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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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Time course of activation of KCC at 37°C after NEM pretreatment at 0°C. Rabbit red cells (not density separated) were treated twice with 2 mM NEM at 0°C as in Fig. 6, then washed in cold medium. One aliquot of cells was suspended in isosmotic flux medium and incubated at 37°C for 20 min before adding 86Rb+ at t = 0 (•). The remaining cells were kept on ice for 20 min, and then suspended in 37°C flux medium containing 86Rb+ at t = 0 (▴; mean ± range of two suspensions). The curve through the points represents  with a lag time of 8.9 min. Control cells were not exposed to NEM (▪).
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Figure 7: Time course of activation of KCC at 37°C after NEM pretreatment at 0°C. Rabbit red cells (not density separated) were treated twice with 2 mM NEM at 0°C as in Fig. 6, then washed in cold medium. One aliquot of cells was suspended in isosmotic flux medium and incubated at 37°C for 20 min before adding 86Rb+ at t = 0 (•). The remaining cells were kept on ice for 20 min, and then suspended in 37°C flux medium containing 86Rb+ at t = 0 (▴; mean ± range of two suspensions). The curve through the points represents with a lag time of 8.9 min. Control cells were not exposed to NEM (▪).

Mentions: For experiments involving transport activation by swelling, low pH, or Mg2+ depletion, cells were separated on Percoll-Renograffin as previously described (Al-Rohil and Jennings 1989) to select the least dense one third of cells. The lower-density fractions are enriched in younger cells, which have a higher volume-dependent KCC activity (Brugnara and Tosteson 1987; Canessa et al. 1987; Al-Rohil and Jennings 1989). The NEM activation experiments in Fig. 6Fig. 7Fig. 8Fig. 9 used unseparated red cells; comparable experiments with density-separated cells gave indistinguishable results. If blood had been stored more than a few hours, cells were washed three times and incubated 60–90 min at 37°C in HEPES-buffered physiological saline (HPS: 150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM Na-phosphate, 10 mM HEPES, pH 7.4) plus 10 mM glucose to try to establish a reproducible steady state.


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

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

Time course of activation of KCC at 37°C after NEM pretreatment at 0°C. Rabbit red cells (not density separated) were treated twice with 2 mM NEM at 0°C as in Fig. 6, then washed in cold medium. One aliquot of cells was suspended in isosmotic flux medium and incubated at 37°C for 20 min before adding 86Rb+ at t = 0 (•). The remaining cells were kept on ice for 20 min, and then suspended in 37°C flux medium containing 86Rb+ at t = 0 (▴; mean ± range of two suspensions). The curve through the points represents  with a lag time of 8.9 min. Control cells were not exposed to NEM (▪).
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Figure 7: Time course of activation of KCC at 37°C after NEM pretreatment at 0°C. Rabbit red cells (not density separated) were treated twice with 2 mM NEM at 0°C as in Fig. 6, then washed in cold medium. One aliquot of cells was suspended in isosmotic flux medium and incubated at 37°C for 20 min before adding 86Rb+ at t = 0 (•). The remaining cells were kept on ice for 20 min, and then suspended in 37°C flux medium containing 86Rb+ at t = 0 (▴; mean ± range of two suspensions). The curve through the points represents with a lag time of 8.9 min. Control cells were not exposed to NEM (▪).
Mentions: For experiments involving transport activation by swelling, low pH, or Mg2+ depletion, cells were separated on Percoll-Renograffin as previously described (Al-Rohil and Jennings 1989) to select the least dense one third of cells. The lower-density fractions are enriched in younger cells, which have a higher volume-dependent KCC activity (Brugnara and Tosteson 1987; Canessa et al. 1987; Al-Rohil and Jennings 1989). The NEM activation experiments in Fig. 6Fig. 7Fig. 8Fig. 9 used unseparated red cells; comparable experiments with density-separated cells gave indistinguishable results. If blood had been stored more than a few hours, cells were washed three times and incubated 60–90 min at 37°C in HEPES-buffered physiological saline (HPS: 150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM Na-phosphate, 10 mM HEPES, pH 7.4) plus 10 mM glucose to try to establish a reproducible steady state.

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