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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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Lack of effect of cell volume on KCC in Mg2+-depleted cells. Cells (not density separated) were suspended in 160 mM NaCl, 10 mM HEPES, 1 mM EDTA, pH 7.5, 0.2% ethanol ± 10 μM A23187, and incubated 10 min at 30°C to deplete Mg2+. Each suspension was then washed once in the above medium (without ethanol and A23187), split in half, and washed once in either the same medium or in medium with 160 mM NaNO3 instead of 160 mM NaCl. Cells were then resuspended in HEPES-buffered NaCl or NaNO3 media with osmolality varied from 185 to 410 mosmol/kg H2O by adding either water or 1 M NaCl (or NaNO3). Suspensions were incubated 20 min at 30°C before adding 1 μCi 86Rb+. Influx was measured for 30 min at 30°C. All flux solutions contained 10−4 M ouabain and 5 mM K+ (added as KCl or KNO3). (Top) No A23187. (Bottom) Mg2+ depletion with A23187. Flux in Cl− media (•). Flux in NO3− media (▪).
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Figure 5: Lack of effect of cell volume on KCC in Mg2+-depleted cells. Cells (not density separated) were suspended in 160 mM NaCl, 10 mM HEPES, 1 mM EDTA, pH 7.5, 0.2% ethanol ± 10 μM A23187, and incubated 10 min at 30°C to deplete Mg2+. Each suspension was then washed once in the above medium (without ethanol and A23187), split in half, and washed once in either the same medium or in medium with 160 mM NaNO3 instead of 160 mM NaCl. Cells were then resuspended in HEPES-buffered NaCl or NaNO3 media with osmolality varied from 185 to 410 mosmol/kg H2O by adding either water or 1 M NaCl (or NaNO3). Suspensions were incubated 20 min at 30°C before adding 1 μCi 86Rb+. Influx was measured for 30 min at 30°C. All flux solutions contained 10−4 M ouabain and 5 mM K+ (added as KCl or KNO3). (Top) No A23187. (Bottom) Mg2+ depletion with A23187. Flux in Cl− media (•). Flux in NO3− media (▪).

Mentions: Fig. 5 shows that, in cells that have been depleted of Mg2+, there is only a very minor effect of cell volume on the KCC flux. A slight volume dependence of the flux in Mg2+-depleted cells is observed even in NO3− medium. Varying the osmolality from 410 to 185 mosmol/kg (2.4–5.4 [osmol/kg]−1) caused the Cl−-dependent flux to change by <20%. The same results were observed in two other experiments. In one earlier experiment, there appeared to be a decrease in the flux in very hypertonic solutions (>400 mosmol/kg), but in the range between 200 and 400 mosmol/kg there is essentially no effect of volume on KCC in Mg2+-depleted rabbit red cells. In contrast, KCC is still volume sensitive in LK sheep red cells, though less so than in cells with normal Mg2+ (Dunham et al. 1993; Dunham 1995). It is impossible to determine whether there is a lag time in the response of KCC to cell swelling in low Mg2+ rabbit red cells, because the flux is not volume dependent.


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

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

Lack of effect of cell volume on KCC in Mg2+-depleted cells. Cells (not density separated) were suspended in 160 mM NaCl, 10 mM HEPES, 1 mM EDTA, pH 7.5, 0.2% ethanol ± 10 μM A23187, and incubated 10 min at 30°C to deplete Mg2+. Each suspension was then washed once in the above medium (without ethanol and A23187), split in half, and washed once in either the same medium or in medium with 160 mM NaNO3 instead of 160 mM NaCl. Cells were then resuspended in HEPES-buffered NaCl or NaNO3 media with osmolality varied from 185 to 410 mosmol/kg H2O by adding either water or 1 M NaCl (or NaNO3). Suspensions were incubated 20 min at 30°C before adding 1 μCi 86Rb+. Influx was measured for 30 min at 30°C. All flux solutions contained 10−4 M ouabain and 5 mM K+ (added as KCl or KNO3). (Top) No A23187. (Bottom) Mg2+ depletion with A23187. Flux in Cl− media (•). Flux in NO3− media (▪).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Lack of effect of cell volume on KCC in Mg2+-depleted cells. Cells (not density separated) were suspended in 160 mM NaCl, 10 mM HEPES, 1 mM EDTA, pH 7.5, 0.2% ethanol ± 10 μM A23187, and incubated 10 min at 30°C to deplete Mg2+. Each suspension was then washed once in the above medium (without ethanol and A23187), split in half, and washed once in either the same medium or in medium with 160 mM NaNO3 instead of 160 mM NaCl. Cells were then resuspended in HEPES-buffered NaCl or NaNO3 media with osmolality varied from 185 to 410 mosmol/kg H2O by adding either water or 1 M NaCl (or NaNO3). Suspensions were incubated 20 min at 30°C before adding 1 μCi 86Rb+. Influx was measured for 30 min at 30°C. All flux solutions contained 10−4 M ouabain and 5 mM K+ (added as KCl or KNO3). (Top) No A23187. (Bottom) Mg2+ depletion with A23187. Flux in Cl− media (•). Flux in NO3− media (▪).
Mentions: Fig. 5 shows that, in cells that have been depleted of Mg2+, there is only a very minor effect of cell volume on the KCC flux. A slight volume dependence of the flux in Mg2+-depleted cells is observed even in NO3− medium. Varying the osmolality from 410 to 185 mosmol/kg (2.4–5.4 [osmol/kg]−1) caused the Cl−-dependent flux to change by <20%. The same results were observed in two other experiments. In one earlier experiment, there appeared to be a decrease in the flux in very hypertonic solutions (>400 mosmol/kg), but in the range between 200 and 400 mosmol/kg there is essentially no effect of volume on KCC in Mg2+-depleted rabbit red cells. In contrast, KCC is still volume sensitive in LK sheep red cells, though less so than in cells with normal Mg2+ (Dunham et al. 1993; Dunham 1995). It is impossible to determine whether there is a lag time in the response of KCC to cell swelling in low Mg2+ rabbit red cells, because the flux is not volume dependent.

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