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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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Steady state KCC flux (•) and inverse lag time for inactivation (○) at 25°C after preactivation in 196 mosmol/kg medium at 37°C as in Fig. 10. Data are the mean ± SD for three experiments, including that in Fig. 10. The flux in 325 mosmol/kg media was subtracted from the steady state flux at all other osmolalities for each preparation of cells to correct for variations in the basal flux.
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Figure 11: Steady state KCC flux (•) and inverse lag time for inactivation (○) at 25°C after preactivation in 196 mosmol/kg medium at 37°C as in Fig. 10. Data are the mean ± SD for three experiments, including that in Fig. 10. The flux in 325 mosmol/kg media was subtracted from the steady state flux at all other osmolalities for each preparation of cells to correct for variations in the basal flux.

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)

Steady state KCC flux (•) and inverse lag time for inactivation (○) at 25°C after preactivation in 196 mosmol/kg medium at 37°C as in Fig. 10. Data are the mean ± SD for three experiments, including that in Fig. 10. The flux in 325 mosmol/kg media was subtracted from the steady state flux at all other osmolalities for each preparation of cells to correct for variations in the basal flux.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 11: Steady state KCC flux (•) and inverse lag time for inactivation (○) at 25°C after preactivation in 196 mosmol/kg medium at 37°C as in Fig. 10. Data are the mean ± SD for three experiments, including that in Fig. 10. The flux in 325 mosmol/kg media was subtracted from the steady state flux at all other osmolalities for each preparation of cells to correct for variations in the basal flux.
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