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Cooperative Binding of Substrate and Ions Drives Forward Cycling of the Human Creatine Transporter-1

Creatine serves as an ATP buffer and is thus an integral component of cellular energy metabolism. Most cells maintain their creatine levels via uptake by the creatine transporter (CRT-1, SLC6A8). The activity of CRT-1, therefore, is a major determinant of cytosolic creatine concentrations. We determ...

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Autores principales: Farr, Clemens V., El-Kasaby, Ali, Erdem, Fatma A., Sucic, Sonja, Freissmuth, Michael, Sandtner, Walter
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9273935/
https://www.ncbi.nlm.nih.gov/pubmed/35837012
http://dx.doi.org/10.3389/fphys.2022.919439
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author Farr, Clemens V.
El-Kasaby, Ali
Erdem, Fatma A.
Sucic, Sonja
Freissmuth, Michael
Sandtner, Walter
author_facet Farr, Clemens V.
El-Kasaby, Ali
Erdem, Fatma A.
Sucic, Sonja
Freissmuth, Michael
Sandtner, Walter
author_sort Farr, Clemens V.
collection PubMed
description Creatine serves as an ATP buffer and is thus an integral component of cellular energy metabolism. Most cells maintain their creatine levels via uptake by the creatine transporter (CRT-1, SLC6A8). The activity of CRT-1, therefore, is a major determinant of cytosolic creatine concentrations. We determined the kinetics of CRT-1 in real time by relying on electrophysiological recordings of transport-associated currents. Our analysis revealed that CRT-1 harvested the concentration gradient of NaCl and the membrane potential but not the potassium gradient to achieve a very high concentrative power. We investigated the mechanistic basis for the ability of CRT-1 to maintain the forward cycling mode in spite of high intracellular concentrations of creatine: this is achieved by cooperative binding of substrate and co-substrate ions, which, under physiological ion conditions, results in a very pronounced (i.e. about 500-fold) drop in the affinity of creatine to the inward-facing state of CRT-1. Kinetic estimates were integrated into a mathematical model of the transport cycle of CRT-1, which faithfully reproduced all experimental data. We interrogated the kinetic model to examine the most plausible mechanistic basis of cooperativity: based on this systematic exploration, we conclude that destabilization of binary rather than ternary complexes is necessary for CRT-1 to maintain the observed cytosolic creatine concentrations. Our model also provides a plausible explanation why neurons, heart and skeletal muscle cells must express a creatine releasing transporter to achieve rapid equilibration of the intracellular creatine pool.
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spelling pubmed-92739352022-07-13 Cooperative Binding of Substrate and Ions Drives Forward Cycling of the Human Creatine Transporter-1 Farr, Clemens V. El-Kasaby, Ali Erdem, Fatma A. Sucic, Sonja Freissmuth, Michael Sandtner, Walter Front Physiol Physiology Creatine serves as an ATP buffer and is thus an integral component of cellular energy metabolism. Most cells maintain their creatine levels via uptake by the creatine transporter (CRT-1, SLC6A8). The activity of CRT-1, therefore, is a major determinant of cytosolic creatine concentrations. We determined the kinetics of CRT-1 in real time by relying on electrophysiological recordings of transport-associated currents. Our analysis revealed that CRT-1 harvested the concentration gradient of NaCl and the membrane potential but not the potassium gradient to achieve a very high concentrative power. We investigated the mechanistic basis for the ability of CRT-1 to maintain the forward cycling mode in spite of high intracellular concentrations of creatine: this is achieved by cooperative binding of substrate and co-substrate ions, which, under physiological ion conditions, results in a very pronounced (i.e. about 500-fold) drop in the affinity of creatine to the inward-facing state of CRT-1. Kinetic estimates were integrated into a mathematical model of the transport cycle of CRT-1, which faithfully reproduced all experimental data. We interrogated the kinetic model to examine the most plausible mechanistic basis of cooperativity: based on this systematic exploration, we conclude that destabilization of binary rather than ternary complexes is necessary for CRT-1 to maintain the observed cytosolic creatine concentrations. Our model also provides a plausible explanation why neurons, heart and skeletal muscle cells must express a creatine releasing transporter to achieve rapid equilibration of the intracellular creatine pool. Frontiers Media S.A. 2022-06-28 /pmc/articles/PMC9273935/ /pubmed/35837012 http://dx.doi.org/10.3389/fphys.2022.919439 Text en Copyright © 2022 Farr, El-Kasaby, Erdem, Sucic, Freissmuth and Sandtner. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Physiology
Farr, Clemens V.
El-Kasaby, Ali
Erdem, Fatma A.
Sucic, Sonja
Freissmuth, Michael
Sandtner, Walter
Cooperative Binding of Substrate and Ions Drives Forward Cycling of the Human Creatine Transporter-1
title Cooperative Binding of Substrate and Ions Drives Forward Cycling of the Human Creatine Transporter-1
title_full Cooperative Binding of Substrate and Ions Drives Forward Cycling of the Human Creatine Transporter-1
title_fullStr Cooperative Binding of Substrate and Ions Drives Forward Cycling of the Human Creatine Transporter-1
title_full_unstemmed Cooperative Binding of Substrate and Ions Drives Forward Cycling of the Human Creatine Transporter-1
title_short Cooperative Binding of Substrate and Ions Drives Forward Cycling of the Human Creatine Transporter-1
title_sort cooperative binding of substrate and ions drives forward cycling of the human creatine transporter-1
topic Physiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9273935/
https://www.ncbi.nlm.nih.gov/pubmed/35837012
http://dx.doi.org/10.3389/fphys.2022.919439
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