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Mutagenic Mapping of the Na-K-Cl Cotransporter for Domains Involved in Ion Transport and Bumetanide Binding
The human and shark Na-K-Cl cotransporters (NKCCs) are 74% identical in amino acid sequence yet they display marked differences in apparent affinities for the ions and bumetanide. In this study, we have used chimeras and point mutations to determine which transmembrane domains (tm's) are respon...
Autores principales: | , , , |
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Formato: | Texto |
Lenguaje: | English |
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The Rockefeller University Press
1998
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2229443/ https://www.ncbi.nlm.nih.gov/pubmed/9806964 |
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author | Isenring, Paul Jacoby, Steven C. Chang, John Forbush, Bliss |
author_facet | Isenring, Paul Jacoby, Steven C. Chang, John Forbush, Bliss |
author_sort | Isenring, Paul |
collection | PubMed |
description | The human and shark Na-K-Cl cotransporters (NKCCs) are 74% identical in amino acid sequence yet they display marked differences in apparent affinities for the ions and bumetanide. In this study, we have used chimeras and point mutations to determine which transmembrane domains (tm's) are responsible for the differences in ion transport and in inhibitor binding kinetics. When expressed in HEK-293 cells, all the mutants carry out bumetanide-sensitive (86)Rb influx. The kinetic behavior of these constructs demonstrates that the first seven tm's contain all of the residues conferring affinity differences. In conjunction with our previous finding that tm 2 plays an important role in cation transport, the present observations implicate the fourth and seventh tm helices in anion transport. Thus, it appears that tm's 2, 4, and 7 contain the essential affinity-modifying residues accounting for the human–shark differences with regard to cation and anion transport. Point mutations have narrowed the list of candidates to 13 residues within the three tm's. The affinity for bumetanide was found to be affected by residues in the same tm 2–7 region, and also by residues in tm's 11 and 12. Unlike for the ions, changes in bumetanide affinity were nonlinear and difficult to interpret: the K (i(bumetanide)) of a number of the constructs was outside the range of sNKCC1 and hNKCC1 K (i)s. |
format | Text |
id | pubmed-2229443 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 1998 |
publisher | The Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-22294432008-04-21 Mutagenic Mapping of the Na-K-Cl Cotransporter for Domains Involved in Ion Transport and Bumetanide Binding Isenring, Paul Jacoby, Steven C. Chang, John Forbush, Bliss J Gen Physiol Article The human and shark Na-K-Cl cotransporters (NKCCs) are 74% identical in amino acid sequence yet they display marked differences in apparent affinities for the ions and bumetanide. In this study, we have used chimeras and point mutations to determine which transmembrane domains (tm's) are responsible for the differences in ion transport and in inhibitor binding kinetics. When expressed in HEK-293 cells, all the mutants carry out bumetanide-sensitive (86)Rb influx. The kinetic behavior of these constructs demonstrates that the first seven tm's contain all of the residues conferring affinity differences. In conjunction with our previous finding that tm 2 plays an important role in cation transport, the present observations implicate the fourth and seventh tm helices in anion transport. Thus, it appears that tm's 2, 4, and 7 contain the essential affinity-modifying residues accounting for the human–shark differences with regard to cation and anion transport. Point mutations have narrowed the list of candidates to 13 residues within the three tm's. The affinity for bumetanide was found to be affected by residues in the same tm 2–7 region, and also by residues in tm's 11 and 12. Unlike for the ions, changes in bumetanide affinity were nonlinear and difficult to interpret: the K (i(bumetanide)) of a number of the constructs was outside the range of sNKCC1 and hNKCC1 K (i)s. The Rockefeller University Press 1998-11-01 /pmc/articles/PMC2229443/ /pubmed/9806964 Text en 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 | Article Isenring, Paul Jacoby, Steven C. Chang, John Forbush, Bliss Mutagenic Mapping of the Na-K-Cl Cotransporter for Domains Involved in Ion Transport and Bumetanide Binding |
title | Mutagenic Mapping of the Na-K-Cl Cotransporter for Domains Involved in Ion Transport and Bumetanide Binding |
title_full | Mutagenic Mapping of the Na-K-Cl Cotransporter for Domains Involved in Ion Transport and Bumetanide Binding |
title_fullStr | Mutagenic Mapping of the Na-K-Cl Cotransporter for Domains Involved in Ion Transport and Bumetanide Binding |
title_full_unstemmed | Mutagenic Mapping of the Na-K-Cl Cotransporter for Domains Involved in Ion Transport and Bumetanide Binding |
title_short | Mutagenic Mapping of the Na-K-Cl Cotransporter for Domains Involved in Ion Transport and Bumetanide Binding |
title_sort | mutagenic mapping of the na-k-cl cotransporter for domains involved in ion transport and bumetanide binding |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2229443/ https://www.ncbi.nlm.nih.gov/pubmed/9806964 |
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