| Popis: |
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 KM and KDapp for different sugars, kobs 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 (Vmax/KM): D-glucose > MDG > D-galactose > 3-OMG > D-xylose. While KM differs 160-fold across tested substrates and plays a major role in substrate specificity, Vmax only varies by a factor of 1.9. Interestingly, D-glucose has the lowest Vmax 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 (KD,Kapp = 210 mM). Affinity for D-glucose increases 14-fold (KD,Naapp = 15 mM) in the presence of sodium as a result of cooperativity. Apparent affinity for D-glucose during transport increases 8-fold (KM = 1.9 mM) compared to KD,Naapp due to optimized kinetics. In contrast, KM and KDapp 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. |
