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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 86Rb+ (*K+) influx into rabbit red cells at 37°C from a medium consisting of (mM): 155 NaCl, 5 KCl, 10 HEPES, 10−4 M ouabain. In two of the suspensions (•, ▪), the pH was initially 7.5 and was lowered to 6.8 at t = 12.5 min by addition of 18 mM MOPS and 5 mM NaHCO3. The solid curve through the data corresponds to a single exponential increase in flux, with a lag time of 14 min. In the other two suspensions (▴, ▾), the pH was lowered to 6.8 by adding 18 mM MOPS and 5 mM NaHCO3 15 min before adding 86Rb+; the pH of these suspensions was raised to 7.9 at t = 12.5 min by addition of NaOH. The curve through these data is , with a lag time of 3.6 min.
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Figure 2: Time course of 86Rb+ (*K+) influx into rabbit red cells at 37°C from a medium consisting of (mM): 155 NaCl, 5 KCl, 10 HEPES, 10−4 M ouabain. In two of the suspensions (•, ▪), the pH was initially 7.5 and was lowered to 6.8 at t = 12.5 min by addition of 18 mM MOPS and 5 mM NaHCO3. The solid curve through the data corresponds to a single exponential increase in flux, with a lag time of 14 min. In the other two suspensions (▴, ▾), the pH was lowered to 6.8 by adding 18 mM MOPS and 5 mM NaHCO3 15 min before adding 86Rb+; the pH of these suspensions was raised to 7.9 at t = 12.5 min by addition of NaOH. The curve through these data is , with a lag time of 3.6 min.

Mentions: The time course of activation of KCC by low pH was determined by adding MOPS from a 1-M stock solution to a final concentration of 15–18 mM. Within a short time after extracellular acidification, the intracellular pH reaches Donnan equilibrium with the extracellular pH (e.g., Funder and Wieth 1966). We estimated intracellular pH in lysed pellets (in 10 vol water, with ionic strength then returned to 100 mM with KCl). This method does not accurately determine the absolute value of intracellular pH, but it is adequate for estimating the time course of changes in pH. We found that the half time for pH equilibration under the conditions of the flux experiment in Fig. 3 (25°C, ambient CO2) is ∼0.8 min in rabbit red cells. At higher temperature (Fig. 1 and Fig. 2), the time course of pH equilibration was not measured, but is believed to be considerably faster, not only because of the high temperature dependence of pH equilibration (Jennings 1978), but because 4–5 mM NaHCO3 was added to facilitate pH equilibration by the Jacobs–Stewart cycle (Jacobs and Stewart 1942).


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 86Rb+ (*K+) influx into rabbit red cells at 37°C from a medium consisting of (mM): 155 NaCl, 5 KCl, 10 HEPES, 10−4 M ouabain. In two of the suspensions (•, ▪), the pH was initially 7.5 and was lowered to 6.8 at t = 12.5 min by addition of 18 mM MOPS and 5 mM NaHCO3. The solid curve through the data corresponds to a single exponential increase in flux, with a lag time of 14 min. In the other two suspensions (▴, ▾), the pH was lowered to 6.8 by adding 18 mM MOPS and 5 mM NaHCO3 15 min before adding 86Rb+; the pH of these suspensions was raised to 7.9 at t = 12.5 min by addition of NaOH. The curve through these data is , with a lag time of 3.6 min.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Time course of 86Rb+ (*K+) influx into rabbit red cells at 37°C from a medium consisting of (mM): 155 NaCl, 5 KCl, 10 HEPES, 10−4 M ouabain. In two of the suspensions (•, ▪), the pH was initially 7.5 and was lowered to 6.8 at t = 12.5 min by addition of 18 mM MOPS and 5 mM NaHCO3. The solid curve through the data corresponds to a single exponential increase in flux, with a lag time of 14 min. In the other two suspensions (▴, ▾), the pH was lowered to 6.8 by adding 18 mM MOPS and 5 mM NaHCO3 15 min before adding 86Rb+; the pH of these suspensions was raised to 7.9 at t = 12.5 min by addition of NaOH. The curve through these data is , with a lag time of 3.6 min.
Mentions: The time course of activation of KCC by low pH was determined by adding MOPS from a 1-M stock solution to a final concentration of 15–18 mM. Within a short time after extracellular acidification, the intracellular pH reaches Donnan equilibrium with the extracellular pH (e.g., Funder and Wieth 1966). We estimated intracellular pH in lysed pellets (in 10 vol water, with ionic strength then returned to 100 mM with KCl). This method does not accurately determine the absolute value of intracellular pH, but it is adequate for estimating the time course of changes in pH. We found that the half time for pH equilibration under the conditions of the flux experiment in Fig. 3 (25°C, ambient CO2) is ∼0.8 min in rabbit red cells. At higher temperature (Fig. 1 and Fig. 2), the time course of pH equilibration was not measured, but is believed to be considerably faster, not only because of the high temperature dependence of pH equilibration (Jennings 1978), but because 4–5 mM NaHCO3 was added to facilitate pH equilibration by the Jacobs–Stewart cycle (Jacobs and Stewart 1942).

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