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) (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.