Bazzone, Andre and Zerlotti, Rocco and Barthmes, Maria and Fertig, Niels (2023) Functional characterization of SGLT1 using SSM-based electrophysiology: Kinetics of sugar binding and translocation. FRONTIERS IN PHYSIOLOGY, 14: 1058583. ISSN , 1664-042X
Full text not available from this repository. (Request a copy)Abstract
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-K (app) = 210 mM). Affinity for D-glucose increases 14-fold (K-D,K-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,K-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.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | NA+-GLUCOSE COTRANSPORTER; SOLID-SUPPORTED MEMBRANES; ELECTROGENIC PROPERTIES; CONFORMATIONAL-CHANGES; ALTERNATING ACCESS; VOLTAGE-CLAMP; TRANSPORT; MECHANISM; HSGLT1; SITE; SGLT1; pre-steady-state kinetics; transport mechanism; solid supported membrane-based electrophysiology; SLC transporters; binding assay; kinetics; membrane transporter |
| Subjects: | 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biochemie, Genetik und Mikrobiologie |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 12 Mar 2024 14:38 |
| Last Modified: | 12 Mar 2024 14:38 |
| URI: | https://pred.uni-regensburg.de/id/eprint/59828 |
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