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Volume-Sensitive K(+)/Cl(−) Cotransport in Rabbit Erythrocytes: Analysis of the Rate-Limiting Activation and Inactivation Events
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-ethylmaleimid...
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Formato: | Texto |
Lenguaje: | English |
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The Rockefeller University Press
1999
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2230653/ https://www.ncbi.nlm.nih.gov/pubmed/10578012 |
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author | Jennings, Michael L. |
author_facet | Jennings, Michael L. |
author_sort | Jennings, Michael L. |
collection | PubMed |
description | 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°C, washed, and then incubated at higher temperatures. The forward rate constant for activation has a very high temperature dependence (E(a) ∼ 32 kCal/mol), but is not affected measurably by cell volume. Inactivation kinetics were examined by swelling cells at 37°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. |
format | Text |
id | pubmed-2230653 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 1999 |
publisher | The Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-22306532008-04-22 Volume-Sensitive K(+)/Cl(−) Cotransport in Rabbit Erythrocytes: Analysis of the Rate-Limiting Activation and Inactivation Events Jennings, Michael L. J Gen Physiol Original Article 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°C, washed, and then incubated at higher temperatures. The forward rate constant for activation has a very high temperature dependence (E(a) ∼ 32 kCal/mol), but is not affected measurably by cell volume. Inactivation kinetics were examined by swelling cells at 37°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. The Rockefeller University Press 1999-12-01 /pmc/articles/PMC2230653/ /pubmed/10578012 Text en © 1999 The Rockefeller University Press This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/). |
spellingShingle | Original Article Jennings, Michael L. Volume-Sensitive K(+)/Cl(−) Cotransport in Rabbit Erythrocytes: Analysis of the Rate-Limiting Activation and Inactivation Events |
title | Volume-Sensitive K(+)/Cl(−) Cotransport in Rabbit Erythrocytes: Analysis of the Rate-Limiting Activation and Inactivation Events |
title_full | Volume-Sensitive K(+)/Cl(−) Cotransport in Rabbit Erythrocytes: Analysis of the Rate-Limiting Activation and Inactivation Events |
title_fullStr | Volume-Sensitive K(+)/Cl(−) Cotransport in Rabbit Erythrocytes: Analysis of the Rate-Limiting Activation and Inactivation Events |
title_full_unstemmed | Volume-Sensitive K(+)/Cl(−) Cotransport in Rabbit Erythrocytes: Analysis of the Rate-Limiting Activation and Inactivation Events |
title_short | Volume-Sensitive K(+)/Cl(−) Cotransport in Rabbit Erythrocytes: Analysis of the Rate-Limiting Activation and Inactivation Events |
title_sort | volume-sensitive k(+)/cl(−) cotransport in rabbit erythrocytes: analysis of the rate-limiting activation and inactivation events |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2230653/ https://www.ncbi.nlm.nih.gov/pubmed/10578012 |
work_keys_str_mv | AT jenningsmichaell volumesensitivekclcotransportinrabbiterythrocytesanalysisoftheratelimitingactivationandinactivationevents |