Bridging the gap between structure and kinetics of human SGLT1

Abstract

The Na(+)-glucose cotransporter hSGLT1 is a member of a class of membrane proteins that harness Na(+) electrochemical gradients to drive uphill solute transport. Although hSGLT1 belongs to one gene family (SLC5), recent structural studies of bacterial Na(+) cotransporters have shown that Na(+) transporters in different gene families have the same structural fold. We have constructed homology models of hSGLT1 in two conformations, the inward-facing occluded (based on vSGLT) and the outward open conformations (based on Mhp1), mutated in turn each of the conserved gates and ligand binding residues, expressed the SGLT1 mutants in Xenopus oocytes, and determined the functional consequences using biophysical and biochemical assays. The results establish that mutating the ligand binding residues produces profound changes in the ligand affinity (the half-saturation concentration, K(0.5)); e.g., mutating sugar binding residues increases the glucose K(0.5) by up to three orders of magnitude. Mutation of the external gate residues increases the Na(+) to sugar transport stoichiometry, demonstrating that these residues are critical for efficient cotransport. The changes in phlorizin inhibition constant (K(i)) are proportional to the changes in sugar K(0.5), except in the case of F101C, where phlorizin K(i) increases by orders of magnitude without a change in glucose K(0.5). We conclude that glucose and phlorizin occupy the same binding site and that F101 is involved in binding to the phloretin group of the inhibitor. Substituted-cysteine accessibility methods show that the cysteine residues at the position of the gates and sugar binding site are largely accessible only to external hydrophilic methanethiosulfonate reagents in the presence of external Na(+), demonstrating that the external sugar (and phlorizin) binding vestibule is opened by the presence of external Na(+) and closes after the binding of sugar and phlorizin. Overall, the present results provide a bridge between kinetics and structural studies of cotransporters.

Stochastic steps in secondary active sugar transport

“…S10), potentially allowing for a tighter seal in mammalian SGLTs. For hSGLT1, approximately two Na + ions are coupled to inward sugar transport, but we have found that mutation of the large hydrophobic side chains of the external gates to cysteine also increases the coupling ratio to as high as 6-1 for F453C (F424 in vSGLT) (29). This decreased coupling ratio suggests that mutations in the outer gate increase the probability that partially transported sugars return to the external solution.…”

Functional identification and characterization of sodium binding sites in Na symporters

“…The selection of mutants at putative Na1 and Na2 sites was guided by previous findings on the kinetics of hSGLT1 mutants (20,21) and the location of the Na1 and Na2 sites in LeuT (6). Fig.…”

Conformational cycle and ion-coupling mechanism of the Na + /hydantoin transporter Mhp1

“…Although we speculate, based on our results, that the role of the conserved Na2 site is to stabilize substrate binding without conformational selection, analysis of other Na + -coupled transporters is needed to test this conjecture. Kinetic and conformational investigations of putative LeuT-fold members, hSGLT and PutP, have begun to tease out such details (23,36,37). Such analysis will also test a similarly tantalizing notion that subclasses of ion-coupled LeuT-fold transporters, defined by their transport modes and/or type and number of symported ions, share commonalities in their structural mechanics of alternating access.…”

“…simulations (20,21) and electron paramagnetic resonance (EPR) analysis (13,22) of LeuT demonstrated that Na + binding favors an outward-facing conformation although it is unclear which Na + site (or both) is responsible for triggering this conformational transition. Similarly, a role for Na + in conformational switching has been uncovered in putative human LeuT-fold transporters, including hSGLT (23). In Mhp1, the sole Na2 site has been shown to modulate substrate affinity (15); however, its proposed involvement in gating of the intracellular side (12, 21) lacks experimental validation.…”