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Perturbation Analysis of the Voltage-sensitive Conformational Changes of the Na(+)/Glucose Cotransporter

Conformational changes of the human Na(+)/glucose cotransporter (hSGLT1) were studied using voltage-jump methods. The cotransporter was expressed in Xenopus laevis oocytes, and SGLT1 charge movements were measured in the micro- to millisecond time scale using the cut-open oocyte preparation and in t...

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Autores principales: Loo, Donald D.F., Hirayama, Bruce A., Cha, Albert, Bezanilla, Francisco, Wright, Ernest M.
Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 2005
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2217483/
https://www.ncbi.nlm.nih.gov/pubmed/15596535
http://dx.doi.org/10.1085/jgp.200409150
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author Loo, Donald D.F.
Hirayama, Bruce A.
Cha, Albert
Bezanilla, Francisco
Wright, Ernest M.
author_facet Loo, Donald D.F.
Hirayama, Bruce A.
Cha, Albert
Bezanilla, Francisco
Wright, Ernest M.
author_sort Loo, Donald D.F.
collection PubMed
description Conformational changes of the human Na(+)/glucose cotransporter (hSGLT1) were studied using voltage-jump methods. The cotransporter was expressed in Xenopus laevis oocytes, and SGLT1 charge movements were measured in the micro- to millisecond time scale using the cut-open oocyte preparation and in the millisecond to second time scale using the two-electrode voltage clamp method. Simultaneous charge and fluorescence changes were studied using tetramethylrhodamine-6-maleimide-labeled hSGLT1 Q457C. In 100 mM external [Na(+)], depolarizing voltage steps evoked a charge movement that rose initially to a peak (with time constant τ = 0.17 ms) before decaying to steady state with two time constants (τ = 2–30 and 25–150 ms). The time to peak (0.9 ms) decreased with [Na(+)], and was not observed in 0 mM [Na(+)]. In absence of Na(+), charge movement decayed monotonically to steady state with three time constants (0.2, 2, and 150 ms). Charge movement was accompanied by fluorescence changes with similar time courses, indicating that global conformational changes monitored by charge movement are reflected by local environmental changes at or near Q457C. Our results indicate that the major voltage-dependent step of the Na(+)/glucose transport cycle is the return of the empty carrier from inward to outward facing conformations. Finally, we observed subtle differences between time constants for charge movement and for optical changes, suggesting that optical recordings can be used to monitor local conformational changes that underlie the global conformational changes of cotransporters.
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spelling pubmed-22174832008-03-21 Perturbation Analysis of the Voltage-sensitive Conformational Changes of the Na(+)/Glucose Cotransporter Loo, Donald D.F. Hirayama, Bruce A. Cha, Albert Bezanilla, Francisco Wright, Ernest M. J Gen Physiol Article Conformational changes of the human Na(+)/glucose cotransporter (hSGLT1) were studied using voltage-jump methods. The cotransporter was expressed in Xenopus laevis oocytes, and SGLT1 charge movements were measured in the micro- to millisecond time scale using the cut-open oocyte preparation and in the millisecond to second time scale using the two-electrode voltage clamp method. Simultaneous charge and fluorescence changes were studied using tetramethylrhodamine-6-maleimide-labeled hSGLT1 Q457C. In 100 mM external [Na(+)], depolarizing voltage steps evoked a charge movement that rose initially to a peak (with time constant τ = 0.17 ms) before decaying to steady state with two time constants (τ = 2–30 and 25–150 ms). The time to peak (0.9 ms) decreased with [Na(+)], and was not observed in 0 mM [Na(+)]. In absence of Na(+), charge movement decayed monotonically to steady state with three time constants (0.2, 2, and 150 ms). Charge movement was accompanied by fluorescence changes with similar time courses, indicating that global conformational changes monitored by charge movement are reflected by local environmental changes at or near Q457C. Our results indicate that the major voltage-dependent step of the Na(+)/glucose transport cycle is the return of the empty carrier from inward to outward facing conformations. Finally, we observed subtle differences between time constants for charge movement and for optical changes, suggesting that optical recordings can be used to monitor local conformational changes that underlie the global conformational changes of cotransporters. The Rockefeller University Press 2005-01 /pmc/articles/PMC2217483/ /pubmed/15596535 http://dx.doi.org/10.1085/jgp.200409150 Text en Copyright © 2005, 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 Article
Loo, Donald D.F.
Hirayama, Bruce A.
Cha, Albert
Bezanilla, Francisco
Wright, Ernest M.
Perturbation Analysis of the Voltage-sensitive Conformational Changes of the Na(+)/Glucose Cotransporter
title Perturbation Analysis of the Voltage-sensitive Conformational Changes of the Na(+)/Glucose Cotransporter
title_full Perturbation Analysis of the Voltage-sensitive Conformational Changes of the Na(+)/Glucose Cotransporter
title_fullStr Perturbation Analysis of the Voltage-sensitive Conformational Changes of the Na(+)/Glucose Cotransporter
title_full_unstemmed Perturbation Analysis of the Voltage-sensitive Conformational Changes of the Na(+)/Glucose Cotransporter
title_short Perturbation Analysis of the Voltage-sensitive Conformational Changes of the Na(+)/Glucose Cotransporter
title_sort perturbation analysis of the voltage-sensitive conformational changes of the na(+)/glucose cotransporter
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2217483/
https://www.ncbi.nlm.nih.gov/pubmed/15596535
http://dx.doi.org/10.1085/jgp.200409150
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