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Functional characterization of SGLT1 using SSM-based electrophysiology: Kinetics of sugar binding and translocation

Beside the ongoing efforts to determine structural information, detailed functional studies on transporters are essential to entirely understand the underlying transport mechanisms. We recently found that solid supported membrane-based electrophysiology (SSME) enables the measurement of both sugar b...

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Autores principales: Bazzone, Andre, Zerlotti, Rocco, Barthmes, Maria, Fertig, Niels
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9941201/
https://www.ncbi.nlm.nih.gov/pubmed/36824475
http://dx.doi.org/10.3389/fphys.2023.1058583
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author Bazzone, Andre
Zerlotti, Rocco
Barthmes, Maria
Fertig, Niels
author_facet Bazzone, Andre
Zerlotti, Rocco
Barthmes, Maria
Fertig, Niels
author_sort Bazzone, Andre
collection PubMed
description Beside the ongoing efforts to determine structural information, detailed functional studies on transporters are essential to entirely understand the underlying transport mechanisms. We recently found that solid supported membrane-based electrophysiology (SSME) enables the measurement of both sugar binding and transport in the Na(+)/sugar cotransporter SGLT1 (Bazzone et al, 2022a). Here, we continued with a detailed kinetic characterization of SGLT1 using SSME, determining K(M) and K(D) (app) for different sugars, k(obs) values for sugar-induced conformational transitions and the effects of Na(+), Li(+), H(+) and Cl(−) on sugar binding and transport. We found that the sugar-induced pre-steady-state (PSS) charge translocation varies with the bound ion (Na(+), Li(+), H(+) or Cl(−)), but not with the sugar species, indicating that the conformational state upon sugar binding depends on the ion. Rate constants for the sugar-induced conformational transitions upon binding to the Na(+)-bound carrier range from 208 s(−1) for D-glucose to 95 s(−1) for 3-OMG. In the absence of Na(+), rate constants are decreased, but all sugars bind to the empty carrier. From the steady-state transport current, we found a sequence for sugar specificity (V(max)/K(M)): D-glucose > MDG > D-galactose > 3-OMG > D-xylose. While K(M) differs 160-fold across tested substrates and plays a major role in substrate specificity, V(max) only varies by a factor of 1.9. Interestingly, D-glucose has the lowest V(max) across all tested substrates, indicating a rate limiting step in the sugar translocation pathway following the fast sugar-induced electrogenic conformational transition. SGLT1 specificity for D-glucose is achieved by optimizing two ratios: the sugar affinity of the empty carrier for D-glucose is similarly low as for all tested sugars (K(D,K) (app) = 210 mM). Affinity for D-glucose increases 14-fold (K(D,Na) (app) = 15 mM) in the presence of sodium as a result of cooperativity. Apparent affinity for D-glucose during transport increases 8-fold (K(M) = 1.9 mM) compared to K(D,Na) (app) due to optimized kinetics. In contrast, K(M) and K(D) (app) values for 3-OMG and D-xylose are of similar magnitude. Based on our findings we propose an 11-state kinetic model, introducing a random binding order and intermediate states corresponding to the electrogenic transitions detected via SSME upon substrate binding.
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spelling pubmed-99412012023-02-22 Functional characterization of SGLT1 using SSM-based electrophysiology: Kinetics of sugar binding and translocation Bazzone, Andre Zerlotti, Rocco Barthmes, Maria Fertig, Niels Front Physiol Physiology Beside the ongoing efforts to determine structural information, detailed functional studies on transporters are essential to entirely understand the underlying transport mechanisms. We recently found that solid supported membrane-based electrophysiology (SSME) enables the measurement of both sugar binding and transport in the Na(+)/sugar cotransporter SGLT1 (Bazzone et al, 2022a). Here, we continued with a detailed kinetic characterization of SGLT1 using SSME, determining K(M) and K(D) (app) for different sugars, k(obs) values for sugar-induced conformational transitions and the effects of Na(+), Li(+), H(+) and Cl(−) on sugar binding and transport. We found that the sugar-induced pre-steady-state (PSS) charge translocation varies with the bound ion (Na(+), Li(+), H(+) or Cl(−)), but not with the sugar species, indicating that the conformational state upon sugar binding depends on the ion. Rate constants for the sugar-induced conformational transitions upon binding to the Na(+)-bound carrier range from 208 s(−1) for D-glucose to 95 s(−1) for 3-OMG. In the absence of Na(+), rate constants are decreased, but all sugars bind to the empty carrier. From the steady-state transport current, we found a sequence for sugar specificity (V(max)/K(M)): D-glucose > MDG > D-galactose > 3-OMG > D-xylose. While K(M) differs 160-fold across tested substrates and plays a major role in substrate specificity, V(max) only varies by a factor of 1.9. Interestingly, D-glucose has the lowest V(max) across all tested substrates, indicating a rate limiting step in the sugar translocation pathway following the fast sugar-induced electrogenic conformational transition. SGLT1 specificity for D-glucose is achieved by optimizing two ratios: the sugar affinity of the empty carrier for D-glucose is similarly low as for all tested sugars (K(D,K) (app) = 210 mM). Affinity for D-glucose increases 14-fold (K(D,Na) (app) = 15 mM) in the presence of sodium as a result of cooperativity. Apparent affinity for D-glucose during transport increases 8-fold (K(M) = 1.9 mM) compared to K(D,Na) (app) due to optimized kinetics. In contrast, K(M) and K(D) (app) values for 3-OMG and D-xylose are of similar magnitude. Based on our findings we propose an 11-state kinetic model, introducing a random binding order and intermediate states corresponding to the electrogenic transitions detected via SSME upon substrate binding. Frontiers Media S.A. 2023-02-07 /pmc/articles/PMC9941201/ /pubmed/36824475 http://dx.doi.org/10.3389/fphys.2023.1058583 Text en Copyright © 2023 Bazzone, Zerlotti, Barthmes and Fertig. 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
Bazzone, Andre
Zerlotti, Rocco
Barthmes, Maria
Fertig, Niels
Functional characterization of SGLT1 using SSM-based electrophysiology: Kinetics of sugar binding and translocation
title Functional characterization of SGLT1 using SSM-based electrophysiology: Kinetics of sugar binding and translocation
title_full Functional characterization of SGLT1 using SSM-based electrophysiology: Kinetics of sugar binding and translocation
title_fullStr Functional characterization of SGLT1 using SSM-based electrophysiology: Kinetics of sugar binding and translocation
title_full_unstemmed Functional characterization of SGLT1 using SSM-based electrophysiology: Kinetics of sugar binding and translocation
title_short Functional characterization of SGLT1 using SSM-based electrophysiology: Kinetics of sugar binding and translocation
title_sort functional characterization of sglt1 using ssm-based electrophysiology: kinetics of sugar binding and translocation
topic Physiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9941201/
https://www.ncbi.nlm.nih.gov/pubmed/36824475
http://dx.doi.org/10.3389/fphys.2023.1058583
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