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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