Coupled Sodium/Glucose Cotransport by SGLT1 Requires a Negative Charge at Position 454

Dı́ez-Sampedro, Ana; Loo, Donald D. F.; Wright, Ernest M.; Zampighi, Guido A.; Hirayama, Bruce A.

doi:10.1021/bi048652d

Cited by 21 publications

(16 citation statements)

References 29 publications

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“…The ligand binding and gating residues identified in vSGLT (3,25) are largely conserved in the six members of the SGLT gene family (47). Table 1 shows the conservation between vSGLT and hSGLT1.…”

Section: Homology Modelingmentioning

confidence: 99%

Bridging the gap between structure and kinetics of human SGLT1

Sala-Rabanal

Hirayama

Loo

et al. 2012

American Journal of Physiology-Cell Physiology

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126

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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.

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Section: Homology Modelingmentioning

confidence: 99%

Bridging the gap between structure and kinetics of human SGLT1

Sala-Rabanal

Hirayama

Loo

et al. 2012

American Journal of Physiology-Cell Physiology

Self Cite

126

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“…The best example is the lactose/proton symporter, LacY, where two carboxyl side chains play irreplaceable roles in proton-coupled sugar translocation (21). Similarly, negatively charged residues are mechanistically involved in other antiporters and symporters (22)(23)(24)(25)(26)(27). Stimulated by these studies, we have examined the role of acidic residues in MdfA.…”

mentioning

confidence: 99%

The Secondary Multidrug/Proton Antiporter MdfA Tolerates Displacements of an Essential Negatively Charged Side Chain

Sigal

Fluman

Siemion

et al. 2009

Journal of Biological Chemistry

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The largest family of solute transporters includes ion motive force-driven secondary transporters. Several well characterized solute-specific transport systems in this group have at least one irreplaceable acidic residue that plays a critical role in energy coupling during transport. Previous studies have established the importance of acidic residues in substrate recognition by major facilitator superfamily secondary multidrug transporters, but their role in the transport mechanism remained unknown. We have been investigating the involvement of acidic residues in the mechanism of MdfA, an Escherichia coli secondary multidrug/ proton antiporter. We demonstrated that no single negatively charged side chain plays an irreplaceable role in MdfA. Accordingly, we hypothesized that MdfA might be able to utilize at least two acidic residues alternatively. In this study, we present evidence that indeed, unlike solute-specific secondary transporters, MdfA tolerates displacements of an essential negative charge to various locations in the putative drug translocation pathway. The results suggest that MdfA utilizes a proton translocation strategy that is less sensitive to perturbations in the geometry of the proton-binding site, further illustrating the exceptional structural promiscuity of multidrug transporters.

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“…The polar residues at position 176 hydrogen bond to the hydroxyl group on the ␤-phenyl ring of phloridzin (18). There is also evidence that D454 in the putative external loop joining TM X-XI is involved in the coupling of Na ϩ and sugar in the transport process (4). The sugar binding domain, on the other hand, has been localized to the COOH-terminal half of SGLT1 (16).…”

mentioning

confidence: 99%

Transmembrane IV of the high-affinity sodium-glucose cotransporter participates in sugar binding

Liu¹,

Lo²,

Speight³

et al. 2008

American Journal of Physiology-Cell Physiology

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Investigation of the structure/function relationships of the sodium-glucose transporter (SGLT1) is crucial to understanding the cotransporter mechanism. In the present study, we used cysteine-scanning mutagenesis and chemical modification by methanethiosulfonate (MTS) derivatives to test whether predicted transmembrane IV participates in sugar binding. Five charged and polar residues (K139, Q142, T156, K157, and D161) and two glucose/galactose malabsorption missense mutations (I147 and S159) were replaced with cysteine. Mutants I147C, T156C, and K157C exhibited sufficient expression to be studied in detail using the two-electrode voltage-clamp method in Xenopus laevis oocytes and COS-7 cells. I147C was similar in function to wild-type and was not studied further. Mutation of lysine-157 to cysteine (K157C) causes loss of phloridzin and ␣-methyl-D-glucopyranoside (␣MG) binding. These functions are restored by chemical modification with positively charged (2-aminoethyl) methanethiosulfonate hydrobromide (MTSEA). Mutation of threonine-156 to cysteine (T156C) reduces the affinity of ␣MG and phloridzin for T156C by ϳ5-fold and ϳ20-fold, respectively. In addition, phloridzin protects cysteine-156 in T156C from alkylation by MTSEA. Therefore, the presence of a positive charge or a polar residue at 157 and 156, respectively, affects sugar binding and sugar-induced Na ϩ currents.cysteine scanning mutagenesis; chemical modification by methanethiosulfonate reagents THE HIGH-AFFINITY sodium-glucose cotransporter (SGLT1) belongs to the homologous family of Na ϩ

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Coupled Sodium/Glucose Cotransport by SGLT1 Requires a Negative Charge at Position 454

Cited by 21 publications

References 29 publications

Bridging the gap between structure and kinetics of human SGLT1

Bridging the gap between structure and kinetics of human SGLT1

The Secondary Multidrug/Proton Antiporter MdfA Tolerates Displacements of an Essential Negatively Charged Side Chain

Transmembrane IV of the high-affinity sodium-glucose cotransporter participates in sugar binding

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