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Severed Channels Probe Regulation of Gating of Cystic Fibrosis Transmembrane Conductance Regulator by Its Cytoplasmic Domains

Opening and closing of a CFTR Cl(−) channel is controlled by PKA-mediated phosphorylation of its cytoplasmic regulatory (R) domain and by ATP binding, and likely hydrolysis, at its two nucleotide binding domains. Functional interactions between the R domain and the two nucleotide binding domains wer...

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Autores principales: Csanády, László, Chan, Kim W., Seto-Young, Donna, Kopsco, David C., Nairn, Angus C., Gadsby, David C.
Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 2000
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2233695/
https://www.ncbi.nlm.nih.gov/pubmed/10962022
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author Csanády, László
Chan, Kim W.
Seto-Young, Donna
Kopsco, David C.
Nairn, Angus C.
Gadsby, David C.
author_facet Csanády, László
Chan, Kim W.
Seto-Young, Donna
Kopsco, David C.
Nairn, Angus C.
Gadsby, David C.
author_sort Csanády, László
collection PubMed
description Opening and closing of a CFTR Cl(−) channel is controlled by PKA-mediated phosphorylation of its cytoplasmic regulatory (R) domain and by ATP binding, and likely hydrolysis, at its two nucleotide binding domains. Functional interactions between the R domain and the two nucleotide binding domains were probed by characterizing the gating of severed CFTR channels expressed in Xenopus oocytes. Expression levels were assessed using measurements of oocyte conductance, and detailed functional characteristics of the channels were extracted from kinetic analyses of macroscopic current relaxations and of single-channel gating events in membrane patches excised from the oocytes. The kinetic behavior of wild-type (WT) CFTR channels was compared with that of split CFTR channels bearing a single cut (between residues 633 and 634) just before the R domain, of split channels with a single cut (between residues 835 and 837) just after the R domain, and of split channels from which the entire R domain (residues 634–836) between those two cut sites was omitted. The channels cut before the R domain had characteristics almost identical to those of WT channels, except for less than twofold shorter open burst durations in the presence of PKA. Channels cut just after the R domain were characterized by a low level of activity even without phosphorylation, strong stimulation by PKA, enhanced apparent affinity for ATP as assayed by open probability, and a somewhat destabilized binding site for the locking action of the nonhydrolyzable ATP analog AMPPNP. Split channels with no R domain (from coexpression of CFTR segments 1–633 and 837–1480) were highly active without phosphorylation, but otherwise displayed the characteristics of channels cut after the R domain, including higher apparent ATP affinity, and less tight binding of AMPPNP at the locking site, than for WT. Intriguingly, severed channels with no R domain were still noticeably stimulated by PKA, implying that activation of WT CFTR by PKA likely also includes some component unrelated to the R domain. As the maximal opening rates were the same for WT channels and split channels with no R domain, it seems that the phosphorylated R domain does not stimulate opening of CFTR channels; rather, the dephosphorylated R domain inhibits them.
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spelling pubmed-22336952008-04-22 Severed Channels Probe Regulation of Gating of Cystic Fibrosis Transmembrane Conductance Regulator by Its Cytoplasmic Domains Csanády, László Chan, Kim W. Seto-Young, Donna Kopsco, David C. Nairn, Angus C. Gadsby, David C. J Gen Physiol Original Article Opening and closing of a CFTR Cl(−) channel is controlled by PKA-mediated phosphorylation of its cytoplasmic regulatory (R) domain and by ATP binding, and likely hydrolysis, at its two nucleotide binding domains. Functional interactions between the R domain and the two nucleotide binding domains were probed by characterizing the gating of severed CFTR channels expressed in Xenopus oocytes. Expression levels were assessed using measurements of oocyte conductance, and detailed functional characteristics of the channels were extracted from kinetic analyses of macroscopic current relaxations and of single-channel gating events in membrane patches excised from the oocytes. The kinetic behavior of wild-type (WT) CFTR channels was compared with that of split CFTR channels bearing a single cut (between residues 633 and 634) just before the R domain, of split channels with a single cut (between residues 835 and 837) just after the R domain, and of split channels from which the entire R domain (residues 634–836) between those two cut sites was omitted. The channels cut before the R domain had characteristics almost identical to those of WT channels, except for less than twofold shorter open burst durations in the presence of PKA. Channels cut just after the R domain were characterized by a low level of activity even without phosphorylation, strong stimulation by PKA, enhanced apparent affinity for ATP as assayed by open probability, and a somewhat destabilized binding site for the locking action of the nonhydrolyzable ATP analog AMPPNP. Split channels with no R domain (from coexpression of CFTR segments 1–633 and 837–1480) were highly active without phosphorylation, but otherwise displayed the characteristics of channels cut after the R domain, including higher apparent ATP affinity, and less tight binding of AMPPNP at the locking site, than for WT. Intriguingly, severed channels with no R domain were still noticeably stimulated by PKA, implying that activation of WT CFTR by PKA likely also includes some component unrelated to the R domain. As the maximal opening rates were the same for WT channels and split channels with no R domain, it seems that the phosphorylated R domain does not stimulate opening of CFTR channels; rather, the dephosphorylated R domain inhibits them. The Rockefeller University Press 2000-09-01 /pmc/articles/PMC2233695/ /pubmed/10962022 Text en © 2000 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
Csanády, László
Chan, Kim W.
Seto-Young, Donna
Kopsco, David C.
Nairn, Angus C.
Gadsby, David C.
Severed Channels Probe Regulation of Gating of Cystic Fibrosis Transmembrane Conductance Regulator by Its Cytoplasmic Domains
title Severed Channels Probe Regulation of Gating of Cystic Fibrosis Transmembrane Conductance Regulator by Its Cytoplasmic Domains
title_full Severed Channels Probe Regulation of Gating of Cystic Fibrosis Transmembrane Conductance Regulator by Its Cytoplasmic Domains
title_fullStr Severed Channels Probe Regulation of Gating of Cystic Fibrosis Transmembrane Conductance Regulator by Its Cytoplasmic Domains
title_full_unstemmed Severed Channels Probe Regulation of Gating of Cystic Fibrosis Transmembrane Conductance Regulator by Its Cytoplasmic Domains
title_short Severed Channels Probe Regulation of Gating of Cystic Fibrosis Transmembrane Conductance Regulator by Its Cytoplasmic Domains
title_sort severed channels probe regulation of gating of cystic fibrosis transmembrane conductance regulator by its cytoplasmic domains
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2233695/
https://www.ncbi.nlm.nih.gov/pubmed/10962022
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