.
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Related In: Results  -  Collection

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

Figure 10: Effect of cell volume on the rate of inactivation of KCC at 25°C. Cells were preincubated in hyposmotic (200 mosmol/kg) medium for 15 min at 37°C to activate KCC and were pelleted and maintained at 37°C before resuspending at 25°C in 86Rb-containing media of the indicated osmolality. All flux mediate contained 5 mM KCl and 10−4 M ouabain. The solid curves through the data represent single exponential decreases in the flux (), with initial flux equal to the initial slope of the curve in 196 mosmol/kg medium. The lag times for the individual curves (from the top) are 17.5, 11.3, 6.0, 3.7, and 1.8 min.
Mentions: The influx data were fit (Sigma Plot; Jandel Scientific) to , with two adjustable parameters, τ and J1, for most experiments. In the NEM activation experiments, the steady state flux J1 was determined independently in a parallel suspension that had been incubated 15–20 min at 37°C to allow KCC to activate. In these experiments, the only adjustable parameter in the curve fits was the lag time τ, which can be estimated accurately when all the other parameters are determined independently. In the inactivation experiments (Fig. 10Fig. 11Fig. 12), the initial flux was estimated in swollen cells, and the lag time was determined in a two-parameter fit (τ and J1). The estimate of τ is reasonably accurate in an inactivation experiment (despite the two-parameter fit) because the final steady state flux is small.

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