Ligand-mediated conformational changes and positioning of tryptophans in reconstituted human sodium/d-glucose cotransporter1 (hSGLT1) probed by tryptophan fluorescence

Kumar, Azad; Tyagi, Navneet K.; Kinne, Rolf

doi:10.1016/j.bpc.2006.12.006

Cited by 8 publications

(8 citation statements)

References 56 publications

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“…The emission spectra obtained by non‐phosphorylated SGLT1 indicated that most of the Trp is buried within the protein. This is in accordance with topology predictions of SGLT1 in which most of the Trps are located in a hydrophobic environment, and corroborates our previous findings on Trp localization [Raja et al, 2003; Kumar et al, 2007b]. In addition, acrylamide quenching experiments provided valuable information regarding the conformational changes of proteins, in this case SGLT1.…”

Section: Discussionsupporting

confidence: 92%

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Protein kinase‐A affects sorting and conformation of the sodium‐dependent glucose co‐transporter SGLT1

Subramanian

Glitz

Kipp

et al. 2008

J of Cellular Biochemistry

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In Chinese hamster ovary cells expressing rabbit sodium-dependent glucose transporter (rbSGLT1) protein kinase A (PKA) activators (forskolin and 8-Br-cAMP) stimulated alpha-methyl D-glucopyranoside uptake. Kinetic analysis revealed an increase in both V(max) and affinity of the transport. Immunohistochemistry and biotinylation experiments showed that this stimulation was accompanied by an increased amount of SGLT1 localized into the plasma membrane, which explains the higher V(max) of the transport. Cytochalasin D only partly attenuated the effect of forskolin as did deletion of the PKA phosphorylation site of SGLT1 in transient transfection studies. Experiments using an anti-phosphopeptide antibody revealed that forskolin also increased the extent of phosphorylation of SGLT1 in the membrane fraction. These results suggested that regulation of SGLT1 mediated glucose transport involves an additional direct effect on SGLT1 by phosphorylation. To evaluate this assumption further, phosphorylation studies of recombinant human SGLT1 (hSGLT1) in vitro were performed. In the presence of the catalytic subunit PKA and [(32)P] ATP 1.05 mol of phosphate were incorporated/mol of hSGLT1. Additionally, phosphorylated hSGLT1 demonstrated a reduction in tryptophan fluorescence intensity and a higher quenching by the hydrophilic Trp quencher acrylamide, particularly in the presence of D-glucose. These results indicate that PKA-mediated phosphorylation of SGLT1 changes the conformation of the empty carrier and the glucose carrier complex, probably causing the increase in transport affinity. Thus, PKA-mediated phosphorylation of the transporter represents a further mechanism in the regulation of SGLT1-mediated glucose transport in epithelial cells, in addition to a change in surface membrane expression.

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Section: Discussionsupporting

confidence: 92%

“…The expression and isolation hSGLT1 from a heterologous expression system offers the possibility to study a possible direct effect of phosphorylation on the conformation of the molecule [Tyagi et al, 2005]. Changes of conformation have recently been analyzed in this system and correlated to different functional states of the transporter [Kumar et al, 2007a,b].…”

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confidence: 99%

Protein kinase‐A affects sorting and conformation of the sodium‐dependent glucose co‐transporter SGLT1

Subramanian

Glitz

Kipp

et al. 2008

J of Cellular Biochemistry

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“…Intrinsic and extrinsic Trp fluorescence studies on the recombinant hSGLT1 identified three different conformational states of the transporter in solution (23) and in reconstituted proteoliposomes (24) confirming directly conclusions derived in previous kinetic and electrophysiological experiments (25,26). Furthermore the intramembrane arrangement of various Trps of the transporter could be determined (24). hSGLT1 contains 14 Trps residues at position 6, 45, 66, 67, 103, 114, 276, 289, 291, 440, 477, 487, 561, and 641.…”

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confidence: 81%

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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“…Previous studies have shown that SGLT1 activity changes in parallel to the changes in fluidity when it is incorporated into liposomes composed of phospholipids with different transition points (54). This strong dependence is probably due to the fact that ϳ40% of the Trps responsible for hSGLT1 fluorescence are in close contact to the membrane phospholipids (23).…”

Section: Discussionmentioning

confidence: 98%

“…Phlorizin acts as a competitive inhibitor of SGLT1 with an apparent K i of 1 M (22). The phlorizin carrier complex represents a dead end conformation of the transporter in which it is locked into a condensed, rigid conformation unable to mediate translocation (23,24). Phlorizin consists of a pyranose ring (sugar residue) and two aromatic rings joined by an alkyl spacer (the aglucon moiety, phloretin) (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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Ligand-mediated conformational changes and positioning of tryptophans in reconstituted human sodium/d-glucose cotransporter1 (hSGLT1) probed by tryptophan fluorescence

Cited by 8 publications

References 56 publications

Protein kinase‐A affects sorting and conformation of the sodium‐dependent glucose co‐transporter SGLT1

Protein kinase‐A affects sorting and conformation of the sodium‐dependent glucose co‐transporter SGLT1

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

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

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