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Structure/Function Studies of the High Affinity Na /Glucose Cotransporter

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Structure/Function Studies of the High Affinity Na /Glucose Cotransporter Structure/Function Studies of the High Affinity Na +/Glucose Cotransporter (SGLT1) by Tiemin Liu A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Institute of Medical Sciences University of Toronto Copyright 2009 by Tiemin Liu Abstract Structure/Function Studies of the High Affinity Na +/Glucose Cotransporter (SGLT1) Tiemin Liu Doctor of Philosophy Institute of Medical Sciences University of Toronto, 2009 The high affinity sodium/glucose cotransporter (SGLT1) couples transport of Na + and glucose. Investigation of the structure/function relationships of the sodium/glucose transporter (SGLT1) is crucial to understanding co-transporter mechanism. In the first project, we used cysteine-scanning mutagenesis and chemical modification by methanethiosulphonate (MTS) derivatives to test whether predicted TM IV participates in sugar binding. Charged and polar residues and glucose/galactose malabsorption (GGM) missense mutations in TM IV were replaced with cysteine. Mutants exhibited sufficient expression to be studied in detail using the two-electrode voltage-clamp method in Xenopus laevis oocytes and COS-7 cells. The results from mutants T156C and K157C suggest that TM IV participates in sugar interaction with SGLT1. This work has been published in Am J Physiol Cell Physiol 295 (1) , C64-72, 2008 . The crystal structure of Vibrio parahaemolyticus SGLT (vSGLT) was recently published (1) and showed discrepancy with the predicted topology of mammalian SGLT1 in the region surrounding transmembrane segments IV-V. Therefore, in the second project, we investigated the topology in this region, thirty-eight residues from I143 to A180 in the N-terminal half of rabbit SGLT1 were individually replaced with cysteine II and then expressed in COS-7 cells or Xenopus laevis oocytes. Based on the results from biotinylation of mutants in intact COS-7 cells, MTSES accessibility of cysteine mutants expressed in COS-7 cells, effect of substrate on the accessibility of mutant T156C in TM IV expressed in COS-7 cells, and characterization of cysteine mutants in TM V expressed in Xenopus laevis oocytes, we suggest that the region including residues 143-180 forms part of the Na +- and sugar substrate-binding cavity. Our results also suggest that TM IV of mammalian SGLT1 extends from residue 143-171 and support the crystal structure of vSGLT. This work has been published in Biochem Biophys Res Commun 378 (1) , 133- 138, 2009 Previous studies established that mutant Q457C human SGLT1 retains full activity, and sugar translocation is abolished in mutant Q457R or in mutant Q457C following reaction with methanethiosulfonate derivatives, but Na + and sugar binding remain intact. Therefore, in the third project, we explored the mechanism by which modulation of Q457 abolishes transport, Q457C and Q457R of rabbit SGLT1 expressed in Xenopus laevis oocytes were studied using chemical modification, the two-electrode voltage-clamp technique and computer model simulations. Our results suggest that glutamine 457, in addition to being involved in sugar binding, is a residue that is sensitive to conformational changes of the carrier. This work has been published in Biophysical Journal 96 (2) , 748-760, 2009. Taken together our study along with previous biochemical characterization of SGLT1 and crystal structure of vSGLT, we propose a limited structural model that attempts to bring together the functions of substrate binding (Na + and sugar), coupling, and translocation. We propose that both Na + and sugar enter a hydrophilic cavity formed III by multiple transmembrane helices from both N-terminal half of SGLT1 and C-terminal half of SGLT1, analogous to all of the known crystal structures of ion-coupled transporters (the Na +/leucine transporter, Na +/aspartate transporter and lactose permease). The functionally important residues in SGLT1 (T156 and K157 in TM 4, D454 and Q457 in TM 11) are close to sugar binding sites. IV Acknowledgements I would like to acknowledge the valuable insights of my committee members, Dr. Peter Backx and Dr. Robert Tsushima. I would also like to thank my lab mates: Steven Huntley and Daniel Krofchick for their help with the Xenopus laevis oocyte expression system, the two-electrode voltage- clamp technique, and computer model simulations; Pam Speight for her excellent technical assistance and mutant preparation; Neil Goldenberg for his help with the COS-7 cell expression system; and Sandy McGugan and Fong Tsang for keeping me organized. To the various members of the Membrane Biology Research Group, especially Dr. David Clarke, Dr. Tip Loo, Claire Bartlett, and Dr. Reinhart Reithmeier, I wish to express my deepest gratitude for their helpful discussions and for guiding me into the world of membrane proteins. Finally, with much gratitude, I would like to thank my supervisor, Mel Silverman for his continuous support and valuable guidance throughout my Ph.D. I only hope the work I will endeavour in the future will make him proud of his investment in me. V Dedication To my parents and family VI Contents Contents 1 Introduction……………………………………………………………………… 1 1.1 Membrane transport in life……………………………………………. 1 1.2 Glucose transporters in humans………………………………………. 3 1.3 Sodium/glucose cotransporters……………………………………….. 6 1.4 Experimental techniques for studies on sodium/glucose cotransporters. 14 1.4.1 Xenopus laevis oocyte expression system…………………… 14 1.4.2 Two-electrode voltage-clamp technique…………………….. 17 1.5 The high affinity sodium/glucose cotransporter 1 (SGLT1)………….. 18 1.5.1 Structure of SGLT1…………………………………………. 19 1.5.2 Function of SGLT1…………………………………………. 22 1.5.2.1 Steady state transport kinetics of SGLT1…………. 23 1.5.2.2 Pre-steady state transport kinetics of SGLT1……… 27 1.5.3 Functional disorders of the SGLT1 (Glucose-galactose malabsorption)……………………………………………………... 28 1.6 Experimental rationale………………………………………………… 32 2 Materials and methods…………………………………………………………... 35 2.1 Molecular biology……………………………………………………... 35 2.2 Oocyte preparation and injection……………………………………… 35 2.3 Electrophysiology using two-microelectrode voltage clamp…………. 38 VII 2.4 Transient current measurements………………………………………. 40 2.5 Phloridzin affinity measurements…………………………………….. 42 2.6 Protocols for chemical modification………………………………….. 43 2.7 State model simulations……………………………………………….. 43 2.8 Cell transfection and western blot detection………………………….. 44 2.9 αMG uptake experiment………………………………………………. 44 2.10 Endo H and PNGase F deglycosylation analysis……………………. 45 2.11 Labeling of surface expressed wild type or mutants in COS-7 cells with biotin-MTSEA……………………………………………………….. 45 2.12 Statistical comparisons of means……………………………………. 46 3 Transmembrane IV of the high affinity sodium/glucose cotransporter participates in sugar binding………………………………………………………. 47 3.1 Summary………………………………………………………………. 47 3.2 Introduction……………………………………………………………. 48 3.3 Results…………………………………………………………………. 50 3.3.1 Characterization of mutant K157C………………………….. 51 3.3.1.1 Pre-steady state behavior of mutant K157C 51 compared to WT…………………………………………… 3.3.1.2 Site-directed alkylation of cysteine 157 (K157C) 53 rescued activity of SGLT1………………………………… 3.3.1.3 Steady state αMG induced Na + currents of K157C and K157C-MTSEA………………………………………. 61 VIII 3.3.2 Characterization of mutant T156C………………………….. 63 3.3.2.1 Steady state transport kinetics of T156C………….. 63 3.3.2.2 Apparent affinity of T156C for phloridzin………… 64 3.3.2.3 Chemical modification of T156C by MTS reagents. 65 3.4 Discussion……………………………………………………………... 68 4. Reanalysis of structure/function correlations in the region of transmembrane segments 4 and 5 of the rabbit sodium/glucose cotransporter……..………………. 71 4.1 Summary………………………………………………………………. 71 4.2 Introduction……………………………………………………………. 72 4.3 Results…………………………………………………………………. 73 4.3.1 Expression and αMG transport activity of mutants in COS-7 cells………………………………………………………………… 73 4.3.2 Determination of topology for TMs IV-V…………………… 76 4.3.2.1 Biotinylation of mutants in intact COS-7 cells……. 76 4.3.2.2 MTSES accessibility of cysteine mutants expressed in COS-7 cells……………………………………………... 78 4.3.3 Determination of functions for TMs IV-V…………………... 81 4.3.3.1 Effect of substrate on the accessibility of mutant T156C in TM IV expressed in COS-7 cells………………... 81 4.3.3.2 Characterization of cysteine mutants I177C, Y178C and A180C in TM V expressed in Xenopus laevis oocytes.. 83 4.4 Discussion…………………………………………………………….. 85 IX 5. Effects on conformational states of the rabbit sodium/glucose cotransporter through modulation of polarity and charge at glutamine 457……………………... 88 5.1 Summary………………………………………………………………. 88 5.2 Introduction……………………………………………………………. 89 5.3 Results…………………………………………………………………. 90 5.3.1 Steady State Kinetics of Mutant Q457C of Rabbit SGLT1…. 90 5.3.2 Pre-Steady State Kinetics of Mutants Q457C and Q457R of Rabbit SGLT1……………………………………………………... 92 5.3.3 Chemical Modification of Mutant Q457C of Rabbit SGLT1 by MTS Reagents………………………………………………….. 96 5.3.3.1 Effect of MTSET on voltage sensitivity and charge transfer of mutant Q457C…………………………………. 96 5.3.3.2 Effects of MTSET on sugar transport and sugar binding of mutant Q457C…………………………………. 97 5.3.3.3 Effect of MTSES or MTSEA on mutant Q457C….. 98 5.3.4 Phloridzin Affinity…………………………………………... 99 5.3.5 Decay Constants of Q457C of Rabbit SGLT1………………. 100 5.3.6 Model Simulations…………………………………………... 107 5.4 Discussion……………………………………………………………... 111 6. Structural implications of structure-function studies in SGLT1..……………….
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