Three Surface Subdomains Form the Vestibule of the Na+/Glucose Cotransporter SGLT1

Puntheeranurak, Theeraporn; Kasch, Myriam; Xia, Xiaobing; Hinterdorfer, Peter; Kinne, Rolf

doi:10.1074/jbc.m704190200

Cited by 22 publications

(30 citation statements)

References 46 publications

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“…The small change in conformation of this area in loop 13 in the presence of D-glucose might be indirect but expected when loop 13 is indeed part of the vestibule for sugar binding of SGLT1 as postulated recently (47).…”

Section: Discussionmentioning

confidence: 93%

d-Glucose-Recognition and Phlorizin-Binding Sites in Human Sodium/d-Glucose Cotransporter 1 (hSGLT1): A Tryptophan Scanning Study

et al. 2007

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In order to gain a better understanding of the structure-function relation in hSGLT1, single Trp residues were introduced into a functional hSGLT1 mutant devoid of Trps at positions that previously had been postulated to be involved in sugar recognition/translocation and/or phlorizin binding. The mutant proteins were expressed in Pichia pastoris, purified, and reconstituted into liposomes. In transport experiments the putative sugar binding site mutants W457hSGLT1 and W460hSGLT1 showed a drastic decrease in affinity toward alpha-methyl-d-glucopyranoside with Km values of 13.3 and 5.26 mM compared to 0.4 mM of the Trp-less hSGLT1. In addition, a strong decrease in the inhibitory effect of phlorizin was observed. In Trp fluorescence studies the position of the emission maxima of the mutants, their sensitivity to N-bromosuccinimide oxidation, and their interaction with water soluble quenchers demonstrate that Trp457 and Trp460 are in contact with the hydrophilic extravesicular environment. In both mutants Trp fluorescence was quenched significantly, but differently, by various glucose analogues. They also show significant protection by d-glucose and phlorizin against acrylamide, KI, or TCE quenching. W602hSGLT1 and W609hSGLT1, the putative aglucone binding site mutants, exhibit normal sugar and phlorizin affinity, and show fluorescence properties which indicate that these residues are located in a very hydrophilic environment. Phlorizin and phloretin, but not d-glucose, protect both mutants against collisional quenchers. Depth-calculations using the parallax method suggest a location of Trp457 and Trp460 at an average distance of 10.8 A and 7.4 A from the center of the bilayer, while Trp602 and Trp609 are located outside the membrane. These results suggest that in the native carrier residues Gln at position 457 and Thr at position 460 reside in a hydrophilic access pathway extending 5-7 A into the membrane to which sugars as well as the sugar moiety of inhibitory glucosides bind. Residues Phe602 and Phe609 contribute by their hydrophobic aromatic residues toward binding of the aglucone part of phlorizin. Thereby in the phlorizin-carrier complex a close vicinity between these two subdomains of the transporter is established creating a phlorizin binding pocket with the previously estimated dimensions of 10 x 17 x 7 A.

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Section: Discussionmentioning

confidence: 93%

d-Glucose-Recognition and Phlorizin-Binding Sites in Human Sodium/d-Glucose Cotransporter 1 (hSGLT1): A Tryptophan Scanning Study

et al. 2007

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“…These results suggest that other residues may be important for binding of sugar. Recently, Puntheeranurak et al (19) expressed rSGLT1 in COS-7 and G6D3 cells and found that mutants C255 (in the putative external loops joining TM VI-VII) and C608 (the putative external loops joining TM XIII-XIV) formed a disulfide bridge. These results suggest that the putative external loops joining TM VIII-IX are involved in sugar binding.…”

Section: Discussionmentioning

confidence: 99%

“…1. The secondary topology model of the high-affinity Na ϩ -glucose cotransporter SGLT1 (19). The functional importance of residues (T156, K157, F163, A166, Q170, L173, D176, D454, and Q457) and domains is indicated.…”

Section: Characterization Of K157cmentioning

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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“…Hirayama et al (60), 2007) reported that 3 residues between transmembrane helices 10 and 13 play a direct role in sugar binding and translocation from the external environment, 3 more face the binding site, and 8 further residues, although not contributing to the binding of sugar directly, affect sugar recognition. Thus, it might be assumed that at 37°C some of the primarily not accessible binding sites further down in the translocation pathway are reached by thioglucose on AA-PEG 5000 (21). A sequential multistep mechanism for sugar recognition and translocation has also been proposed based on prior AFM investigations (20).…”

Section: Sglt1 Sensorsmentioning

confidence: 99%

“…They form a vestibule for the entry of the sugar into the translocation pathway and contain the first of several sugar recognition sites. This vestibule is accessible to the sugar only in the presence of sodium (20,21). Phlorizin acts as a competitive inhibitor of SGLT1 with an apparent K i of 1 M (22).…”

mentioning

confidence: 99%

Forces and Dynamics of Glucose and Inhibitor Binding to Sodium Glucose Co-transporter SGLT1 Studied by Single Molecule Force Spectroscopy

Neundlinger

Puntheeranurak

Wildling

et al. 2014

Journal of Biological Chemistry

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Background: SGLT1 functions in intestinal glucose absorption and renal reabsorption. Results: With increasing temperature, width of energy barrier and average life time increased for glucose binding to SGLT1 but decreased for phlorizin binding. Conclusion: Sugar translocation and inhibitor binding involves several steps with different temperature sensitivity. Significance: Force spectroscopy can be used to study dynamics and structure of membrane transporter.

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Three Surface Subdomains Form the Vestibule of the Na+/Glucose Cotransporter SGLT1

Cited by 22 publications

References 46 publications

d-Glucose-Recognition and Phlorizin-Binding Sites in Human Sodium/d-Glucose Cotransporter 1 (hSGLT1): A Tryptophan Scanning Study

d-Glucose-Recognition and Phlorizin-Binding Sites in Human Sodium/d-Glucose Cotransporter 1 (hSGLT1): A Tryptophan Scanning Study

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

Forces and Dynamics of Glucose and Inhibitor Binding to Sodium Glucose Co-transporter SGLT1 Studied by Single Molecule Force Spectroscopy

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