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

Home , Antiporter, Choline transporter, GLUT5, Phloretin, SLC2A10, SLC2A11

Structure/Function Studies of

the High Affinity

(SGLT1)

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

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.

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

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..……………….. 120

7. Future directions………………………………………………………………… 128

7.1 Background overview…………………………………………………. 128

7.2 Biophysical characterization of SGLT1……………………………….. 130

7.2.1 Sodium interaction domain of SGLT1………………………. 130

7.2.2 Sugar interaction domain of SGLT1………………………… 130

7.3 Biophysical characterization of SGLT2……………………………….. 131

7.4 Rationale of experimental strategy…………………………………….. 132

7.5 Research plan………………………………………………………….. 136

7.5.1 Investigation of the function/structure of SGLT1…………… 136

7.5.1.1 Investigation of the Na +- and substrate-binding

domain for transmembrane segments II-IV in human

SGLT1 and vSGLT………………………………………... 136

7.5.1.2 Investigation of the topology and important residues

for Na +- and substrate-binding in transmembrane segments

II-III of the human SGLT1 by scanning cysteine

accessibility methods……………………………………… 139

7.5.1.3 Investigation of a negative residue that forms a

charge pair with K157 in TM IV for regulating sugar

binding…………………………………………………….. 140

7.5.2 Investigation of the function/structure of SGLT2…………… 141

XI 7.5.2.1 Investigation of the non-conserved charged or polar

residues in transmembrane segments II-IV of human

SGLT1 and human SGLT2 by scanning cysteine

accessibility methods………………………………………. 141

7.5.2.2 Investigation of the essential Na +- and substrate-

binding residues in human SGLT2………………………… 142

7.6 Materials and methods………………………………………………… 143

7.6.1 Construction of Chimeric Transporter cDNAs……………… 143

7.6.2 Transport assay……………………………………………… 144

7.7 Preliminary data for human SGLT1-Cell Transfection and Western

Blot Detection……………………………………………………………... 145

XII List of Figures

Figure 1.1: Passive and active transport……………………………………….. 2

Figure 1.2: The multi-functions for sodium/glucose cotransporter family.……. 13

Figure 1.3: Xenopus laevis oocytes expression system and two-electrode-

voltage clamp technique (TEVC)………………………………….. 16

Figure 1.4: The proposed membrane topology of rabbit Na +/glucose

cotransporter (rSGLT1)……………………………………………. 21

Figure 1.5: The apparent affinities of ligands for human, rabbit and rat

Na +/glucose cotransporter 1 (SGLT1)…………………………….. 26

Figure 1.6: Model for sugar absorption………………………………………... 29

Figure 2.1: Equipments for Xenopus oocyte injection and Model P-97

micropipette puller………………………………………………… 36

Figure 2.2: Two-microelectrode voltage clamp protocol…..………………….. 39

Figure 2.3: Q vs. V t curve was fit to a 2-state Boltzmann relation…………….. 40

Figure 2.4: Sugar-induced sodium currents as a function of substrate

concentration for SGLT1………………………………………… 41

Figure 3.1: The secondary topology model of SGLT1………………………… 50

Figure 3.2: Representative pre-steady state transient currents (nA) generated

with a voltage clamp over a range of –150 mV to +70 mV in

oocytes expressing WT and mutant K157C……………………….. 52

Figure 3.3: Rescue strategy for activity of SGLT1 by site-directed alkylation

of cysteine 157 (K157C)…………………………………………... 54

XIII Figure 3.4: Representative pre-steady state transient currents (nA) generated

with a voltage clamp over a range of –150 mV to +70 mV in

oocytes expressing mutant K157C after reaction with MTSEA ….. 55

Figure 3.5: Results demonstrating the effects on K157C charge transfer in

oocytes treated with various 1 mM sulfhydryl-specific reagents

(MTS derivatives) in 100 mM Na +………………………………… 57

Figure 3.6: Comparison of the effects of 100 mM Na + ( ■), 40 mM Na + ( ●) and

10 mM Na + ( ) on mutant K157C reacted with 1 mM MTSEA in

the same oocyte……………………………………………………. 59

Figure 3.7: Typical results demonstrating the effects of pretreatment with 1

mM MTSEA on mutant K157C charge transfer in the absence of

Na + (choline replacement) or in the presence of 100 mM Na +……. 60

Figure 3.8: MTSEA rescue of K157C activity..……………………………….. 62

Figure 3.9: αMG-induced Na + currents in presence of 100 mM Na + in an

oocyte expressing mutant T156C…………………...……………... 64

Figure 3.10: (A) Specificity of biotin-MTSEA reaction to the cysteine at 156.

(B) Phloridzin protection of MTSEA accessibility/reactivity to

cysteine 156 in mutant T156C……………………………………. 67

Figure 4.1: Model of a modified secondary structure of rabbit SGLT1….……. 74

Figure 4.2: Biotin-MTSEA reactivity to mutants and a representative

experiment for deglycosylation of WT or mutants expressed in

COS7 cells…………………………………………………….…. 77

Figure 4.3: MTSES pretreatment protects against biotin-MTSEA reactivity to

XIV mutants…………………………………………………………… 80

Figure 4.4: Effect of substrate on biotin-MTSEA reactivity to mutant T156C.. 82

Figure 4.5: Typical results demonstrating the effects on WT or mutants charge

+ + transfer in oocytes in 100 mM Na , and the αMG K0.5 and Na K0.5

of WT and mutants for voltage dependence……………………….. 84

Figure 4.6: Amino acid sequence alignment and secondary structure of the

transmembrane segments 4-5 in SGLT1 and vSGLT……………... 86

+ Figure 5.1: The αMG K0.5 and The Na K0.5 of WT rabbit SGLT1 and Q457C

rabbit SGLT1 for voltage dependence…………………………….. 91

Figure 5.2: Comparison of the effects of 100 mM Na + and 40 mM Na + on pre-

steady state kinetics and steady state kinetics…………………….. 93

Figure 5.3: Estimation of the phloridzin K0.5 of rabbit Q457C and Q457C-

MTSET for voltage dependence with the transferred charge…….. 99

Figure 5.4: The kinetic models for SGLT1 in the presence of sugar or absence

of sugar……………………………………………………………. 101

Figure 5.5: The fit residuals for mutant rabbit Q457C before or after reaction

with MTSET……………………………………………………….. 103

Figure 5.6: Voltage- and Na +-dependence of the three decay constants………. 106

Figure 5.7: Simulated transient currents, charge transfer and decay constants

in 100 mM Na + were predicted by the model solution in Table 3… 108

+ Figure 5.8: Occupancy probability ( Ci) in 100 mM Na as a function of time

as calculated by the four-state kinetic model for WT rabbit SGLT1

and mutant Q457C before or after exposed to MTSET…………… 115

XV Figure 5.9: Simulation predictions on occupancy probabilities ( Ci) in 100 mM

Na + as a function of voltage as calculated by the four-state kinetic

model……………………………………………………………… 118

Figure 6.1: A proposed structural model for substrate binding in SGLT1 and

Structure of vSGLT………………………………………………... 123

Figure 6.2: Amino acid sequence alignment and secondary structure of the

transmembrane segments 2-11 in vSGLT, hSGLT1, NIS and PutP. 125

Figure 6.3: Topology of vSGLT……………………………………………… 127

Figure 7.1: Blocking glucose absorption into blood by inhibiting SGLT1 or

excretion of excessive glucose out of the blood to the urine by

inhibiting SGLT2…………………………………………………. 129

Figure 7.2: Crystal structure of vSGLT……………………………………… 133

Figure 7.3: Amino acid sequence alignment and secondary structure of the

transmembrane segments II-IV in human SGLT1, human SGLT2

and vSGLT………………………………………………………… 135

Figure 7.4: Secondary structure models of chimeric transporters used in this

study………………………………………………………………. 138

Figure 7.5: Western blot analysis of WT-myc human SGLT1, detected with an

anti-myc antibody………………………………………………… 146

XVI List of Tables

Table 1.1: The facilitative Na +-independent sugar transporters (SLC2A)…….. 5

Table 1.2: The members of the SGLT (SLC5A) gene family………………… 7

Table 1.3: The secondary active Na +-dependent sugar cotransporters

(SLC5A)..…………………………………………………………… 11

Table 1.4: Mutations in SGLT1………………………………………………… 30

Table 4.1: Na +-dependent αMG uptake of single cysteine-substituted rabbit

SGLT1 mutants……………………………………………………… 75

Table 5.1: The pre-steady state parameters for WT rSGLT1, mutant Q457C

rSGLT1 before or after reaction with MTSET and mutant Q457R

rSGLT1……………………………………………………………… 95

Table 5.2: Summary of the time constants for WT and mutants……………….. 104

Table 5.3: Rate constants of a four-state kinetic model used for the pre-steady

state current simulations of WT rabbit SGLT1 and mutants in 100

mM Na +……………………………………………………………… 110

XVII Chapter 1

Introduction

1.1 Membrane transport in life

The cell membrane is a flexible lipid bilayer that separates the inner cellular environment from the external cellular environment (3, 4 ). The different proteins on the membrane surface have hydrophilic and hydrophobic regions and are used for several functions such as cell surface receptors, surface antigens, enzymes, and transporters.

The lipid bilayer of the cell membrane is relatively permeable to water molecules and a few other small, uncharged, molecules like oxygen (O 2) and carbon dioxide (CO 2) that can pass this barrier by diffusion. But the lipid bilayer is impermeable to most essential molecules and ions in the surrounding extracellular fluid including ions (Na +,

+ 2+ - - K , Ca , Cl , HCO 3 ), small hydrophilic molecules (glucose), and macromolecules

(proteins and RNA). These molecules and ions can pass this barrier by diffusion or active transport (Fig. 1.1), facilitated by proteins or assembled proteins embedded in the plasma membrane that form a water-filled pore. According to the needs of the cell, the hydrophilic molecules and ions can move down their concentration gradient with no cost of energy (Fig. 1.1A) or move through the membrane against their concentration gradient at an energy cost (Fig. 1.1B).

1 A

Figure 1.1: Passive and active transport (5). (A) Passive transport with no cost of energy , including the diffusion of oxygen and carbon dioxide, osmosis of water, and facilitated diffusion. (B) Active transport with the cost of energy, including transport of large molecules (non-lipid soluble) and the sodium-potassium pump.

2 As shown in Fig. 1.1B, to drive active transport, the transmembrane protein can either directly use the energy of ATP hydrolysis or indirectly use the energy already stored in the gradient of a directly-pumped ion by facilitated diffusion. In indirect active transport, a molecule or ion moves against its gradient by using the downhill flow of another ion. The driving ion is usually sodium with its gradient established by the Na +/K +

ATPase. As shown in Fig. 1.1B, the symporters drive ion and molecule passage through the membrane pump in the same direction, and the antiporters drive ion and molecule passage through the membrane pump in the opposite direction. Active transport pathways include channels and transporters (6-9). The major function for ion channels is to maintain the membrane potential by allowing the flow of ions down their electrochemical gradient (10-12 ). The function for transporters is to move molecules (e.g., glucose) across cell membranes (13-17 ). The transport rate for transporters is at least 10,000 fold slower than that of ion channels.

1.2 Glucose transporters in humans

Normal activities in humans require glucose to provide energy. However, it is very important to maintain the blood glucose concentrations in a narrow range. For example, low blood glucose concentrations in the brain can cause seizures, loss of consciousness, and death. High blood glucose concentrations may cause diabetes, resulting in blindness, renal failure, cardiac and peripheral vascular disease, and neuropathy. Because the lipid bilayer of the cell membrane is impermeable to carbohydrates, transporters in two structurally and functionally distinct gene families are

3 involved in the transfer of glucose (and other hexoses, including fructose and lactose) across the plasma membrane of different cells.

One glucose transporter, the facilitative Na +-independent sugar transporter

(GLUT family, gene name SLC2A; solute carrier family 2), is subdivided into thirteen subtypes, the glucose transporters (GLUT) 1–12 and the H +-myo -inositol cotransporter

(HMIT) (18, 19 ). Solute carrier family 2 functions as a simple carrier and transports glucose by facilitated diffusion down glucose-concentration gradients. Cytochalasin-B or phloretin can inhibit this facilitative transport. The amino acid multiple sequence alignment of the thirteen human GLUT family members shows ~28% to ~65 % identity to GLUT1 (20 ). The transporters have a common primary structure, predicted to have twelve transmembrane domains (TM) with both the amino and carboxy-terminal ends in the cytoplasmic side and a single N -linked oligosaccharide side-chain located either in the first or the fifth extracellular loop. These thirteen subtypes have been divided into three subclasses based on a multiple sequence alignment of the GLUT families (class 1:

GLUT1–4; class 2: GLUT5, 7, 9 and 11, which lack the tryptophan equivalent to W388 of GLUT1; and class 3: GLUT6, 8, 10, 12 and HMIT, which have a much shorter extracellular loop between TM I-II and a longer extracellular loop between TM X-XI that contains the only N -glycosylation site) (18 ). The tissue distributions and substrate specificities of solute carrier family 2 are summarized in Table 1.1.

Following several decades of study (21 ), a growing number of human diseases

(GLUT deficiency syndrome) have been discovered to be caused by inherited mutations in genes encoding glucose transporters (16, 17, 22-28 ). Therefore, it is important to understand the functional roles of GLUT proteins.

4 Isoform Class Main tissue localization Functional Insulin Present in Present in characteristics sensitive? skeletal white adipose (transport) muscle? tissue? GLUT 1 (29, 30 ) I Erthyrocytes, brain, Glucose No Yes Yes ubiquitous GLUT 2 (30-32 ) I Liver, pancreas, intestine, Glucose (low affinity); No No No kidney fructose GLUT 3 (30, 33 ) I Brain Glucose (high affinity) No No Yes (m) GLUT 4 (16, 34, 35 ) I Heart, muscle, WAT, Glucose (high affinity) Yes Yes Yes BAT, brain GLUT 5 (36, 37 ) II Intestine, testes, kidney Glucose (very low No Yes Yes affinity); fructose GLUT 6 (38, 39 ) III Brain, spleen, leucocytes Glucose No No n. d. GLUT 7 (40-42 ) II Intestine Glucose; fructose n. d. n. d. n. d. GLUT 8 (39, 43-45 ) III Testes, brain and other Glucose No (yes in Yes (m) Yes (m) tissues blastocytes) GLUT 9 (46, 47 ) III Brain, spleen, liver, Glucose/? n. d. No n. d. kidney GLUT 10 (48, 49 ) III Liver, pancreas Glucose No Yes (m) n. d. GLUT 11 (50-53 ) II Heart, muscle Glucose (low affinity); No Yes (m) No fructose (long form) GLUT 12 (54 ) III Heart, prostate, muscle, Glucose; galactose; Yes Yes Yes small intestine, WAT fructose HMIT (55 ) III Brain H+-myo -inositol n. d. No (m) Yes (m)

Table 1.1: The facilitative Na +-independent sugar transporters (SLC2A).

WAT, white adipose tissue; BAT, brown adipose tissue; m, mRNA only; n. d., not determined.

5 1.3 Sodium/glucose cotransporters

Another group of glucose transporters in humans is the Na +-dependent glucose co-transporter (SGLT family, gene name SLC5A; solute carrier family 5), which is subdivided into 7 subtypes, the Na +-dependent glucose co-transporters (SGLT) 1–6 and the Na + -myo -inositol cotransporter (SMIT) (56, 57 ). The identified members of the

SGLT (SLC5A) gene family are listed in Table 1.2. Eleven members of the SLC5 human gene subfamily have been identified (58 ), including SLC5A1, 2, 9 and 11 (transport sugars); SLC5A3 and 11 (transport myo-inositols); SLC5A8 (transport monocarboxylate); SLC5A5 (transport anions); SLC5A6 (transport vitamins); SLC5A7

(transport choline); SLC5A10 (not known); and SLC5A4 (one glucose sensor). Using the basic local alignment search tool (BLAST), the amino acid multiple sequence alignment of the eleven human SGLT family members show more than 59% identity to SGLT1.

The gene for human SLC5A (containing 580 to 718 residues) codes for 60 kDa to 80 kDa of membrane protein. This group functions as a secondary active transporter that utilizes the sodium electrochemical gradient to transport sugar substrates uphill against a concentration gradient (56 ). The Na +/K + ATPase pump provides the Na +-electrochemical gradient. The transporters in this group are mainly restricted to the epithelial cell brush border of the small intestine for glucose absorption and the proximal convoluted tubules of the kidney for glucose re-absorption. Phloridzin can inhibit this active transport. The transporters in this group have a common primary structure, predicted to have fourteen transmembrane domains (TM) and a single N -linked oligosaccharide side-chain located in the putative external loop joining TM VI-VII (59 ).

6 Isoform Protein ID Source Amino acid

Human SGLT1 (60 ) P13866 Homo sapiens (human) 664

Rabbit SGLT1 (61 ) P11170 Oryctolagus cuniculus (rabbit) 662

Rat SGLT1 (62 ) P53790 Rattus norvegicus (Norway rat) 665

Mouse SGLT1 (63 ) Q8C3K6 Mus musculus (house mouse) 665

Sheep SGLT1 (64 ) P53791 Ovis aries (sheep) 664

Pig SGLT1 (65 ) P26429 Sus scrofa (pig) 605

Horse SGLT1 (66 ) CAC35538 Equus caballus (horse) 662

Bovine SGLT1 (67 ) AAM34274 Bos taurus (cattle) 664

Chicken SGLT1 (68 ) CAB93941 Gallus gallus (chicken) 323

Dog SGLT1 AAV41055 Canis lupus familiaris (dog) 662

Human SGLT2 (69 ) P31639 Homo sapiens (human) 672

Rat SGLT2 (70 ) P53792 Rattus norvegicus (Norway rat) 670

Mouse SGLT2 (71 ) Q923I7 Mus musculus (house mouse) 670

Bovine SGLT2 (72 ) AAP43921 Bos taurus (cattle) 673

Shark SGLT2 (73 ) CAJ41168 Squalus acanthias (spiny dogfish) 673

Skate SGLT2 (74 ) CAJ70644 Leucoraja erinacea (little skate) 662

Trout SGLT2 CAJ77221 Oncorhynchus mykiss (rainbow 667

trout)

Dog SGLT2 AAV41056 Canis lupus familiaris (dog) 672

Human SGLT3 (75 ) Q9NY91 Homo sapiens (human) 659

Pig SGLT3 (76 ) P31636 Sus scrofa (pig) 660

Mouse SGLT3 (77 ) Q9ET37 Mus musculus (house mouse) 656

7 Mouse SGLT3b (71 ) AAL06342 Mus musculus (house mouse) 660

Human SGLT4 (78 ) NP_001011 Homo sapiens (human) 669

547

Mouse SGLT4 (78 ) NP_663526 Mus musculus (house mouse) 685

Human SGLT5 (79 ) A0PJK1 Homo sapiens (human) 596

Rabbit SGLT5 (80 ) Q28610 Oryctolagus cuniculus (rabbit) 597

Mouse SGLT5 (63 ) Q5SWY8 Mus musculus (house mouse) 596

Bovine SGLT5 (67 ) Q6R4Q5 Bos taurus (cattle) 597

Pig SGLT5 Q5FY69 Sus scrofa (pig) 597

Human SGLT6 (57, 81 ) NP_443176 Homo sapiens (human) 675

Rabbit SGLT6 (82, 83 ) NP_001075 Oryctolagus cuniculus (rabbit) 674

662

Mouse SGLT6 (57 ) NP_666310 Mus musculus (house mouse) 673

Human SMIT1 (84 ) P53794 Homo sapiens (human) 718

Mouse SMIT1 (85 ) Q9JKZ2 Mus musculus (house mouse) 718

Bovine SMIT1 (86, 87 ) P53793 Bos taurus (cattle) 718

Dog SMIT1 (88 ) P31637 Canis lupus familiaris (dog) 718

Rat SMIT1 (89 ) AAO73471 Rattus norvegicus (Norway rat) 718

Human NIS (90, 91 ) Q92911 Homo sapiens (human) 643

Mouse NIS (92, 93 ) Q99PN0 Mus musculus (house mouse) 618

Rat NIS (94, 95 ) Q63008 Rattus norvegicus (Norway rat) 618

Pig NIS (96 ) NP_999575 Sus scrofa (pig) 643

Frog NIS (97 ) NP_001086 Xenopus laevis (African clawed 684

8 891 frog)

Human SMVT (98 ) Q9Y289 Homo sapiens (human) 635

Rabbit SMVT (99 ) Q9XT77 Oryctolagus cuniculus (rabbit) 636

Mouse SMVT (100 ) Q5U4D8 Mus musculus (house mouse) 634

Rat SMVT (101 ) O70247 Rattus norvegicus (Norway rat) 634

Bovine SMVT (102, ABM06081 Bos taurus (cattle) 638

103 )

Human CHT (104, 105 ) Q9GZV3 Homo sapiens (human) 580

Mouse CHT (106 ) Q8BGY9 Mus musculus (house mouse) 580

Rat CHT (107 ) Q9JMD7 Rattus norvegicus (Norway rat) 580

Human SMCT (108- NP_666018 Homo sapiens (human) 610

115 )

Mouse SMCT (110, NP_663398 Mus musculus (house mouse) 611

116-118 )

Table 1.2: The members of the SGLT (SLC5A) gene family.

SGLT: Sodium/glucose cotransporter; SMIT: Sodium/myo-inositol cotransporter; NIS:

Sodium/iodide cotransporter; SMVT: Sodium/ multivitamin cotransporter; CHT: Choline transporter; SMCT: Sodium/monocarboxylate cotransporter.

The substrate specificities, substrate apparent affinities, and tissue distributions in

SGLT family are summarized in Table 1.3. SGLT1 transports glucose and galactose.

SGLT1 is mainly distributed in the epithelial cell brush border of the small intestine, but is also found in the renal proximal tubule of kidney and other organs such as the brain and heart. Gene defects in SGLT1 cause malabsorption of glucose and galactose (56,

119-125 ). SGLT2 plays a major role in the re-absorption of glucose from the glomerular filtrate. SGLT2 is mainly distributed in the renal proximal tubule of kidney, but is also found in other tissues throughout the body such as the brain and liver. Gene defects in

SGLT2 cause familial renal glycosuria (126-133 ). The difference between SGLT1 and

SGLT2 is that SGLT2 is a lower affinity transporter for glucose and does not transport galactose. The coupling ratio for SGLT1 is 2 Na + ions : 1 glucose molecule (134, 135 ) and for SGLT2 is 1 Na + ion : 1 glucose molecule (70, 133 ). SGLT3 is mainly distributed in the cholinergic neurons of the small intestine and the skeletal muscle at the neuromuscular junctions, and is also found in other tissues such as kidney and uterus.

Recent research for SGLT3 suggests that SGLT3 does not transport glucose, but is a glucosensor involved in the regulation of muscle activity (136 ). SGLT4 is a lower affinity transporter for mannose and glucose, and is distributed in a variety of tissues. SGLT5 is mainly distributed in the renal proximal tubule of kidney, but its functions are still unknown. SGLT6 (SMIT2) has a high-affinity for myo-inositol and a low-affinity for glucose. SGLT6 is mainly distributed in brain, kidney, and intestine. SMIT1 has the similar features as SGLT6.

10 Isoform Main tissue localization Functional K0.5 Link to disease Human gene Sequence characteristics (mM) locus Accession (transport) ID SGLT1 (SLC5A1) Intestine, trachea, kidney, Glucose, 0.5 Glucose galactose 22q13.1 NM_0003 (61 ) heart, brain, testis, prostate galactose malabsorption G 43 MIM 182380 SGLT2 (SLC5A2) Kidney, brain, liver, Glucose, 2 Familial renal 16p12-p11 NM_0030 (69) thyroid, muscle, heart galactose # glycosuria G 41 MIM 233100 SGLT3 (SLC5A4) Small Intestine, skeletal Glucose n. d. n. d. 22q12.2- NM_1422 (76 ) muscle, kidney, uterus and activated Na + q12.3 7 testis; plasma membranes (H +) channel SGLT4 (SLC5A9) Intestine, kidney liver, Glucose, 2.4 n. d. 17p11.2 XM_0644 (56 ) brain, lung, trachea, uterus, mannose 87 pancreas SGLT5 (SLC5A10) Kidney n. d. n. d. n. d. 16p12.1 NM_0529 (56 ) 44 SGLT6 (SLC5A11, Brain, kidney, intestine Myo-inositol, 0.12 n. d. 12q23.1 NM_1459 SMIT2) (57, 137, glucose 35 13 138 ) SMIT1 (SLC5A3) Brain, heart, kidney, lung Myo-inositol, 0.05 Down’ s syndrome? 21q22.12 NM_0069 (84, 139 ) glucose >30 OMIM 600444 33

Table 1.3: The secondary active Na +-dependent sugar cotransporters (SLC5A) (56, 58 ). n. d., not determined. #, not transported; G, Gene defect.

11 Members of the SGLT (SLC5A) gene family have several functions (Fig. 1.2).

All members tightly cotransport of sodium with sugars, amino acids, vitamins and ions such as iodide.

SGLT1 can act as a water pump (140-142 ). Different groups have proposed two mechanisms of sugar-coupled water flow. One mechanism is that SGLT1 behaves as a secondary active water transporter and glucose triggers the water transport that couples

Na +, glucose and water with a fixed stoichiometry (2 Na + : 1 glucose : 260 water molecules) (140 ). In the steady state, the authors (140 ) estimate that one-third of the total water transport in isotonic water flow is directly transported by SGLT 1, one-third is transported by osmosis through SGLT 1 and the remaining one-third is transported by osmosis through the plasma membrane. Another mechanism proposed that the water transport is dependent on the osmotic gradients and the intracellular accumulation of glucose triggers the water transport immediately after cotransport of Na + and glucose occurs (141, 142 ). Until now, it is unclear which hypothesis is correct. Whether water transport is dependent or independent of the osmotic gradients, both mechanisms suggest that SGLT1 can pump water across the intestinal brush border membrane. SGLT1 also behaves as a channel for water and small hydrophilic solutes (140, 143 ). The urea influx through SGLT1 is blocked by phloridzin (143 ).

SGLT3 does not transport glucose, but is a glucosensor. Glucose causes a specific, phloridzin-sensitive, inward Na + current that depolarizes the membrane potential

(136 ).

12 SGLT1, pig SGLT3, SMIT1 and NIS behave as a uniport through uncoupled Na + transport in the absence of sugar (144 ). The uncoupled Na + transport through SGLT1 is blocked by phloridzin (the high-affinity, non-transported, competitive inhibitor).

Figure 1.2: The multi-functions for sodium/glucose cotransporter family

13 The SGLT family is also referred to as the sodium/substrate symporter family

(SSSF, TC 2.A. 21), and has more than 230 members based on sequence similarities

(145 ). Members in the sodium/substrate symporter family transport a wide variety of solutes including sugars, proline, pantothenate, and iodide across cytoplasmic membranes of pro- and eukaryotic cells. The common topological motif of the sodium/substrate symporter family is predicted to have thirteen transmembrane domains. The SGLT family has an additional fourteenth TM in the end of C-terminal.

1.4 Experimental techniques for studies on sodium/glucose cotransporters

The substituted cysteine accessibility method (SCAM) and the two-electrode voltage-clamp method in Xenopus laevis oocytes have been used to study on sodium/glucose cotransporter.

1.4.1 Xenopus laevis oocyte expression system

Xenopus laevis (African clawed frog) is a species of South African aquatic frog of the genus Xenopus and provides a simple, low-cost and well-controlled system for expression of exogenous proteins in large quantities for electrophysiological studies (Fig.

1.3A ) (146, 147 ). The ovaries of a mature female can contain over 100,000 oocytes between 50 µm and 1.2 mm in diameter. Oocytes contain the animal pole (dark brown in

14 color) and the vegetable pole (yellow in color), and are classified from stage I to stage VI based on their developmental state. The stage V and VI oocytes (~1.2 mm in diameter) are generally used for electrophysiological studies. John Gurdon and co-workers first expressed globin protein on the membrane surface by injection of globin mRNA into

Xenopus laevis oocyte (148 ). Subsequently, this functional expression system has been used for studied on recepters, channels, or transporters in a native biological membrane environment by injection of cDNAs into nucleus of Xenopus laevis oocytes or mRNAs into cytoplasm of Xenopus laevis oocytes (149-154 ). In contrast to mammalian cells,

Xenopus laevis oocytes express membrane protein complexes formed by multiple subunits by injection of multiple species of cDNAs or mRNAs. The function of expressed proteins on the membrane of Xenopus laevis oocytes can be easily studied by standard electrophysiological techniques because the large size and spherical shape of the oocytes can easily insert electrodes and control voltage (155 ). Research for SGLT1 is focussed on the relationships between structure and function, dynamics and regulation (56, 156-159 ).

Xenopus laevis oocytes only express a very low number of endogenous membrane channels and transporters because they are virtually independent from exogenous nutrients. Therefore, the critical rule for study on SGLT1 using Xenopus laevis oocytes is that oocytes do not contain endogenous channels or cotransporters with properties similar to SGLT1 (156, 160 ). Especially when the expression level of chimeras or site-directed mutants of SGLT1 are very low, it is essential to demonstrate that Xenopus laevis oocytes do not contain small numbers of endogenous channels (such as endogenous sodium channels) with properties similar to those being investigated.

15 A

Figure 1.3: Xenopus laevis oocytes expression system and two-electrode-voltage clamp

technique (TEVC) (146, 147, 161 ).

16 1.4.2 Two-electrode voltage-clamp technique

The most widely used electrophysiological technique to measure currents flowing through channels or transporters expressed in Xenopus laevis oocytes, is the two- electrode-voltage clamp (TEVC) technique (161 ). Four parts of equipment used to record signals from the oocytes includes: the stereomicroscope equipped with a cold light source and oocyte stage, both placed on a vibration isolation table under a Faraday cage (to shield from any external electrical noise); the oocyte voltage clamp amplifier; an analogue to digital converter (A/D converter); and the computer/software (Fig. 1.3B).

The Xenopus laevis oocyte membrane voltage is controlled (clamped) while currents flowing through channels or electrogenic transporters are measured by the voltage clamp amplifier in voltage-clamp mode. The analogue to digital converter will convert the continuous signals sent from the voltage clamp amplifier into digital signals for recognition by computer. As shown in Fig. 1.3C , four electrodes are connected to voltage clamp. The electrodes for voltage and current are held in place by micromanipulators with 3 knobs for moving the electrodes in 3 dimensions of space (up and down, back and forth, in and out). Two electrodes are made of glass and filled with 3 M KCl solution to obtain an electric resistance between 0.2 and 3 M Ω. The voltage offset between electrodes and bath is cancelled before impaling an oocyte. The two electrodes are then inserted through the Xenopus laevis oocyte membrane. The successful penetration is confirmed by reading the resting membrane potential on the amplifier display. The resting potential of a healthy oocyte (with follicle cells intact) is between -40 to -60 mV, mainly due to K + leak out of the cell though expression of endogenous ion channels. The

17 resting membrane potential will move to more negative values with expression of K + selective channels or move to more positive values with expression of constitutively active Na + or Ca 2+ channels. The electrodes that are connected to electrode-holders include a silver wire coated with a layer of AgCl 2 and transfer the signal from the KCl solution through the silver wire to the amplifier. The voltage-electrode measures the membrane potential of the Xenopus laevis oocyte, and then transfers this signal to a feedback amplifier where it is compared with the command potential. According to the difference between the measured and the desired membrane potentials, the current- electrode injects the currents across the membrane to the ground electrodes. As shown in

Fig. 1.3D, under voltage clamp, the voltage across the membrane is changed and held based on the command potential, resulting in the changed resistance when channels or transporters were opened allowing ions to move across the membrane. Therefore, the equal amount of injected current in the opposite direction for holding the membrane at this new voltage (against the flow of these ions) is directly related to the number of channels or transporters opened. By measuring the injected current, the number of channels or transporters opened can be calculated. Two ground electrodes (one for the voltage-electrode and one for the current-electrode) made of chloride silver wires, are placed in the bath and send the signals to the oocyte clamp module.

1.5 The high affinity sodium/glucose cotransporter 1

(SGLT1)

18 In 1987, the high affinity sodium/glucose cotransporter 1 (SGLT1, SLC5A1) was the first member in the SLC5 family to be cloned from rabbit intestine (61 ). Human

SGLT1 was cloned in 1989 (60 ). Until now, ten members of SGLT1 have been identified

(Table 1.2). The amino acid multiple sequence alignment of nine SGLT1 members

(excludes Chicken SGLT1) using BLAST revealed about more than 80% identity to human SGLT1. SGLT1 is mainly found in the apical membranes of small-intestinal absorptive cells (enterocytes) and renal proximal straight tubules of kidney (S3 cells)

(56 ). Investigation of the structure/function relationships of the sodium/glucose transporter (SGLT1) is crucial to understanding co-transporter mechanism.

1.5.1 Structure of SGLT1

In the past twenty years, the structure and function of SGLT1 has been intensively characterized (59, 159, 162-164 ). The data from homology cloning and the Human

Genome Project has indicated that human SGLT1 and rabbit SGLT1 includes 664 and

662 amino acids, respectively. About 75 kDa proteins are coded by the SGLT1 genes.

Based on the primary sequence of SGLT1, the secondary structure model of human

SGLT1 was first proposed to have eleven transmembrane helices (61 ). Subsequently proposed secondary structures either deleted or added more helices (60, 62, 165 ). The secondary structure model of human SGLT1 was revised by the neural network

PredictProtein (166 ) and the program MEMSAT (167 ) in 1996 (168 ) and 1997 (59 ), which predicted that human SGLT1 had fourteen transmembrane helices. All transmembrane helices are presumably alpha-helical. The hydrophilic N-terminus of

19 human SGLT1 is located on the extracellular side of the membrane. The hydrophobic C- terminus of human SGLT1 forms the fourteenth transmembrane helix ending abruptly at the membrane extracellular interface. The only native N-glycosylation site, asparagine

248, is found at the putative external loop joining TM VI-VII, but is not necessary for activity. The predicted model of the membrane topology of the human SGLT1 is supported by a variety of experiments including N-glycosylation scanning mutagenesis with Xenopus oocytes expression system (168 ), in vitro membrane insertion of the translation products of truncated mRNAs (168 ), freeze-fracture studies of cloned SGLT1 expressed in the plasma membranes of Xenopus oocytes (169 ), immunogold electron microscopy (170 ), antibody recognition (171 ), and labeling with membrane impermeant

MTS compounds or maleimides (163 ). However, the 3D crystal structure for SGLT1 has not been identified. Fig. 1.3 is the predicted model of the membrane topology of rabbit

SGLT1, based on the membrane topology of human SGLT1.

Common architecture found in over 230 members of the solute symporter family

(SSF) gene family consists of G57 in the internal loop joining TM I-II to G492 in TM XII

(172 ). The sodium:SSF signature sequence is located in TM V: [GS]-x (2)-[LIY]-x (3)-

[LIVMFYWSTAG] (7)-x (3)-[LIV]-[STAV]-x (2)-G-G-[LMF]-x-[SAP]. The motif shared by the sodium/glucose cotransporter (SGLTs) and Na +-myoinositol cotransporters

(SMITs) is indicated in the internal loop joining TM I-II and TM II: R-x-T-x (4)-F-L-A-

G-x (4)-W-W-x (2)-G-A-S.

Figure 1.4: The proposed membrane topology of rabbit Na +/glucose cotransporter (rSGLT1) (59 )

21 The first proposed model suggested that the end of the fourteenth transmembrane helix faces intracellularly. This is in contrast to most subsequent models that suggest the fourteenth transmembrane helix ends abruptly at the membrane extracellular interface. A second model suggested that the end of the fourteenth transmembrane helix is a dynamic helical structure and alternates between inside-facing and outward-facing states during substrate translocation by SGLT1 conformational changes. In both models, the large hydrophilic internal loop joining TM XIII-XIV is a reentrant pore-loop structure with accessibility depended on the conformation of the cotransporter, similar as other transporters (173-176 ). Only definitive structural data will resolve the contradictory results of models proposing the membrane topology of SGLT1.

1.5.2 Function of SGLT1

The functions of the isoforms of the high affinity sodium/glucose cotransporter 1

(SGLT1) expressed in Xenopus laevis oocytes (61, 177, 178 ) or the mammalian cell lines including COS7 cells (179, 180 ), HEK293 cells (181 ) and G6D3 (182 ), were extensively studied in this laboratory by using electrophysiological techniques (156, 183, 184 ), radioactive tracing (180 ), fluorescence measurements (163, 164, 185 ), and atomic force microscopy (182, 186-188 ). The members of SGLT1 cloned from human intestine (60 ), small intestine (61 ) and kidney (189 ), and rat kidney (62 ) are similar in function as tightly coupled cotransporters and show different kinetics for substrate recognition (190 ).

SGLT1 has a stoichiometry such that two Na + ions are simultaneously transported with one sugar molecule.

1.5.2.1 Steady state transport kinetics of SGLT1

Steady state inward currents associated with the cloned SGLT1 were studied in

Xenopus laevis oocytes by electrophysiological methods. Analysis of the

sugar sugar cation electrophysiological data has indicated kinetic information ( K0.5 , Imax , K0.5 ,

cation PZ Imax , K i ) for a single oocyte in a wide range of membrane potentials (+90 mV to –

150 mV). The different values in steady state inward currents measured before and after the addition of sugar were studied as a function of the test potential (I / V curves).

Sugar induced steady state inward currents represent the function of the external sugar concentration at fixed external cation concentrations. To determine the affinities of human, rabbit and rat SGLT1 for sugar substrate α-methyl-D-glucose ( αMG, a nonmetabolized substrate for SGLT1), αMG induced steady state currents were measured at various sugar concentrations in 100 mM Na + buffer over a range of holding potentials and the resulting curves were fitted to the Michaelis-Menten relationship. From −150 mV to −10 mV, the K0.5 of human and rat SGLT1 exhibits a marked voltage-dependence whereas rabbit SGLT1 exhibits a slight voltage-dependence (Fig. 1.5A). The order of

αMG apparent K0.5 at −50 mV is human (0.49 ± 0.03 mM) > rat (0.31 ± 0.02 mM) >

αMG rabbit (0.17 ± 0.01 mM) (190 ). The K0.5 values presented with the natural logarithm scale versus the test potentials by least squares linear analysis shows a rate of e-fold (66.7

%) / 28 ± 2 mV for human or e-fold (66.7 %) / 18 ± 1 mV for rat. The external Na +

αMG αMG concentrations affect K0.5 values. K0.5 values of rabbit SGLT1 at -150 mV increase from 0.073 mM to 0.36 mM when the external Na + concentrations decrease from

23 100 mM to 10 mM (156 ). The members of SGLT1 family show selectivity for natural monosaccharides. The substrate specificity for human SGLT1 and rabbit SGLT1 is αMG

> D-glucose > D-galactose >> L-galactose ≈ D-mannose (190-192 ). The substrate specificity for rat SGLT1 is D-glucose > αMG ≈ D-galactose >> L-galactose ≈ D- mannose (193 ). The kinetics of transport of glucose analogues, each modified at one position of the pyranose ring, were also studied in oocytes expressing human SGLT1 (the affinities for glucose analogues: 4-deoxy-D-galactose > 6-deoxy-D-galactose > 5-thio-D- galactose > 1-deoxy-D-galactose >>2-deoxy-D-galactose >> 3-deoxy-D-galactose) (191,

194 ).

Sugar induced steady state inward currents will represent the function of the external cation concentration at fixed external sugar concentrations. To determine the affinities of human, rabbit and rat SGLT1 for Na +, αMG induced steady state currents

(obtained at saturating concentration of αMG) were measured at various Na + concentrations over a range of holding potentials and the resulting curves were fitted to

+ the Hill relationship. The Na apparent K0.5 of human, rabbit and rat SGLT1 exhibited voltage-dependent in the test range from -150 mV to -30 mV (Fig. 1.5B) (190 ). The

Na+ order of K0.5 at -150 mV is rabbit (7.0 ± 4.4 mM) > rat (4 ± 0.6 mM) > human (2.5 ±

0.1 mM), at -30 mV is human (44 ± 8 mM) > rat (40 ± 2 mM) > rabbit (16.5 ± 0.5 mM).

Na+ The corresponding values for “n”, the Hill coefficient ranged from 1.0 to 2.0. The K0.5 values presented with the natural logarithm scale versus the test potentials by least squares linear analysis shows a rate of e-fold/28 ± 2 mV for human, or e-fold /33 ± 11 mV for rabbit, or e-fold /46 ± 11 mV for rat. The external αMG concentrations affect

24 Na+ Na+ K0.5 values. K0.5 values of rabbit SGLT1 at -150 mV decrease from 14 mM to 1 mM when the external αMG concentrations increase from 0.1 mM to 20 mM (156 ).

Previous research found that while cotransporters were highly specific for Na + cation above all others, but H + or Li + could partially substitute for Na + to generate the energy of the transmembrane electrochemical ion gradient for driving the accumulation of a substrate against its concentration gradient into the cell (195 ). Therefore, the effect of cation substitution (H + and Li +) on kinetics of rabbit SGLT1 was also studied in detail using the two-electrode voltage-clamp method in Xenopus laevis oocytes (192 ). The

αMG + + order of K0.5 in different cation buffers at -150 mV is H (3.8 ± 1 mM) > Li (1.7 ±

0.6 mM) > Na + (0.17 ± 0.01 mM), at -50 mV is Li + (28 ± 8 mM) > H + (6.8 ± 0.4 mM) >

Na + (0.17 ± 0.01mM). These results show that even the electrochemical gradient of Li + and H + could drive sugar transport, but the substitution of Li + or H + for Na + decreased the apparent affinity for αMG by an order of magnitude or more.

For these three isoforms of SGLT1, the affinities of human and rat SGLT1 for

αMG are much more sensitive to voltage than that of rabbit. Although the direction of cotransport is reversible, the apparent affinities of human SGLT1 for ligands are different in the forward and backward directions (196 ). In the absence of Na + or sugar on the opposite side of the membrane, the apparent affinities for Na + and sugar are one order of magnitude lower for influx than for efflux (Na +: 1.3 mM versus 12 mM; αMG: 0.15 mM versus 56 mM).

25 A

Figure 1.5: The apparent affinities of ligands for human ( ), rabbit ( ) and rat ( )

+ Na +/glucose cotransporter 1 (SGLT1) (190 ). (A) αMG apparent K0.5 (B) Na apparent

K0.5

26 Phloridzin (glucose, 1-[2-(β-D-glucopyranosyloxy)-4,6-dihydroxyphenyl]-3-(4- hydroxyphenyl)-1-propanone) belongs to the chalcone class of organic compounds, consisting of a glucose moiety and two aromatic rings joined by an alkyl spacer. It is a high-affinity, non-transported, competitive inhibitor for SGLT1 (156, 178, 197-199 ).

Phloridzin can reversibly block αMG induced steady state inward currents. To determine the affinities of human, rabbit and rat SGLT1 for phloridzin, αMG induced steady state currents were measured in various phloridzin concentrations at different concentrations of

αMG with 100 mM Na + buffer over a range of holding potentials. The results were plotted as the reciprocal of the currents against the phloridzin concentration (Dixon plot).

The intercept of the l/ I versus the phloridzin concentration slopes was the Ki of phloridzin. The Ki of three isofroms exhibits a slight (~2 fold) voltage-dependence. The order of Ki at -150 mV is rabbit (0.76 ± 0.17 µM) > human (0.22 ± 0.07 µM) > rat (0.012

± 0.002 µM).

1.5.2.2 Pre-steady state transport kinetics of SGLT1

Pre-steady state currents provide insight on the partial reactions of the transport cycle including ion binding/debinding and a conformational change of the empty carrier.

Pre-steady state currents associated with the cloned SGLT1 in the absence of sugar have been studied in Xenopus laevis oocytes by electrophysiological methods in the millisecond time scale. Analysis of the electrophysiological data has indicated the maximum kinetic information ( Qmax , maximal charge transferred; V 0.5 , the potential at which half of the total charge transfer is complete; z , apparent valence of the movable

27 charge) for a single oocyte in a wide range of membrane potentials (+90 mV to –150 mV). Phloridzin and αMG can reversibly eliminate pre-steady state transient currents.

To determine the pre-steady state kinetic parameters of human, rabbit and rat

+ SGLT1 in 100 mM Na buffer, the pre-steady state currents for each Vt were integrated over the entire course of the trace to calculate the total charge transferred by the

cotransporter. The charge, Q, was plotted as a function of the test pulses, and these Q (Vt) curves were fitted to the two-state Boltzmann relation. The V0.5 values of human and rat

SGLT1 are similar [-47 ± 0.4 mV (200 ) and -43 ± 3 mV (193 ), respectively], whereas rabbit SGLT1 is ~45 mV more positive [-2.54 ± 0.7 mV (162 )]. The z values of human, rabbit and rat SGLT1 are identical [0.9 ± 0.1 (200 ), 1.07 ± 0.03 (162 ) and 1.0 ± 0.15

(193 ), respectively]. The maximal rate of transporter turnover, k, was calculated by the

steady state Imax and the pre-steady state Qmax . The turnover values of human, rabbit and rat SGLT1 are 28 s –1 (164 ), 23 s –1 (162 ) and 30 s –1 (193 ), respectively.

The magnitude of pre-steady state currents ( Qmax ) and apparent valence of the movable charge ( z) for rabbit SGLT1 are independent of the holding potential (between 0 mV and –100 mV) and temperature (20-30 oC), and dependent of Na + concentration

(184 ). The V0.5 of rabbit SGLT1 will move to more negative values if the temperature increases or Na + concentration decreases.

1.5.3 Functional disorders of the SGLT1 (Glucose- galactose malabsorption)

28 Glucose is the energy source for normal activities in humans. Fig. 1.6 shows a model for glucose absorption (18, 56, 201 ). Na +/K-pump pumps Na + out of the cell, resulting in a lower Na + concentration inside and a negative membrane potential. SGLT1 utilizes the Na + electrochemical gradient to drive transport of sugar across the brush border membrane (left panel of Fig. 1.6). Glucose diffuses out from another side of the cell, through the basolateral membrane of cells lining the small intestine or the proximal tubules of the kidneys into the blood by GLUT2 or GLUT1. SGLT1 is important to humans since mutations in the SGLT1 gene cause malabsorption of glucose and galactose

(GGM) (121, 202, 203 ). Congenital defects result in severe diarrhea and dehydration in the newborn (119, 204 ). The severe diarrhea associated with glucose-galactose malabsorption could be corrected by replacing dietary glucose with fructose.

Fig. 1.6 Model for glucose absorption (left panel: SGLT1 in intestine; right panel:

SGLT1 in kidney) (18, 56, 201 ).

29 Molecular basis of GGM has been extensively studied after SGLT1 was cloned in

1987 (61 ). Thirty-five missense, seven nonsense, seven frame-shift and seven splice-site mutations of SGLT1 have been identified from different GGM patients (Table 1.4). A relatively high incidence of GGM has been reported in Sweden. Most missense mutations have been studied using Xenopus laevis oocyte expression system and exhibited trafficking defects during vesicular transport to the plasma membrane. Only Arg135

→Trp mutant (205 ) and Gln457 →Arg mutant (124, 163, 206, 207 ) undergo normal trafficking to the plasma membrane but are non-functional.

Missense mutation AA Residue % uptake* Kindred Genotype 1 Ala → Val 12 nd 40 Het 2 Asp → Asn 28 (121 ) 0 1 Hom 3 Asp → Gly 28 (165, 208 ) 0 12 Hom/Het 4 Gly → Gln 43 nd 71 Het 5 Asn → Ser 51 70 45/9 Hom/Het 6 Gly → Val 100 nd 61 Hom 7 Ala → Pro 123 nd 62 Hom 8 Arg → Trp 135 (205 ) 0 16 Het 9 Leu → Arg 147 (123 ) 0 28 Het 10 Ser → Pro 159 0 6/24 Het 11 Ala → Thr 166 (162, 206, 209 ) 9 11 Hom 12 Cys → Trp 255 nd 60/67 Hom 13 Asp → Gly 273 (210 ) nd 44 Het 14 Trp → Leu 276 4 30 Hom 15 Cys → Tyr 292 0 8/48 Hom 16 Gln → Arg 295 0 5 Hom 17 Arg → Ser 300 0 23 Het 18 Ala → Val 304 (205 ) 2 41 Hom 19 Ile → Leu 315 nd 71 Het 20 Gly → Arg 318 (122 ) 0 56 Het 21 Cys → Ser 355 (123 ) 0 10/28/69 Het 22 Tyr → Cys 366 nd 70 Hom 23 Leu → Ser 369 0 46 Het 24 Arg → Gln 379 0 24 Het 25 Ala → Val 388 0 15 Het 26 Phe → Ser 405 0 15 Het

30 27 Ala → Thr 411 66 30/66/73 Hom 28 Gly → Arg 426 0 6 Het 29 Gln → Arg 457 (163, 207 ) 0 40/49/50 Het 30 Thr → Pro 460 (207, 211 ) 0 12 Hom 31 Ala → Val 468 (122, 206, 207 ) 4 56 Het 32 Val → Asn 470 (205 ) 10 10 Het 33 Arg → His 499 (205, 207 ) 30 38 Het 34 Arg → Cys 558 nd 73 Hom 35 His → Gln 615 67 45 Hom

Nonsense mutation AA Residue Exon Kindred Genotype 36 Arg → Stop 63 2 64 Hom 37 Tyr → Stop 191 (125 ) 6 39 Hom 38 Lys → Stop 254 8 2 Hom 39 Arg → Stop 267 (212 ) Hom/Het 40 Trp → Stop 276 8 54 Hom 41 Arg → Stop 379 (125 ) 11 38/72 Het 42 Trp → Stop 477 12 69 Het

Frame shift AA Base Exon Kindred Genotype 43 FS → Stop 248delG 3 47 Home 44 FS → Stop 273delC 3 55 Het 45 FS → Stop 799delC 8 3 Hom 46 Ins LVGCT 1098ins 15 10 44 Het 47 FS → Stop 1265del4 11 7/9 Hom 48 FS → Stop 1268insT 11 45 Hom 49 FS → Stop 1404insT 12 35 Hom

Splice site Base Intron Kindred Genotype 50 145 + 1 1 26 Hom 51 382 + 2(del/ins) 4 23 Het 52 382 + 1(Dupli) 4 55 Het 53 497 + 2 5 73 Het 54 593 + 1 6 43 Hom 55 1458 +5 12 18 Hom 56 1458 +2 12 16 Het Table 1.4: Mutations in SGLT1

*Sugar uptakes for the mutants were determined in oocytes and are given as a percentage of that obtained for the wild type SGLT1.

1.6 Experimental rationale

Coupled transport of Na + and glucose is controlled by the high affinity sodium/glucose cotransporter (SGLT1). The primary amino acid sequence of SGLT1 predicts a membrane topology involving 14 transmembrane domains. Investigation of the structure/function relationships of the sodium/glucose transporter (SGLT1) is crucial to understanding co-transporter mechanism.

In previously published work, application of the substituted cysteine accessibility method (SCAM) and the two-electrode voltage-clamp method in Xenopus laevis oocytes has shown that the Na + interaction domain is located in the N-terminal half of SGLT1 and involves residues 163, 166, 170, and 173 in the putative external loop joining TM IV-

V (159, 162, 213, 214 ). Interestingly, there is also evidence that A166 is important for the interaction between the Na + and sugar and that helices TMIV-V are close to TMX-XI

(209 ). Previous studies on D204 in the putative internal loops joining TM V-VI, K321

TM VIII and the C-terminal half of SGLT1 (TM X-XIII) also suggest that the Na + interaction domain is located in the N-terminal half of SGLT1 (215, 216 ).

The sugar binding domain, on the other hand, has been proposed to be located only in the C-terminal half of SGLT1, TM X-XIII (215 ). Evidence to support this assumption includes i) the sugar ( αMG) affinity of the C-terminal half of rabbit SGLT1

(TM X-XIV) is about 50 mM and reduces by ~300–fold compared with WT rabbit

SGLT1 (~0.15 mM) (159 ); ii) C-terminal half of SGLT1 (TM X-XIV) totally abolishes the binding of the specific inhibitor, phloridzin ( Ki for WT is about 0.76 µM) (190 ), but shows the same phloretin (the aglucone moiety of phloridzin) affinity as WT (~500 µM).

32 However, we believe that all of results (215 ) suggest that the C-terminal half of SGLT1

(TM X-XIV) likely participates in binding of the aglucone moiety of phloridzin, but not sugar. The sugar binding of SGLT1 requires TM I-IX of SGLT1. Further support for our assumption came from various studies conducted by different groups (182, 217-220 ):

1) Kinne et al suggest that a hydrophobic region located in the C-terminal loop 13

(between TM XIII-XIV) is critically involved in the binding of the aglucone moiety of phloridzin (220-226 ). Recently, Tyagi et al (220 ) reconstituted recombinant hSGLT1 in proteoliposomes and found that mutants F602 and F609 in the putative external loops joining TM XIII-XIV were protected against NBS by phloridzin and phloretin, but not by

D-glucose and L-glucose. They proposed that residues F602 and F609 might form part of the binding sites for the aglucone moiety of phloridzin.

2) Lo et al have identified the functionally important mutants (F163C and A166C) by the X. laevis oocyte expression system and the two-electrode voltage clamp technique

(159, 162 ). The affinities of F163C and A166C for αMG were decreased about 5-fold compared to WT. Reaction of MTS reagents with F163C and A166C significantly decreased sugar transport by 70% -80%.

3) Panayotova-Heiermann et al have shown that phloridzin (176) recognition is located in the putative external loop joining TM IV-V in the N-terminal half of the transporter (227 ). They also suggest that K321 in TM VIII plays a major role in the Na + and sugar binding during cotransporter activity (210 ).

4) Coady et al suggest the first 69 amino acids of SGLT1 enhance access to the sugar binding site (228 ).

33 5) Puntheeranurak et al (182 ) 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. Gagnon et al (217 ), using the Xenopus laevis oocytes expression system, showed that mutants C255 and C511 form a disulfide bridge, suggesting the putative loop TM

VI-VII is close to TM XI-XIII. Despite the discrepancy between these two sets of observations (which may be due to the different experimental systems used), both studies suggest that the putative loop TM VI-VII also participates as part of the extracellular binding pocket for sugar.

These various studies have led us to hypothesize that the binding of sugar and phloridzin requires TM I-IX of SGLT1. Therefore, the aim of the present investigation was to explore whether residues within predicted TMs IV-V and the putative external loop joining TM IV-V participate in sugar interaction.

34 Chapter 2

Materials and methods

2.1 Molecular biology

The eukaryotic expression vector pMT3 (kindly provided by the Genetics Institute,

Boston, MA)) was treated with Pst I and Kpn I to extract the multiple cloning site, generating pMT4. The cDNA of rSGLT1 (kindly provided by M. A. Hediger) was subcloned into the remaining Eco RI site. The cysteine mutations were prepared by the site-directed mutagenesis using polymerase chain reaction protocol mutagenesis as described previously and confirmed by sequencing (229 ).

2.2 Oocyte preparation and injection

X. laevis were prepared as described previously (2). X. laevis were anesthetized in a 0.2% aqueous solution of 3-aminobenzoic acid ethyl ester. Stage V or VI oocytes were then surgically removed and digested for 20-25 min with 2 mg/ml type II collagenase

(C6885, Sigma, Oakville, Ontario, Canada) prepared in modified Barth's saline (MBS) containing MgCl. MBS/Mg 2+ solution consists of 0.88 mM NaCl, 1.0 mM KCl, 2.4 mM

NaHCO 3, 15.0 mM HEPES, 1.0 mM MgCl 2, pH 7.4 with NaOH. The digested oocytes for the injection were stored in Leibovitz solution (L5520, Sigma, Oakville, Ontario,

Canada) consisting of 10 mM HEPES, 20 mg gentamycin, and 0.184 g L-glutamine, pH

7.4 with 10 mM NaOH at 16-18 °C. The present study was reviewed and approved by the

35 Committee on Animal Research at the University of Toronto, Toronto, and was conducted in accordance with the committee’s guidelines.

Figure 2.1: (A) Equipments for Xenopus oocyte injection. (B) Model P-97 micropipette puller.

36 The equipments for oocyte injection includes: (i) Standard dissecting scope equipped with a cold light source (Fig. 2.1A, 1: dissecting microscope, 2: cold light source power supply, 3: cold light source goose-necks). The dissecting scope standard illumination system could not be used because the untransparent Xenopus oocytes could not be clearly viewed when illuminated from the bottom and overheating caused by transmitted light may damage the oocytes. (ii) Nanoject II/Auto Nanoliter Injector

(Drummond Scientific Co., Broomall, PA, USA. Cat. No. 3-000-204) with a range between 2.3 nanoliters and 69.0 nanoliters for microinjection mounted on a coarse micromanipulator (Fig. 2.1A, 4). The important feature for this injector is virtually vibration-free. The Injector requires the use of needles pulled from the glass provided by

Drummond Scientific Co (3.5" capillaries, 100 pcs. Cat. No. 3-000-203-G/X). (iii)

Control box controls the injection volumes, injection speeds, and filling the micropipet

(Fig. 2.1A, 5). (iv) Foot Switch is for hands-free operation. It plugs into the control box enables injectors to inject using the foot switch (Drummond Scientific Co., Cat. No. 3-

000-026). (v) Oocyte injection chamber (filter paper for holding oocytes during injection is attached at the bottom of a Petri dish). (vi) Model P-97 micropipette puller (Fig. 2.1B, the Flaming/Brown type puller for fabrication of micropipettes, patch pipettes and microinjection needles from Sutter Instruments Company, Novato, CA, USA). Different pipettes with a wide range of glass compositions and sizes could be designed by using the sophisticated, programmable microprocessor controller. The program for oocyte injection is P=300, Heat=630, Pull=60, VEL=80, and Time=200. The program for oocyte voltage clamp is P=300, Heat=600, Pull=30, VEL=30, and Time=180, requiring the use of needles pulled from the thin wall glass capillaries provided by World Presision

37 Instruments, Inc. [Cat. No. TW150F-4, 4 in. (100 mm), 1.5 / 1.12 OD/ID (mm),

Filament].

After needle pulled for oocyte injection, forceps was used to break off the tip end with 10-30 microns in size. Then the needle was filled with mineral oil by using 30g x 2" needle and a syringe before attached to the injector. The air inside the needle was pushed out by by pushing the "empty" button on the control box before filling the DNA solution drop. Each oocyte was injected with 60 ng cDNA (empty vector pMT4, WT rSGLT1 or mutants rSGLT1). The injected oocytes for the electrophysiology were stored in

Leibovitz solution (L5520, Sigma, Oakville, Ontario, Canada) consisting of 10 mM

HEPES, 20 mg gentamycin, and 0.184 g L-glutamine, pH 7.4 with 10 mM NaOH at 16-

18 °C for 4 days or more before study.

2.3 Electrophysiology using two-microelectrode voltage clamp

Voltage clamping and recordings were performed using a GeneClamp 500 amplifier, Digidata 1200B interface, and pClamp 9.0 data acquisition software (Axon

Instruments Inc., Union City, CA) as described previously (Fig. 2.2) (2). The oocytes were constantly superfused with a voltage clamping solution consisting of 100 mM NaCl,

2 mM KCl, 1 mM MgCl 2, 1 mM CaCl 2, and 10 mM HEPES-Tris base (pH 7.4) and held

at a holding potential, Vh, of –50 mV, then was subjected to a series of voltage test pulses,

Vt. The current responses were recorded with a sampling interval of 20 µs for pre-steady state and steady state experiments. The sampling frequency was 50 KHz. Results were

38 filtered via a 1-kHz, 5-point Gaussian filter. Additional curve fitting was performed in

ORIGIN 7.0 with the Levenberg-Marquardt algorithm; n is the number of observations.

Figure 2.2: Two-microelectrode voltage clamp protocol

39 2.4 Transient current measurements

The rSGLT1 pre-steady state currents were determined as described previously

(2). As shown in Fig. 2.3, on the left hand side, the top figure is the currents from an expressing wild type oocyte. In order to isolate the SGLT1 specific currents, the well- known nontransported sugar inhibitor, phloridzin, is used to block the SGLT1 specific currents. After subtracting these two, the bottom figure is the SGLT1 specific currents

responding to the test voltage. The pre-steady state currents for each Vt were integrated over the entire course of the trace to calculate the total charge transferred by the

cotransporter. The charge, Q, was plotted as a function of the test pulses, and these Q (Vt) curves were fitted to the two-state Boltzmann relation,

Q = -N * e * z / (1+exp (z* u*(V t - V 0.5 )) + Qdep ) (1)

Figure 2.3: Q vs. V t curve was fit to a 2-state Boltzmann relation

Where Q is the total charge transferred, Q dep is the charge due to depolarizing

pulses, e is the elementary charge, z is the apparent valence of the movable charge, V 0.5 is the potential at which half of the total charge transfer is complete, and N is the number of cotransporters expressed at the surface. The term u = F/RT; F is Faraday's constant, R is the gas constant, and T is absolute temperature.

Figure 2.4: Sugar-induced sodium currents as a function of substrate concentration for

SGLT1

41 Steady state parameters were determined with the difference in the steady state currents obtained before and after exposure to the substrate as described previously (Fig.

2.4) (2). Steady state currents were acquired with test pulses of 300 ms duration. The final

100 ms of a test pulse were selected and the average current value of this range was acquired. The average current values were plotted versus [substrate] and the following equation was fit to the curve,

n n n I = Imax * [S] / ([S] + K0.5 ) (2)

+ Where S is the substrate of investigation (Na , αMG), Imax is the maximal current

induced at saturating [substrate], n is the Hill coefficient, and K0.5 is the Michaelis

constant, which is the [S] at which the I = Imax /2, which serves as an approximation of

substrate affinity. The calculation of substrate affinity values used the Imax values of –150 mV test pulses.

2.5 Phloridzin affinity measurements

Phloridzin affinity was determined as described previously (230 ). Transient current measurements were made for an array of voltage steps ranging from −150 to 70 mV in 10 mV steps for mutants. The current recordings acquired in the presence of phloridzin (at the appropriate concentration) were subtracted from the recordings acquired in the absence of phloridzin (P3449, Sigma, Oakville, Ontario, Canada) to obtain the phloridzin sensitive charge movement due to SGLT1. Subtracted data were then base line adjusted from 145–150 ms and integrated to give a Q/V distribution. The standard holding potential was −50 mV. At each membrane potential, charge versus phloridzin concentration was plotted and fit to the Hill equation to find K0.5 .

2.6 Protocols for chemical modification

1 mM of Cysteine-specific reagents, MTSEA, MTSES, or MTSET (Toronto

Research Chemicals, Toronto, ON, Canada), were dissolved in a voltage-clamping

solution consisting of 100 mM NaCl, 2 mM KCl, 1 mM MgCl 2, 1 mM CaCl 2, and 10 mM

HEPES-Tris base (pH 7.4) immediately before use. The oocytes expressing mutant were labeled with the bath solution including cysteine-specific reagents for 10 min, with membrane clamped at 50 mV. The final concentration of ethanol or DMSO should be less than 0.2% in experiment. Previous studies show that under this concentration has no effect on pre-steady state currents or steady state currents or on the stability of the oocytes (190 ).

2.7 State model simulations

State model simulations were performed as described previously (158 ). Transient currents of -130, -90, -10, 30, and 50 mV were simulated and fit simultaneously from 0 to

150 ms (ON currents) and from 150 to 300 ms (OFF currents). Eyring rate theory was used to calculate voltage dependence, V, of the rate constants. The overall χ2 was minimized by optimizing 12 parameters: 6 rate constants ( k) and 6 valences ( z).

43 2.8 Cell transfection and western blot detection

N-terminal myc-tagged WT and mutants used for COS-7 transfections were prepared as described previously (229 ). Non-transfected cells and COS-7 cells transfected with vector alone, served as controls. Proteins samples were resolved on 10%

SDS-PAGE and transferred to nitrocellulose. The myc-epitope was detected with mouse monoclonal 9E10 (anti-c-myc, 1:1000) antibody (Berkley Antibody Company), followed by peroxidase conjugated anti-mouse IgG (1:200,000) (Sigma) Immunoblots were developed by chemiluminescence, and area analysis was performed using the public domain NIH Image program (developed at the US National Institutes of Health). Western blot for β-actin was performed to check equal loading.

2.9 αMG uptake experiment

αMG ( α-methyl-D-glucopyranoside) uptake was prepared as described previously (231 ). Uptake was gauged with [ 14 C] αMG (Amersham Health, Oakville, ON,

Canada) with a specific radioactivity of 293 mCi/mmol. Culture medium was aspirated, and replaced with 500 µl of incubation medium containing either 140 mM NaCl or 140 mM KCl, 20 mM mannitol, 10 mM HEPES/Tris, pH 7.4 and 1 mM [ 14 C] αMG. After 10 minutes at room temperature, the incubation medium was aspirated and the wells were washed three times with 3 ml of ice-cold stop buffer, consisting of 140 mM KCl, 20 mM mannitol, 10 mM HEPES/Tris, pH 7.4, and 200 µM phloridzin. The cells were solubilized with 500 ml of PBS buffer with 0.1% SDS. Solubilization proceeded for 20 minutes, then the solution was removed and prepared for liquid-scintillation counting.

2.10 Endo H and PNGase F deglycosylation analysis

The cell lysates (30 µl) were diluted to 0.5% SDS with water, containing 1%

Nonidet P-40 (for PNGase F analysis only), 1% β-mercaptoethanol, 50 mM sodium citrate, pH 5.5 (for Endo H analysis) or 50 mM sodium phosphate, pH 7.5 (for PNGase F

analysis) and then digested with Endo H f (2 µl) or PNGase F (2 µl) (New England

BioLabs, Inc.) for 3 hours at 37 °C. The samples were then dissolved to 100 mM dithiothreitol and 3.5% SDS before resolving by SDS-PAGE (10 % gel) and analyzed by

Western blot.

2.11 Labeling of surface expressed wild type or mutants in COS-7 cells with biotin-MTSEA

MTS compounds (Toronto Research Chemicals) were prepared fresh in (DMSO) and diluted to 1 mM concentrations in PBS (pH 7.4) immediately before use. Various protocols were used as described in the results section. Typically, cells in each well

(1x10 5 cells/well, 12 wells plate) were first preincubated for varying times at room temperature with either 500 µl PBS (control), 1.35 mM phloridzin, 1 mM MTSEA-alone or in different combinations. The cells in each well were then washed (over 10–20 sec) and immediately exposed to 500 µl 1 mM biotin-MTSEA for varying times up to 10 min at room temperature. Cells in each well were then washed three times with 2 ml cold PBS and individual wells were scraped into 0.5 ml lysis buffer, (50 mM Tris-HCL, 150 mM

45 NaCl, 1% Triton, 1% SDS, 1 mM EDTA and protease inhibitor cocktail). Samples were rocked at 4 °C for 30 min, and the insoluble protein removed by centrifugation at 14,000 rpm for 15 min. Biotin labeled proteins were isolated from the cell lysates with immobilized streptavidin-agarose (Sigma) (10% total volume, about 50 ul) by incubating overnight at 4 °C with gentle agitation. The beads were washed and the biotinylated protein eluted from the beads by the addition of 50 µl SDS-PAGE sample buffer (4%

SDS) at 100 °C for 3 minutes.

2.12 Statistical comparisons of means

Data are presented with mean ± SEM. A one-way analysis of variance (ANOVA) followed by Tukey’s HSD post hoc test was applied to the entire dataset using SPSS

(SPSS, Chicago, IL) to determine if significant differences existed between mean values.

Statistical significance was accepted at an alpha level of p < 0.05.

46 Chapter 3

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

3.1 Summary

Investigation of the structure/function relationships of the sodium/glucose transporter (SGLT1) is crucial to understanding co-transporter mechanism. In the present study, we used cysteinen scanning mutagenesis and chemical modification by methanethiosulphonate (MTS) derivatives to test whether predicted TM IV participates in sugar binding. Five charged and polar residues (K139, Q142, T156, K157 and D161) and two residues that are mutated in missense mutations (I147 and S159) leading to glucose/galactose malabsorption (GGM) 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 WT and was not studied further. Mutation of lysine 157 to cysteine

(K157C) causes loss of phloridzin and αMG binding ( α-methyl-D-glucopyranoside).

Binding was restored by chemical modification with positively charged (2-aminoethyl) methanethiosulphonate hydrobromide (MTSEA). Mutation of threonine 156 to cysteine

(T156C) reduces the affinity of αMG and phloridzin for T156C by ~5–fold and ~20-fold,

47 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 + currents.

3.2 Introduction

The high affinity sodium/glucose co-transporter (SGLT1) belongs to the homologous family of Na +/solute symporters, SLC5 (56 ). It is a secondary active transporter that utilizes the sodium electrochemical gradient to transport sugar substrates uphill against a concentration gradient (56 ). SGLT1 is expressed most abundantly at the mucosal surface of the small intestine and serves as the principal uptake pathway for glucose derived from dietary sources (74, 232-234 ). Dysfunctional mutations in SGLT1 cause intestinal glucose/galactose malabsorption (56 ). For many years, SGLT1 has served as a model system for studying the molecular basis of ion-coupled co-transporters. In pursuit of this objective, structure/function studies have formed an important experimental strategy to identify key residues participating in co-transporter function.

Application of the substituted cysteine accessibility method (SCAM) has shown that the Na + interaction domain is located in the N-terminal half of SGLT1 and involves residues 163, 166, 170, and 173 in the putative external loop joining TM IV-V (2, 214,

235, 236 ). The polar residues at position 176 is suggested to form a hydrogen bond with the hydroxyl group on the β–phenyl ring of phloridzin (237 ). There is also evidence that

D454 in the putative external loop joining TM X-XI is involved in the coupling of Na + and sugar in the transport process (238 ). The sugar binding domain, on the other hand, has been localized to the C-terminal half of SGLT1 (239 ). Q457, located in the putative

48 external loop joining TM X-XI appears to be particularly important because sugar transport is abolished by reaction of MTS reagents and maleimides with Q457C (240 ).

However, under these conditions, the transporter still binds Na + and sugar (240 ). These results suggest that although residue 457 is at or near the sugar-translocation site, other unidentified residue(s) must be involved in the interaction with sugar ligand.

Interestingly, there is evidence that A166 is important for the interaction between the Na + and sugar pathways and that helices TMIV-V are close to TMX-XI (241 ). Therefore, the aim of the present investigation was to explore whether residues within predicted TMIV participate in sugar interaction.

In the 34 different missense mutations that were identified for GGM (56 ), four mutations are located in TMIV and the putative loop joining TM IV-V (R135W, L147R,

S159P and A166T). In the region of TM IV, eighteen of twenty-four residues (138-161) are conserved in all members of SGLT1 and SGLT2. Previous studies have shown that several residues important for substrate and cation recognition are located in the putative external loop joining TM IV-V in the N-terminal half of the transporter (2, 214, 235, 236,

241 ). Therefore, it is possible that TM IV may be important for SGLT1 function.

The present manuscript describes the results from detailed investigation of two single cysteine mutants, K157C and T156C, which are conserved across species in

SGLT1 and SGLT2. Our results suggest that manipulation of positive charge and polarity at positions 157 and 156, respectively, significantly affects sugar binding and sugar-induced Na + currents.

Figure 3.1: The secondary topology model of SGLT1 (182 ). The functional importance of residues (T156, K157, F163, A166, Q170, L173, D176, D454 and Q457) and domains are indicated.

3.3 Results

In the present study we used SCAM and chemical modification by MTS derivatives to study the role of residues of K139-D161 that lie within predicted TM IV

(Fig. 3.1). Five charged and polar residues (K139, Q142, T156, K157 and D161) and two glucose/galactose malabsorption (GGM) missense mutations (I147 and S159) (56 ) were replaced with cysteine. Several of these (I147C, T156C and K157C) exhibited sufficient expression to be studied in detail using the two-electrode voltage-clamp

50 αMG method in Xenopus laevis oocytes. I147C was similar in function to WT (V 0.5 and K ) and was not studied further (unpublished data).

3.3.1 Characterization of K157C

3.3.1.1 Pre-Steady State Behavior of K157C Compared to

Fig. 3.2 shows representative pre-steady state currents from oocytes expressing

WT or mutant K157C in the presence of 100 mM Na +. Saturating concentrations of the competitive inhibitor phloridzin (200 µM) (Figs. 3.2A and 3.2B) or sugar substrate, αMG

(10 mM) (Figs. 3.2A and 3.2C), eliminate WT transient currents, indicating that binding of phloridzin and αMG are intact. Transfection with empty vector (pMT4) does not give rise to pre-steady state currents (Fig. 3.2D). The specific phloridzin-sensitive transient currents for WT are shown in Fig 3.2E.

Figure 3.2: Representative pre-steady state transient currents (nA) generated with a voltage clamp over a range of –150 mV to +70 mV in oocytes expressing WT and mutant K157C, in the presence and absence of 0.2 mM phloridzin or 10 mM αMG. In all cases, the Na + concentration was 100 mM, except for K which were determined at 0 mM Na +. (A) WT transient currents. (B) WT transient currents after exposure to phloridzin. (C) WT transient currents after exposure to αMG. (D) Transient currents from an oocyte injected with empty pMT4 plasmid. (E) The WT specific phloridzin-sensitive transient currents. (F) The K157C specific phloridzin-sensitive transient currents. (G) Charge transfer characteristics of WT rSGLT1 (n=3) compared to K157C rSGLT1 (n=5). (H) K157C transient currents. (I) K157C transient currents after exposure to phloridzin. (J) K157C transient currents after exposure to αMG. (K) K157C transient currents in 0 mM Na +.

52 In contrast to WT, the transient currents of mutant K157C remain essentially unaffected by exposure to either phloridzin (Figs. 3.2H and 3.2I) or αMG (Figs. 3.2H and

3.2J). The phloridzin-sensitive transient currents for the mutant K157C are shown in Fig.

3.2F and are significantly lower than WT (Fig. 3.2E). The phloridzin binding of mutant

K157C could be studied by the effect of the external phloridzin on the pre-steady state charge movement. The pre-steady state charge movement of mutant K157C that shows the loss of phloridzin binding for mutant K157C, was obtained by integrating the phloridzin-sensitive pre-steady state currents (Fig. 3.2G).

3.3.1.2 Site-Directed Alkylation of Cysteine 157 (K157C)

Rescued Activity of SGLT1

Since mutation of positively-charged lysine 157 to neutral cysteine, abolishes both phloridzin and αMG binding, we developed a rescue strategy designed to recover phloridzin and αMG binding by restoring a positive charge at position 157. Accordingly, we exposed oocytes expressing K157C to the positively-charged MTS derivative,

MTSEA, which mimics the lysine side chain at position 157 (Fig. 3.3). Previous work has established that exposure of oocytes expressing WT to various MTS derivatives does not alter SGLT1 function (9).

Figure 3.3: Rescue strategy for activity of SGLT1 by site-directed alkylation of cysteine

157 (K157C).

Fig. 3.4 shows that following exposure to MTSEA (Fig. 3.4A), phloridzin (Fig.

3.4B) and αMG (Fig. 3.4C) inhibition of mutant K157C transient currents are restored, demonstrating that exposure of K157C to MTSEA rescues phloridzin and αMG binding.

The net phloridzin-sensitive K157C transient currents (obtained by subtracting the currents in Fig. 3.4A from Fig. 3.4B) are shown in Fig. 3.4E. We attempted to study phloridzin and αMG binding in mutant K157R. We found that there was insufficient expression of mutant K157R in oocytes to permit further investigation (confirmed by

Western Blots in COS-7 cells, unpublished data).

54 The pre-steady state currents of mutant K157C-MTSEA for each Vt (Fig 3.4E) were integrated over the entire course of the trace to calculate the total charge transferred by the cotransporter. The charge, Q, was then plotted as a function of the test pulses, and

these Q (Vt) curves were fitted to the two-state Boltzmann relation. The V 0.5 value of

K157C-MTSEA is -9.2 ± 1.4 mV (n = 5), while the V 0.5 value of WT is -1.5 ± 5.1 mV (n

= 5). The z-values of the two are also comparable (0.88 for K157C-MTSEA versus 1.01 for WT). The pre-steady state Boltzmann parameters in 100 mM Na + for both WT and rescued K157C-MTSEA are very similar, suggesting equivalent voltage sensitivity.

Figure 3.4: Representative pre-steady state transient currents (nA) generated with a voltage clamp over a range of –150 mV to +70 mV in oocytes expressing mutant K157C after reaction with MTSEA, in presence and absence of 0.2 mM phloridzin or 10 mM

αMG. In all cases, the Na + concentration was 100 mM, except for D which were determined at 0 mM Na +. (A) K157C-MTSEA transient currents. (B) K157C-MTSEA transient currents after exposure to phloridzin. (C) K157C-MTSEA transient currents after exposure to αMG. (D) K157C-MTSEA transient currents in 0 mM Na + . (E) The

K157C-MTSEA specific phloridzin-sensitive transient currents.

55 To determine the extent of the rescue achieved by restoring a positive charge at position 157, we estimated mutant K157C expression relative to WT. SGLT1 pre-steady state currents yields three time constants τ, ( τfast , τmedium , τslow ) (242, 243 ) and the oocyte capacitive membrane current which is not relevant to transporter activity is less than 1 ms

(243, 244 ). Loo DD et al (242 ) estimated WT hSGLT1 expression by adding the charge transferred associated with the medium and slow decays (Q med and Q slow ). We used the same approach to calculate the charge transferred in nC from the medium and slow decays for K157C and rWT (K157C 3.8 ± 0.7 nC, n=8 versus WT 12.8 ± 0.4 nC, n=3).

From this ratio, the estimated expression of mutant K157C is ~30% of WT. Thus we estimate the rescue of K157C sugar binding activity following chemical modification by

MTSEA to be greater than 85% (Fig. 3.5).

Fig. 3.5 also shows the results following chemical modification of cysteine 157 in

K157C by two membrane-impermeant MTS derivatives (positively charged MTSET and negatively charged MTSES). In both cases, rescue of phloridzin and αMG dependent charge transfer is much less than that achieved following treatment with MTSEA (Fig.

3.5; lines 7 and 11 versus line 5; lines 9 and 13 versus line 6), but pretreatment with

MTSET or MTSES blocks the ability of MTSEA to restore K157C phloridzin and αMG binding (Fig. 3.5; lines 8 and 12 versus line 5; lines 10 and 14 versus line 6). Since the

MTS reagents did not affect WT rSGLT1 (2), the changes observed in Fig. 3.5 must arise from alkylation of cysteine 157 after exposure to MTSEA. Therefore, the inability of

MTSET or MTSES to rescue K157C sugar binding is not due to inaccessibility to cysteine 157 because MTSEA rescued sugar binding. Since MTSET and MTSES are both membrane-impermeant and since chemical modification of cysteine 157 in K157C

56 by either derivative blocks reactivity to MTSEA, this indicates that cysteine 157 is located exofacially.

Figure 3.5: Results demonstrating the effects on K157C charge transfer in oocytes treated with various 1 mM sulfhydryl-specific reagents (MTS derivatives) in 100 mM Na + (n ≥3 for each compound tested). The error bars represent SEM. A one-way analysis of variance (ANOVA) was conducted to determine if significant differences existed in the

Qmax levels of K157C (capacitive currents removed), empty pMT4 + PZ, K157C + PZ, K157C + αMG, and K157C reacted with various MTS reagents + PZ or αMG. Following ANOVA, Tukey’s HSD post hoc test was applied to the entire dataset (lines 1- 14) given in Fig. 5. Statistical significance was accepted at an alpha level of ρ< 0.05. The significant statistical comparisons denoted by asterisks are the pairwise comparisons between lines 3 and 5, and between lines 4 and 6.

57 Figs. 3.6A and 3.6B show that the charge transfer following reaction of K157C with MTSEA (i.e. of K157C-MTSEA) depends on the sodium concentration in the bath solution. αMG-induced Na + currents are also Na + dependent (Fig. 3.6C). Na + dependence of K157C-MTSEA thus mirrors Na + dependence of WT rSGLT1 (236 ), and provides further evidence that MTSEA exposure restores sugar-induced Na + currents and phloridzin binding.

To determine whether reactivity of K157C with MTSEA depends on sodium, oocytes were exposed to MTSEA in either 100 mM Na + or 0 mM Na + (choline buffer).

As shown in Figs. 3.7A and 3.7B, reactivity of 1 mM MTSEA with cysteine 157 in

K157C is not influenced by the presence of Na +. K157C-MTSEA, labeled in the absence of Na +, restores both phloridzin and αMG binding. Further, when the same oocyte is pretreated with MTSEA in the absence of Na +, and then treated with MTSEA in the presence of 100 mM Na +, there is no additive effect on rescue of phloridzin and αMG binding.

Figure 3.6: Comparison of the effects of 100 mM Na + ( ■), 40 mM Na + ( ●) and 10 mM

Na + ( ) on mutant K157C reacted with 1 mM MTSEA in the same oocyte. (A) Effects on phloridzin dependent charge transfer. (B) Effects on αMG dependent charge transfer.

Figure 3.7: Typical results demonstrating the effects of pretreatment with 1 mM MTSEA on mutant K157C charge transfer in the absence of Na + (choline replacement) or in the presence of 100 mM Na +. (A) Effects on charge transfer. (B) Effects on αMG binding.

60 3.3.1.3 Steady State αMG-Induced Na + Currents of K157C and K157C-MTSEA

Fig. 3.8A shows the maximal current induced by 10 mM αMG for K157C and for

K157C reacted with various MTS reagents. Neither K157C-MTSET nor K157C-MTSES rescues sugar binding and sugar-induced Na + currents (Fig. 3.8A, lines 6 and 8 versus line 4). This is in contrast to the effect of chemical modification by MTSEA. As shown in

Fig. 3.6C, reaction with MTSEA (K157C-MTSEA) restores Na + dependent sugar- induced Na + currents and the Na + leak is very small (unpublished data). However, when the same oocyte that was pretreated with MTSET or MTSES is then reacted with

MTSEA in the presence of Na +, sugar binding and sugar-induced Na + currents, were not restored (Fig. 3.8A; lines 7 and 9 versus line 5). Although MTSET or MTSES does not affect sugar binding and sugar-induced Na + currents, pretreatment with these agents is able to block reactivity of MTSEA, thus indicating that MTSET and MTSES are able to alkylate cysteine 157 in K157C.

To determine the affinity of rescued K157C-MTSEA for sugar substrate αMG, we measured αMG-induced steady state currents at various sugar concentrations over a range of holding potentials and the resulting curves were fitted to the Michaelis-Menten relationship. As shown in Fig. 3.8B, the K0.5 of restored mutant K157C-MTSEA activity

exhibited voltage-dependence from −150 mV to −50 mV, similar to WT rSGLT1 ( K0.5 is

0.15 ± 0.02 mM at −150 mV) (2). The αMG apparent K0.5 of K157C-MTSEA was 2.65 ±

0.93 mM ( V = -150 mV) and 5.81 ± 2.46 mM ( V = -50 mV) (significantly different, p ≅

0.05). K157C-MTSEA Imax was 28.99 ± 5.99 nA ( V = -150 mV) and 6.48 ± 1.68 nA

61 (V = -50 mV), (significantly different, p < 0.05). Therefore the αMG affinity of K157C-

MTSEA is decreased about 18-fold compared to WT.

Figure 3.8: (A) MTSEA rescue of K157C activity. Figure displays maximal αMG- induced Na + currents measured at –150 mV in oocytes expressing WT whose expression is ~3 time higher than that of K157C, or empty pMT4 plasmid or mutant K157C before or after reaction with MTS compounds. The error bars represent SEM. A one-way analysis of variance (ANOVA) was conducted to determine if significant differences existed in the I max levels of WT, empty pMT4, empty pMT4-MTSEA, K157C and K157C reacted with various MTS reagents. Following ANOVA, Tukey’s HSD post hoc test was applied to the entire dataset (lines 1-9) given in Fig. 8A. Statistical significance was accepted at an alpha level of ρ< 0.05. The * shows significant difference compared with

Imax of K157C before and following rescue with MTSEA at –150 mV ( p < 0.05). (B) The

αMG apparent K0.5 of WT (2), T156C and K157C-MTSEA for voltage dependence (n ≥3). The error bars represent SEM.

In summary, mutation of the positively charged lysine residue at position 157, abolishes sugar and phloridzin binding. Replacement of the positive charge by MTSEA, however, restores activity.

3.3.2 Characterization of T156C

Given the substantial effects on sugar binding and sugar-induced Na + currents that arise following mutation of lysine 157 to neutral cysteine, we investigated whether mutation of the neighbouring residue, threonine 156, would affect SGLT1- sugar interaction.

3.3.2.1 Steady State Kinetics of T156C

A plot of the apparent αMG affinity as functions of V is shown in Fig. 3.8B. The affinity of T156C for αMG (at V = −50 mV, K0.5 = 0.95 ± 0.06 mM) was decreased about

5–fold compared to WT. When Na + dependence of T156C sugar-induced Na + currents was analyzed by fitting the Hill equation, K0.5 was ranged from 28.9 ± 1.4 mM at –30 mV to 22.9 ± 3.7 mM at –150 mV. The corresponding values for “n”, the Hill coefficient ranged from 1.0 to 2.0. The Na + affinities of T156C was decreased about 6–fold at –150 mV and same at –30 mV compared to WT (235, 245 ).

Figure 3.9: αMG-induced Na + currents in presence of 100 mM Na + in an oocyte expressing mutant T156C (A) Steady-state currents induced by 20 mM αMG before and after MTSEA exposure and also after MTSEA exposure in the presence of 2 mM phloridzin in an oocyte expressing mutant T156C. (B) Steady-state currents induced by

20 mM αMG before and after TMR6M (tetramethylrhodamine-6-maleimide) exposure in an oocyte expressing mutant T156C.

3.3.2.2 Apparent Affinity of T156C for Phloridzin

Binding of phloridzin to WT SGLT1 inhibited αMG-induced currents by competing for the sugar binding site, reduced the leak current by locking the transporter in a non-transporting state and eliminated the charge movements that give rise to the transient currents. By measuring the amount of transient current eliminated as a function of phloridzin concentration, an estimate of the apparent affinity of the transporter for phloridzin could be made.

64 We have determined that the phloridzin binding affinity of T156C was decreased about 20–fold compared to WT (230 ). These values for phloridzin binding are consistent with the apparent reduced affinity for αMG demonstrated in Fig. 3.8B. Therefore, a threonine to cysteine mutation at position 156 causes a significant reduction in binding affinity for both sugar and phloridzin.

3.3.2.3 Chemical Modification of T156C by MTS Reagents

The T156C I–V curves in the absence or presence to 2 mM MTSEA are shown in

Fig. 3.9A. After reacting mutant T156C with MTSEA, the Na + leak current is increased

~10-fold and the αMG-induced Na + current is almost abolished (Fig. 3.9A). These results are similar to those reported for mutant Q457C (240 ). This effect was observed in the absence of Na + (choline + replacing Na +) as well as in its presence (230 ). The fluorophore

TMR6M (tetramethylrhodamine-6-maleimide), which blocks sugar-induced Na + currents in Q457C (11), also abolishes αMG-induced Na + currents in T156C (Fig. 3.9B), but the

Na + leak currents of T156C are very small (230 ). A similar exposure (10 min) to 2 mM

MTSEA was previously shown to have no effect on the function of WT SGLT1 (2).

When phloridzin was infused at the same time as MTSEA, phloridzin conferred partial protection against MTSEA modification of T156C. Phloridzin protection was only observed when inhibitor was present together with MTSEA in the infusing solution. It has not been possible to definitively test whether saturating concentrations of αMG could provide similar protection since prolonged exposure to high concentrations of αMG leads to paradoxical inactivation of the transporter.

65 We then studied the apparent competition between phloridzin and MTSEA with respect to MTSEA reactivity with cysteine 156 (229 ). COS-7 cells expressing WT

SGLT1 and T156C were each reacted with biotin-MTSEA. Fig. 3.10A demonstrates that biotin-MTSEA specifically labels cells expressing T156C, but not WT SGLT1. These results demonstrate that MTSEA has reacted with cysteine 156 in T156C. We then developed an assay system based on competition of MTSEA and biotin-MTSEA for cysteine 156. Fig. 3.10B shows that MTSEA prevents biotin-MTSEA from reacting with

T156C, but it takes ~6–7 min for maximal MTSEA effects to be observed. Phloridzin was able to protect the mutant T156C from reacting with MTSEA because after removal of phloridzin/MTSEA, then cells could still react with biotin-MTSEA.

In summary, mutation of the polar amino acid to a neutral residue (T156C) reduces the affinity for both αMG and phloridzin. Conversely, phloridzin protects cysteine 156 from alkylation by MTSEA.

Figure 3.10: (A) Specificity of biotin-MTSEA reaction to the cysteine at 156. Upper panel: COS-7 cells were transfected with empty plasmid (pMT4), N-terminal myc-tagged WT, or N-terminal myc-tagged mutant T156C. Forty-eight hours post transfection, cells were reacted with biotin-MTSEA for 10 min at room temperature. The reaction was stopped by washing with phosphate buffered saline. Lysates were collected, reacted with streptavidin-agarose and bound proteins were subjected to immunoblot analysis with anti- myc antibody. Lower panel: Prior to reacting with streptavidin, aliquots of each lysate were also immunoblotted with anti-myc antibody to verify expression and are shown directly below the corresponding sample. (B) Phloridzin protection of MTSEA accessibility/reactivity to cysteine 156 in mutant T156C. Upper panel: COS-7 cells expressing myc-tagged mutant T156C were pre-exposed to either MTSEA alone or MTSEA in the presence of 1.35 mM phloridzin, for the indicated times. Cells were then washed and treated for 10 min with biotin-MTSEA. The reaction was stopped by washing with PBS, and cells were allowed to recover for 10 min at 37°C. Cell lysates were collected, reacted with streptavidin-agarose, and bound proteins subjected to immunoblot analysis with anti-myc antibody. Lower panel: Aliquots of each cell lysate were also immunoblotted with anti-myc antibody prior to reacting with streptavidin in order to verify equal expression of the myc-tagged mutants and are shown directly below the corresponding sample.

67 3.4 Discussion

We identify two residues (threonine 156 and lysine 157) in predicted TM IV that are important for sugar binding. Wright and coworkers (239 ) have previously proposed the sugar domain to be established by TM X-XIII in the C-terminal half of SGLT1. Their laboratory also demonstrated that reacting Q457C with MTSEA inhibited sugar transport, but the mutant could still bind sugar (11). These results suggest that other residues might be important for binding of sugar. Recently, Puntheeranurak et al (182 ) 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. Gagnon et al (217 ), using the Xenopus laevis oocytes expression system, showed that mutants C255 and C511 form a disulfide bridge, suggesting the putative loop TM VI-VII is close to TM XI-XIII. Despite the discrepancy between these two sets of observations (which might be due to the different experimental systems used), both studies suggest that the putative loop TM VI-VII also participates as part of the extracellular binding pocket for sugar.

Lysine 157 is conserved in all members of both SGLT1 and SGLT2. The finding that mutation of lysine 157 to alanine in hSGLT1 apparently results in “impaired transport” (246 ) supports the notion that lysine 157 is an important residue in sugar binding. In the present study, we also found that lysine 157 is important for sugar binding. Mutation of positively-charged lysine to cysteine abolishes sugar and phloridzin binding. Restoration of the positive charge, however, by chemical modification of cysteine 157 with MTSEA rescues sugar binding and sugar-induced Na + currents. These

68 results suggest that sugar-induced Na + currents may indirectly represent sugar transport or could represent sensing of glucose by SGLT1 as shown for human SGLT3 by Diez-

Sampedro A et al (247 ). In contrast to the functional rescue by MTSEA, reaction of

K157C with either anionic MTSES or bulky cationic MTSET did not lead to rescue of sugar binding even though that both were able to label cysteine 157 (Fig. 3.5). We suggest the rescue of sugar binding by chemical modification of K157C with MTSEA is a result of MTSEA’s ability to mimic the guanidino group of lysine (Fig. 3.3).

The properties of mutant T156C also suggest that TM IV participates in sugar binding. We observe an approximate 5-fold decrease in αMG affinity and at least a 20- fold reduction in phloridzin affinity. The mutant shows a modest reduction in Na + affinity primarily at hyperpolarizing potentials, and reduced voltage dependency (unpublished data). Interestingly, phloridzin protects mutant T156C from alkylation by MTSEA.

Finally, αMG-induced Na + currents for the mutant T156C are blocked after exposure to

TMR6M. These results are similar to those observed for Q457C (240 ).

Alkylation of mutants T156C and K157C with MTSEA is independent of Na + whereas alkylation of Q457C with MTSEA depends on the presence of Na +. Similarly,

MTSEA also reacts with A166C in the absence or the presence of Na+ (235 ). In this regard, alkylation of mutants T156C, K157C and A166C is similar to that of mutant

G527 (TM XIII) of hSGLT1 (207 ) and the sodium/dicarboxylate cotransporter (248 ), all of which are also independent of Na +. However phloridzin binding and sugar-induced

Na + currents of mutants T156C, K157C-MTSEA and Q457C are all dependent on sodium. Binding of Na + appears to produce a conformational change in the TM XI region of mutant Q457 compared to TM IV and/or the putative external loop between TM IV

69 and V region of mutants T156, K157 and A166. A large conformational change in the region of TM XI (Q457) might be required to allow access to extracellular ligand in the late stages of the transport cycle.

Finally, there are two alternative interpretations of the collective data presented in this study. One possibility is that both lysine 157 and threonine 156 directly participate in binding of sugar substrate. This is plausible in view of the evidence derived from the crystallographic structure of lactose permease, that positively-charged residues can participate in hydrogen bonding with sugar ligands (249 ). Another possibility is the mutation of the charge and polarity at positions 157 and 156 causes a conformational change in SGLT1, which alters sugar interaction with its binding pocket in the protein.

Whichever interpretation is correct, there seems to be reciprocity in that phloridzin protects against alkylation of MTSEA at position 156 due to either the glycoside moiety of phloridzin competing directly for binding to position 156 or phloridzin occupancy of the sugar binding site at some other location which induces a conformational change that prevents MTSEA accessibility to position 156. Taken together, these results suggest that

TM IV participates in sugar interaction with SGLT1.

70 Chapter 4

Reanalysis of structure/function correlations in the region of transmembrane segments 4 and 5 of the rabbit sodium/glucose cotransporter

4.1 Summary

The predicted topology of the mammalian high affinity sodium/glucose cotransporter (SGLT1), in the region surrounding transmembrane segments 4 and 5, disagrees with the recent published crystal structure of bacterial SGLT from Vibrio parahaemolyticus (vSGLT). To investigate this issue further, thirty-eight residues from

I143 to A180 in the N-terminal half of rabbit SGLT1 were each replaced with cysteine and then expressed in COS-7 cells or Xenopus laevis oocytes. The membrane orientations of the substituted cysteines were determined by treatment with the thiol-specific reagent

N-biotinoylaminoethyl methanethiosulfonate (biotin-MTSEA), combined with the membrane impermeant thiol-specific reagent sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES). The present results combined with previous structure/function studies of SGLT1, suggest that transmembrane domain (TM) 4 of mammalian SGLT1 extends from residue 143-171 and support the topology observed in the crystal structure of vSGLT.

71 4.2 Introduction

The high affinity sodium/glucose co-transporter (SGLT1) (120 ) is an active secondary transporter that utilizes the sodium electrochemical gradient to transport sugar substrates uphill against a concentration gradient. SGLT1 belongs to the homologous family of

Na +/solute symporters (SLC5) and is expressed most abundantly at the mucosal surface of the small intestine (233, 234 ). It serves as the principal uptake pathway for glucose derived from dietary sources. Mutations that result in dysfunctional SGLT1 affect intestinal glucose/galactose absorption (120 ). Recently, SGLT1 has been a target protein for diabetes treatment (250 ).

The transporter functions as a monomer with 14 transmembrane domains (Fig. 1) and exhibits a stoichometry of 2 Na + ions : 1 sugar molecule (59 ). Investigation of the structure/function relationships of SGLT1 is crucial to understanding co-transporter mechanism (159, 182, 207, 217, 230 ).

The recently published crystal structure of Vibrio parahaemolyticus SGLT

(vSGLT) contains a core structure formed by multiple transmembrane helices from both

N-terminus (TMs 2-6) and C-terminus (TMs 7-11) (1). Galactose and Na + are bound in the center of the core. Both SGLT1 and vSGLT belong to the solute sodium symporters family, have a sequence similarity of 60%, contain 14 transmembrane domains and share an alternating-access mechanism with tight coupling between sodium and solute transport

(1). This suggests that they may have similar structure and function. However, the validity of extrapolating the structure of vSGLT to mammalian SGLT1 is brought into question. Previous biochemical characterization of SGLT1 showed that TMs 10-13 in the

C-terminal half of SGLT1 retained sufficient tertiary structure to transport sugar downhill

72 in a stereospecific and selective manner (207 ).On the other hand, studies from other groups suggest that the putative external loop joining TM 4-5 in the N-terminal half of

SGLT1 is involved in the Na + interaction (159, 162, 213, 214 ) and TM 4 participates in sugar binding (230 ).

The aim of present study was to reinvestigate the topology of rabbit SGLT1 in the region between residues 143-180 using the scanning cysteine accessibility method. We show that by accessibility criteria, multiple residues in this region are extracellularlly and face an aqueous extracellular environment. We also provide functional data confirming a role for residues in TM 4 in the interaction of sugar substrate. Together with the previous structure/function characterization of SGLT1 (159 ) and molecular dynamic simulation studies (251 ), our data indicate that the region including residues 162-171 in SGLT1 is a part of transmembrane segment rather than existing as an extracellular connecting loop.

This is consistent with the recently reported structure of vSGLT (1).

4.3 Results

4.3.1 Expression and αMG transport activity of mutants in COS-7 cells

It has been previously demonstrated that N-terminal myc-tagged WT rabbit

SGLT1 is expressed at the cell surface of transiently transfected COS-7 cells and its activity is similar to the non-myc-tagged WT rabbit SGLT1 in terms of sugar transport and phloridzin binding (180 ). In the present study, we used immunoblotting of myc-

73 tagged mutants in COS-7 cells, chemical modification by MTS derivatives and the two- electrode voltage-clamp method in Xenopus laevis oocytes to study the role of residues

I143-A180 (Fig. 4.1).

Figure 4.1: Model of a modified secondary structure of rabbit SGLT1 (59, 182 ). The conserved N-glycosylation site is shown by a Y. The proposed sugar- binding/translocation domain (residues 407-664) is indicated (172 ). The thirty-eight cysteine-substituted mutants in this study were made from I143 to A180 between TM IV and TM V.

Table 4.1 summarizes the Na +-dependent αMG uptake from COS-7 cells expressing mutants as a percentage of the WT control. The thirty-eight mutants studied retained varying degrees of activity compared with WT. Most mutants exhibited more than 20% of transport activity. Three mutants (Y153, I154C and G165C) retained only

10–15% activity compared with WT. Another three mutants (K157C, A160C and D161C)

74 were inactive or had activities less than 5% of the parent transporter despite expression at the cell surface (Fig. 4.3), suggesting that these three residues may play functionally important roles in glucose transport in SGLT1.

Mutants % Uptake Mutants % Uptake Mutants % Uptake Mutants % Uptake I143C 58 Y153C 14 F163C 33 L173C 66 Y144C 86 I154C 9 S164C 26 G174C 32 L145C 100 F155C 64 G165C 12 L175C 74 S146C 92 T156C 50 A166C 49 D176C 100 I147C 96 K157C 2 I167C 64 I177C 75 L148C 91 I158C 27 F168C 31 Y178C 83 S149C 22 S159C 60 I169C 47 V179C 100 L150C 70 A160C 0 Q170C 75 A180C 100 L151C 74 D161C 1 L171C 63 L152C 77 I162C 26 T172C 69

Table 4.1: Na +-dependent αMG uptake of single cysteine-substituted rabbit SGLT1 mutants. COS-7 cells were transfected with empty vector (pMT4), WT or mutants (n ≥2).

The uptake of αMG was measured as described in the experimental procedures section.

The external medium contained 140 mM NaCl, 20 mM mannitol, 10 mM Hepes/Tris, pH

7.4, and 1 mM [ 14 C] αMG. The Na +-independent uptake of 1 mM [ 14 C] αMG was measured in equivalent medium containing 140 mM KCl. For clarity the Na +- independent uptake values have been subtracted. Sugar uptakes for the mutants are given as a percentage of that obtained for the wild type rabbit SGLT1.

75 4.3.2 Determination of topology for TMs IV-V

4.3.2.1 Biotinylation of mutants in intact COS-7 cells

Previous work had demonstrated that biotin-MTSEA does not react with COS-7 cells expressing myc-tagged WT rabbit SGLT1 or empty vector (pMT4) (231 ).

Therefore, COS-7 cells transfected with vector (pMT4) or WT were used as a negative control in each experiment. The cell surface expression of the thirty-eight mutants in

COS-7 cells was measured directly by labeling with biotin-MTSEA. Mutant T156C in

TM VI was chosen to measure the time course necessary to fully react with biotin-

MTSEA because it exhibited relatively high protein expression in Xenopus laevis oocytes

(Fig. 4.2A) (230 ). While both WT and T156C expressed at the cell surface, biotin-

MTSEA only reacted with cells expressing myc-tagged T156C, suggesting that biotin-

MTSEA specifically reacted with the cysteine residue at position 156 rather than with any of the native cysteines (230 ). Maximum reaction was reached after ~10 minutes of exposure. Of the thirty-eight mutants studied, fourteen mutants (I143C-F155C, A180C;

Fig. 4.1) were not labeled by biotin-MTSEA (data not shown). In contrast, twenty mutants between T156C and V179C specificly bound to biotin-MTSEA (Fig. 4.2B).

Finally, five mutants (I162C, G165C, F168C and D176C) between T156C and V179C were difficult to detect after labeling with MTSEA-biotin, perhaps due to the low expression and/or size of the reagent.

76 A

C mature 66 KDa unglyc 55 KDa

Figure 4.2: (A) COS-7 cells expressing T156C-myc were exposed to biotin-MTSEA for the

indicated times (n=3). Cell lysates were collected, reacted with streptavidin-agarose, and bound

proteins subjected to immunoblot analysis with anti-myc-antibody. Densitometric tracings for

each experiment were analyzed and expressed as a percent of maximum biotin –MTSEA bound

(over the course of 10 min). The error bars represent SEM. (B) Biotin-MTSEA reactivity to

mutants. COS-7 cells expressing empty vector (pMT4), WT, or mutants, were exposed to

biotin-MTSEA at room temperature for 10 minutes. Cell lysates were collected, reacted

with streptavidin-agarose, and bound proteins subjected to immunoblot analysis with

anti-myc-antibody. (C) A representative experiment for deglycosylation of WT or

mutants expressed in COS7 cells. Immunoblot of enzymatic deglycosylation of N-linked

WT or mutants. The samples were left untreated (-), digested with Endo H, or PNGase F

and separated by SDS-PAGE (12%). Mature and unglycosylated ( unglyc ) samples are

indicated as determined by enzymatic deglycosylation.

77 To verify whether mature mutants expressed on the surface of COS-7 cells, the maturity of N-linked glycoproteins labeled with Biotin-MTSEA were studied using two different glycosidases: Endo H and PNGase F. Because PNGase F removes all N-linked oligosaccharides and Endo H is unable to remove N-linked oligosaccharides once the protein reaches the medial Golgi in the biosynthetic pathway, enzymatic deglycosylation assays can reveal if Biotin-MTSEA has reacted with mature mutants expressed in the surface of COS-7 cells. Previous studies have found that treatment of WT SGLT1 with

PNGase F reduced the ~64-66-kDa band to ~54-55 kDa, indicating that the higher molecular mass band represented the mature SGLT1 (168, 231 ). As shown in Fig. 4.2C, treatment of mutants with PNGase F reduced the higher molecular mass band to lower molecular mass band, while treatment with Endo H had no effect on the higher molecular mass band. These results indicate that mature mutants were expressed on the surface of

COS-7 cells

4.3.2.2 MTSES accessibility of cysteine mutants expressed in COS-7 cells

We have shown that mutants between T156C and V179C could be specifically labeled by biotin-MTSEA. Biotin-MTSEA is relatively membrane impermeant (252 ). To determine if residues accessible to alkylation by biotin-MTSEA are located in a hydrophilic vs a hydrophobic environment, we tested whether pre-incubation with membrane impermeant MTSES prevents biotin-MTSEA labeling to mutants. Eleven mutants between residues T156 and I177 were chosen for pretreatment with membrane

78 impermeant MTSES. Since WT SGLT1 expressed in COS-7 cells was previously shown to be insensitive to biotin-MTSEA and MTSES (180 ), COS-7 cells transfected with vector (pMT4) or WT were used as a negative control in each experiment. Pretreatment of mutants with MTSES showed nine out of eleven mutants to be highly sensitive to

MTSES and reactivity with biotin-MTSEA significantly decreased 90%; mutant S159C was also sensitive to MTSES and reactivity with biotin-MTSEA decreased 65%; mutant

T156C was not sensitive to MTSES (Fig. 4.3).

Figure 4.3: MTSES pretreatment protects against biotin-MTSEA reactivity to mutants.

(A) COS-7 cells expressing empty vector (pMT4), WT, or mutants, were preincubated in sodium buffer with (+) or without (-) 1 mM MTSES at room temperature for 10 minutes in order to label the accessible cysteines, washed, and then exposed to biotin-MTSEA at room temperature for 10 minutes. Cell lysates were collected, reacted with streptavidin- agarose, and bound proteins subjected to immunoblot analysis with anti-myc-antibody.

(B) Densitometric tracings for each experiment were analyzed and expressed as a percent of maximum biotin–MTSEA bound (10 min. value). The error bars represent SEM

(n ≥3).

80 4.3.3 Determination of functions for TMs IV-V

4.3.3.1 Effect of substrate on the accessibility of mutant

T156C in TM IV expressed in COS-7 cells

We were interested in investigating whether this region of rabbit SGLT1 participates in sugar interaction/binding. Accordingly, we studied the apparent competition between sugar substrates and biotin-MTSEA with respect to biotin-MTSEA reactivity with cysteine mutant-T156C.

Fig. 4.4 illustrates the results of the relative inhibition of biotin-MTSEA reactivity to mutant T156C in the presence of sugar analogue, SGLT1 specific inhibitor-phloridzin

(including the sugar moiety and aglucone part) or phloretin (which lacks the sugar moiety of phloridzin). Inspection of Fig. 4.4 indicates that mutant T156C was only sensitive to

D-glucose, αMG and phloridzin, and decreased biotin-MTSEA binding by 80%, 70% and 75%, respectively. In contrast, phloretin and L-glucose had no effect on biotin-

MTSEA binding of mutant T156C. These results are consistent with previous studies that demonstrated the order of sugar specificity of WT rabbit SGLT1 to be D-glucose > αMG

>> L-glucose (180 ).

Figure 4.4: Effect of substrate on biotin-MTSEA reactivity to mutant T156C. COS-7 cells expressing mutant T156C were preincubated with either PBS (control), 20 mM D- glucose, 20 mM αMG, 1.35 mM phloridzin, 1.35 mM phloretin or 20 mM L-glucose at room temperature for 10 minutes, then exposed to biotin-MTSEA at room temperature for 10 minutes. Cell lysates were collected, reacted with streptavidin-agarose, and bound proteins subjected to immunoblot analysis with anti-myc-antibody. Densitometric tracings for each experiment were analyzed and expressed as a percent of maximum biotin–MTSEA bound (10 min. value). The error bars represent SEM (n ≥3).

82 4.3.3.2 Characterization of cysteine mutants I177C, Y178C and A180C in TM V expressed in Xenopus laevis oocytes

To extend the functional characterization of rabbit SGLT1 in TM V, we investigated three residues (I177, Y178 and A180) in more detail. The normal residues were individually replaced with cysteine and studied using the two-electrode voltage- clamp method in Xenopus laevis oocytes.

Pre-steady state currents for mutants were obtained as a function of voltage and

integrated over 300 ms ( Vt) to calculate total charge ( Q) transferred by the cotransporter

(V h was -50 mV, V t varied from -150 mV to +70 mV). The charge ( Q) was then plotted

as a function of the test pulses, and the Q(Vt) curves were fit to the two-state Boltzmann equation. This protocol was carried out at 100 mM saturating Na + concentrations and

+ compared to WT rabbit SGLT1. In 100 mM Na , the V0.5 values of mutants I177C,

Y178C and A180C are shifted to negative potentials compared to WT [-28.4 ± 2.3 mV

(n=4), -29.5 ± 2.0 mV (n=5), -32.4 ± 0.9 mV (n=3), and -1.5 ± 5.1 mV (n=5), respectively] (Fig. 4.5A).

αMG The apparent affinity of mutants for sugar substrate αMG, ( K0.5 ), was determined by measuring αMG induced steady state currents at various sugar concentrations and over a range of holding potentials (159 ). The results were then fitted

αMG according to the Michaelis-Menten equation (Fig. 4.5B). At –150 mV, the K0.5 of mutants I177C, Y178C and A180C is 0.15 ± 0.01 mM, 0.04 ± 0.01 mM, and 0.08 ± 0.02

αMG αMG mM, respectively; while for WT, K0.5 is 0.16 ± 0.01 mM. At –50 mV, the K0.5 of

83 mutants I177C, Y178C and A180C is 0.12 ± 0.02 mM, 0.04 ± 0.01 mM, and 0.10 ± 0.03

αMG mM, respectively; while for WT, K0.5 is 0.19 ± 0.02 mM.

Figure 4.5: (A) Typical results demonstrating the effects on WT or mutants charge

+ transfer in oocytes in 100 mM Na . The Q(V t) curves were adjusted to zero at hyperpolarizing voltages (-150 mV) and normalized with respect to the extrapolated

Qmax . (B) The αMG K0.5 of WT (n=5) and mutants (n ≥3) for voltage dependence. The

+ error bars represent SEM. (C) The Na K0.5 of WT (n=4) and mutants (n=3) for voltage dependence. The error bars represent SEM.

84 To determine the Na + affinity of mutants, we measured αMG induced steady state currents (obtained at saturating 5 mM αMG) at various Na + concentrations over a range of holding potentials and the resulting curves were analyzed using the Hill relationship

Na+ (Fig. 4.5C) (159 ). At –150 mV, the K0.5 of mutants I177C, Y178C and A180C is 11.4

αMG ± 1.0 mM, 10.1 ± 0.5 mM, and 14.5 ± 0.1 mM, respectively; while for WT, K0.5 is 8.3

Na+ ± 0.8 mM. At –50 mV, the K0.5 of mutants I177C, Y178C and A180C is 25.9 ± 0.8

αMG mM, 11.8 ± 0.8 mM, and 34.2 ± 6.8 mM, respectively; while for WT, K0.5 is 34.7 ±

1.5 mM.

4.4 Discussion

We have used scanning cysteine accessibility methods with thiol-reactive MTS reagents to investigate the topology in the region surrounding predicted TMs IV-V.

MTSES protects mutants (between T156 and V179) from reacting with biotin-

MTSEA, suggesting that these residues are on the extracellular side of the membrane or facing an aqueous pore. The results obtained from functional characterization of mutant

T156C suggest that TM IV participates in sugar binding because sugar substrates (D- glucose, αMG and phloridzin), but not phloritin and L-glucose protect mutant T156C from reactivity to biotin-MTSEA (Fig. 4.4). Further, since mutants I177C, Y178C and

A180C exhibited altered voltage sensitivity and increased Na + and sugar affinity of the co-transporter, this suggests that while this region of TM V is not directly involved in binding of sugar substrate, residues at this location are likely involved in determining

85 accessibility to the Na +-and sugar-binding domain. Similar effects by mutation on other residues of SGLT1 have been observed by Loo et al (159 ) and Gagnon et al (217 ).

The 3D structure of vSGLT, a homolog of SGLT1, was recently determined and provides important insights into the structure/function of this bacterial co-transporter (1).

Although there are significant sequence differences between vSGLT and mammalian

SGLT1 (the Na +/glucose stoichiometry is 1:1 and 2:1, respectively), it is informative to explore how much of the information obtained from vSGLT structure can be extrapolated to SGLT1. The crystal structure of vSGLT contains a hydrophilic core structure formed by multiple transmembrane helices from both N-terminus (TMs II-VI) and C-terminus

(TMs VII-XI). Na + and galactose are bound in the center of the core. The theme of N- and C-terminal segments participating in formation of a common cavity for Na + and co- transported substrate is also emerging from studies of other ion coupled co-transporters, such as the Na +/leucine transporter (253 ), Na +/aspartate transporter (254 ), Na +/glutamate transporter (255 ), Na +/H + antiporter (256 ) and lactose permease (14 )). For example, crystal studies of lactose permease complexed with lactose demonstrate the involvement of TM I, IV and V in sugar binding (257 ).

Figure 4.6: Amino acid sequence alignment and secondary structure of the transmembrane segments 4-5 in SGLT1 and vSGLT. The previously predicted topology of SGLT1 is shown on top. The secondary structure of vSGLT is shown on bottom.

86 In the region (143-180), vSGLT has a sequence similarity of 76% to all members of SGLT1 (Fig. 4.6). Therefore, it is possible that the topologies in this region surrounding predicted TMs 4-5 of vSGLT and SGLT1 are similar. Previous cysteine scanning mutagenesis (amino acids 162-173 of rabbit SGLT1) in Xenopus laevis oocytes indicated that this region might be α-helix with one face exposed to the extracellular aqueous environment (159 ). Molecular dynamics (MD) simulations at low dielectric constant that were carried out for a 42-residue peptide (amino acids 147-188 of human

SGLT1) also were consistent with the conclusion that the segment 162-173 has an α- helical conformation (251 ). Taken together, the present study along with previous biochemical characterization of SGLT1 (159, 162, 213, 214, 230 ), shows that segment

143-171 is part of TM 4 and that TMs 4-5 in the N-terminal half of SGLT1 form part of aqueous Na +- and sugar-binding cavity. Collectively, our results support the conclusion that the published crystal structure of vSGLT can be extended to mammalian SGLT1.

87 Chapter 5

Effects on conformational states of the rabbit sodium/glucose cotransporter through modulation of polarity and charge at glutamine

457

5.1 Summary

The high affinity sodium/glucose cotransporter (SGLT1) couples transport of Na + and glucose. 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. To explore the mechanism by which modulation of Q457 abolishes transport, Q457C and Q457R of rabbit SGLT1 were studied using chemical modification and the two-electrode voltage-clamp technique. Compared to WT SGLT1, Q457C exhibits ~20 fold reduction in phloridzin affinity and preferential occupancy of an inward–facing state. Alkylation of Q457C by [(2-trimethylammonium) ethyl] methanethiosulphonate bromide, (MTSET), reverses these changes while blocking transport. Analysis of pre-steady state currents in the absence of sugar, yields three decay constants for each of Q457C, Q457C-MTSET and Q457R. Comparison of Q457C-

88 MTSET and Q457R with Q457C and WT, reveals that inhibition of transport is accompanied by a decrease in magnitude and voltage-independence of the slow decay constant at negative potentials. But fast and medium decays remain unchanged.

Computer simulation of transient currents suggests that introduction of positive charge at position 457 leads to a predominant outward rather than inward-facing conformational state. Taken together, the 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.

5.2 Introduction

One polar residue at the carboxy-terminal part of TM XI (Q457 of human

SGLT1) has been extensively investigated and proposed to be essential for binding of sugar through hydrogen bond interactions with O1 and O5 of the pyranose ring (163, 185,

194, 207, 258 ). In addition, cysteine-scanning mutagenesis reveals that Q457R (a naturally occurring mutation in patients with glucose/galactose malabsorption) or Q457C reacting with thiol-reactive reagents (methanethiosulfonates and maleimides) abolishes sugar transport. However, under these conditions, it has been noted that the transporter still binds Na + and sugar (215, 240 ).

The objective of the present study, therefore, was to better understand the role of glutamine 457 in affecting sugar transport. Mutants Q457C and Q457R of rabbit SGLT1 were expressed in Xenopus laevis oocyte system and their functions were studied by the two-microelectrode voltage-clamp technique with a millisecond to second time scale.

Glutamine to cysteine mutation at residue 457 in rabbit SGLT1 causes ~20 fold reduction

in phloridzin affinity and decreases the relative charge contribution of Qdep , suggesting a

89 predominant inward-facing state. However, alkylation of cysteine 457 through chemical modification by MTSET reverses the changes caused by this mutation at residue 457, while at the same time, blocking sugar transport. Analysis of pre-steady state currents and computer simulation using a four state model shows that abolishing of sugar translocation correlates with an altered empty carrier transition state such that Q457C-MTSET is predominant in the outward-facing state. The results of the present study suggest that changes in polarity and charge at position 457 are associated with a minor modification of the orientation of the free carrier and a complete abolition of the translocation of the fully loaded carrier.

5.3 Results

5.3.1 Steady State Kinetics of Mutant Q457C of Rabbit

SGLT1

αMG The apparent affinity of mutant Q457C for sugar substrate αMG, ( K0.5 ), was determined by measuring αMG induced steady state currents at various sugar concentrations and over a range of holding potentials. The results were then fitted

αMG according to the Michaelis-Menten equation. As shown in Fig. 5.1A, the K0.5 for mutant Q457C exhibits more voltage-dependence from -150 mV to -30 mV, compared to

αMG WT rSGLT1. The K0.5 of mutant Q457C is 0.7 ± 0.1 mM ( V = -150 mV) and 1.5 ± 0.2

αMG mM ( V = -50 mV); while for WT, K0.5 is ~0.2 mM (from -150 mV to -50 mV).

Figure 5.1: (A) The αMG K0.5 of WT rabbit SGLT1 (n=3) and Q457C rabbit SGLT1

αMG (n=3) for voltage dependence. The error bars represent SEM. Q457C K 0.5 was 0.7 ±

0.1 mM ( V = -150 mV) and 1.5 ± 0.2 mM ( V = -50 mV), representing significantly

+ different values at -150 mV and -50 mV ( p < 0.05). (B) The Na K0.5 of WT (n=3) and

Na+ Q457C (n=5) for voltage dependence. The error bars represent SEM. Q457C K0.5 was

17.3 ± 2.6 mM ( V = -150 mV) and 26.2 ± 3.2 mM ( V = -30 mV), representing significantly different values at -150 mV and -30 mV ( p < 0.05).

91 To determine the Na + affinity of mutant Q457C, we measured αMG induced steady state currents (obtained at saturating 20 mM αMG) at various Na + concentrations over a range of holding potentials and the resulting curves were analyzed using the Hill

+ relationship (159 ). The Na apparent K0.5 of mutant Q457C exhibits relatively voltage- independent at hyperpolarizing membrane potentials from -150 mV to -80 mV and

Na+ voltage-dependent from -70 mV to -30 mV (Fig. 1B). The K0.5 of mutant Q457C is

17.3 ± 2.6 mM ( V = -150 mV) and 26.2 ± 3.2 mM ( V = -30 mV). The corresponding

Na+ values for “n”, the Hill coefficient, ranges from 1.0 to 2.0. The K0.5 of WT as previously determined is 8.3 ± 0.8 mM (V = -150 mV) and 41.9 ± 2.0 mM (V= -30 mV).

These results show that the glutamine to cysteine mutation at residue 457 in

SGLT1 causes ~4 fold reduction in αMG affinity ( V = -150 mV) and ~ 2 fold reduction in Na + affinity ( V = -150 mV).

5.3.2 Pre-Steady State Kinetics of Mutants Q457C and

Q457R of Rabbit SGLT1

Pre-steady state currents for mutant Q457C were obtained as a function of voltage

and integrated over 300 ms ( Vt) to calculate total charge ( Q) transferred by the cotransporter (V h was -50 mV, V t varied from -150 mV to +70 mV). The charge ( Q) was

then plotted as a function of the test pulses, and the Q(Vt) curves were fit to the two-state

Boltzmann equation. This protocol was carried out at 100 mM saturating Na + concentrations or 40 mM low Na + concentrations, and compared to WT rSGLT1.

Figure 5.2: (A) Comparison of the effects of 100 mM Na + and 40 mM Na + on charge transfer of mutant rabbit Q457C before and after exposure to MTSET (n ≥3). The error bars represent SEM. (B) Typical results demonstrating the effects on mutant Q457C charge transfer in oocytes treated with various 1 mM sulfhydryl-specific reagents

+ (MTSET) in 100 mM Na . The Q(V t) curves were adjusted to zero at hyperpolarizing voltages (-150 mV) and normalized with respect to the extrapolated Qmax . (C)

93 Comparison of the effects on maximal αMG-induced Na + currents measured at –150 mV in oocytes expressing of WT, or empty pMT4 plasmid before or after reaction with

MTSEA, or mutant Q457C before or after reaction with MTSET (n ≥3). The error bars represent SEM. The * shows significant difference compared with I max of mutant Q457C before or after reaction with MTSET at –150 mV ( p < 0.05). The # shows no significant difference compared with I max of empty pMT4 plasmid after reaction with MTSEA and mutant Q457C after reaction with MTSET at –150 mV ( p > 0.05). (D) Comparison of the

Na + leak currents measured at –150 mV in oocytes expressing of WT, or mutant Q457C before or after reaction with MTSET or mutant Q457R (n ≥3). The error bars represent

SEM. (E) The effect of sugar binding to mutant Q457C-MTSET on Q/V relations. The binding of sugar to mutant Q457C-MTSET can be studied from the effect of external sugar on the pre-steady state charge movement. Sugar shifted the V0.5 of the Q/V relation.

Normalized Q/V curves at 0 and 20 mM αMG. The V0.5 of the Q/V curve at 20 mM

αMG shifted ~44 mV. (F) The effect of sugar binding to mutant Q457R on Q/V relations.

Normalized Q/V curves at 0 and 10 mM αMG. The V0.5 of the Q/V curve at 10 mM

αMG shifted ~25 mV.

In the presence of Na + and no sugar, the distribution of the cotransporter in outward-facing conformational states can be explained by the difference in the relative

charge contribution of Qdep (214 ). For WT rSGLT1 we have previously shown that in 40 mM Na + over the range -150 to +70 mV, there is ~80% charge recovery compared to 100 mM Na + (213 ), suggesting that WT SGLT1 exists primarily in outward-facing conformational states (substrate binding site exposed to the extracellular side) (214, 259 ).

94 In mutant Q457C, however, there is less than 50% charge recovery in the presence of 40 mM Na + compared that in 100 mM Na + (Fig. 5.2A), suggesting preferential occupancy of the inward–facing state.

The mutation at residue 457 has altered voltage sensitivity (Table 5.1, Fig. 5.2B).

+ In 100 mM Na , the V0.5 values of mutants Q457C and Q457R are shifted to negative potentials compared to WT (-20.0 ± 0.9 mV, -6.0 ± 5.2 mV, and -1.5 ± 5.1 mV, respectively).

Transporter turnover, k, was calculated using the empirical values for steady state Imax

–1 and the pre-steady state Qmax . The turnover of mutant Q457C and WT are 7.8 ± 0.9 s ( n

= 3) and 9.8 ± 1.2 s –1 ( n = 4), respectively. Therefore, the turnover rate for the mutant

Q457C is decreased by ~20 % compared to WT.

WT Q457C Q457C-MTSET Q457R 100 mM Na + 100 mM Na + 40 mM Na + 100 mM Na + 40 mM Na + 100 mM Na + (n=5) (n=10) (n=6) (n=11) (n=3) (n=7) Qdep /Q max (%) 86 80 46 86 64 83 V0.5 (mV) -1.5±5.1 -20.0±0.9 -50.9±1.0 -0.9±0.5 -33.3±0.5 -6.0±5.2 dV (mV) 25.7±2.5 20.7±0.8 17.7±0.9 28.0±0.4 27.6±0.5 27.3±3.9 z 1.01 1.22 1.43 0.91 0.94 0.94

Table 5.1: The pre-steady state parameters for WT rSGLT1, mutant Q457C rSGLT1 before or after reaction with MTSET and mutant Q457R rSGLT1. The pre-steady state parameters were calculated by integrating the transient currents, and then fitted to a two- state Boltzmann relation.

95 5.3.3 Chemical Modification of Mutant Q457C of Rabbit

SGLT1 by MTS Reagents

We next investigated the functional consequence of alkylation of cysteine 457 through chemical modification by MTS reagents. Oocytes expressing mutant Q457C were exposed separately to positively charged MTS derivatives MTSET, MTSEA, and negatively charged MTSES, for 10 min in 100 mM Na +, with the membrane clamped at -

50 mV.

5.3.3.1 Effect of MTSET on voltage sensitivity and charge transfer of mutant Q457C

Fig. 5.2 shows the functional changes in Q457C after chemical modification by membrane-impermeant cationic MTSET. Representative normalized Q versus V curves obtained by integrating pre-steady state currents in an oocyte expressing WT, mutant

Q457C, mutant Q457C reacted with MTSET (Q457C-MTSET), or mutant Q457C reacted first with MTSET and then MTSEA. As shown in Fig. 5.2B, following exposure to MTSET, the V 0.5 of Q457C is shifted towards more positive potentials (V 0.5 = -0.9 ±

0.5 mV, n = 11), so that the Q/V curve for Q457C-MTSET nearly superimposes on that of WT. These results suggest chemical modification of Q457C by MTSET has reversed the changes in pre-steady state behavior caused by the glutamine to cysteine mutation

(see preceding section). These results are summarized in Table 5.1 where at 100 mM

Na +, the pre-steady state parameters of WT and mutant Q457C-MTSET are almost equivalent. Furthermore, when mutant Q457C in the presence of Na + is sequentially

96 reacted with MTSET, and then MTSEA, there is no additive effect on pre-steady state behavior (data not shown).

We have previously shown that in 40 mM Na + there is ~80% transferred charge recovery for WT and ~50% transferred charge recovery for mutant Q457C compared to

100 mM Na +. Alkylation of Q457C by MTSET substantially reverses the effect caused by the glutamine to cysteine mutation. The Q/V of mutant Q457C-MTSET in 40 mM Na + represent ~80% of the charge transferred in 100 mM Na + (Fig. 5.2A), suggesting an increase in occupancy of the outward-facing state.

5.3.3.2 Effects of MTSET on sugar transport and sugar binding of mutant Q457C

As observed previously for mutant Q457C hSGLT1 (163 ), alkylation of cysteine

457 in mutant Q457C rSGLT1 following exposure to MTSET results in inhibition of steady state αMG induced Na + currents (Fig. 5.2C).

Fig. 5.2D shows that the Na + leak of Q457C-MTSET is also reduced (about 65% of mutant Q457C). The Na + leak values of WT, mutants Q457C, Q457C-MTSET and

Q457R for rabbit SGLT1 are -47.8 ± 6.3 nA (n = 17), -57.6 ± 4.1 nA (n = 12), -37.4 ± 4.1 nA (n = 8) and –513 ± 68 nA (n = 7), respectively. Similarly, previous studies showed that alkylation of mutant Q457C with MTSEA (Q457C-MTSEA) for human SGLT also had relatively large Na + leak (Q457C and Q457C-MTSEA are ~131 nA and ~314 nA, respectively) (163 ).

The effect of the external αMG on pre-steady state charge movement can be used to study sugar-binding characteristics of mutants Q457C-MTSET and Q457R. As

97 described by Gagnon et al. (259 ), transporter specific pre-steady state currents were obtained by subtracting currents in the presence of 200 µM phloridzin from currents measured in the absence or presence of αMG. Although alkylation of Q457C by MTSET inhibits sugar transport, Fig. 5.2E reveals that addition of 20 mM αMG causes the V0.5 of mutant Q457C-MTSET to shift ~44 mV along the voltage axis. This result indicates that sugar binding of mutant Q457C-MTSET is still present. Fig. 5.2F reveals that addition of

10 mM αMG causes the V0.5 of mutant Q457R to shift ~25 mV along the voltage axis and also suggests that sugar binding of mutant Q457R is still present.

These results obtained for mutants Q457C-MTSET and Q457R rSGLT1 are consistent with those reported for mutants Q457C-MTSET and Q457R from human

SGLT1 (163, 215 ).

5.3.3.3 Effect of MTSES or MTSEA on mutant Q457C

MTSES altered pre-steady state currents such that at both depolarizing (+70 mV) and hyperpolarizing (-150 mV) potentials there was no saturation of charge transfer and it was not possible to obtain a fit of the data to a two-state Boltzmann relation. MTSEA caused a marked increase in Na + leak substantially reducing the observable charge transfer associated with cotransporter activity (data not shown). As a consequence of these findings, the effects of chemical modification by MTSES and MTSEA were not further analyzed.

Figure 5.3: Estimation of the phloridzin K0.5 of rabbit Q457C and Q457C-MTSET for voltage dependence with the transferred charge (n ≥3). Voltage dependent transferred charge in the presence of various concentrations of phloridzin was fit to the Hill relationship. The error bars represent SEM.

5.3.4 Phloridzin Affinity

The specific inhibitor, phloridzin, binds to SGLT1 and blocks sugar translocation

(164 ). As described in Methods, the ability of phloridzin to reduce transient charge

99 movement of SGLT1 can been used to measure its apparent affinity for the co-transporter

(164, 230, 259, 260 ). As shown in Fig. 5.3, the phloridzin K0.5 of Q457C is 30.7 ± 0.7

µM, indicating that the glutamine to cysteine mutation at 457 has reduced phloridzin affinity by ~20-fold, compared to WT (1.4 ± 0.2 µM, data not shown). When a positive charge is introduced at position 457, by reacting cysteine 457 with MTSET, however, the phloridzin K0.5 of mutant Q457C is 4.5 ± 0.1 µM, indicating that alkylation with MTSET

(Q457C-MTSET) has rescued the phloridzin affinity of mutant Q457C (which is now reduced by ~3-fold, compared to WT).

5.3.5 Decay Constants of Q457C of Rabbit SGLT1

A simple six-state model has been used to explain the kinetics of SGLT1 function in a limited time range (Fig. 5.4A) (240 ). States C 1 and C 6, C 2 and C 5, and C 3 and C 4

+ + represent the empty [C], Na -bound [CNa 2], and Na - and sugar-bound conformations

[SCNa 2] of the cotransporter at the external and internal membrane surfaces.

The pre-steady state currents in an oocyte expressing SGLT1 consist of a nonspecific component (due to oocyte membrane capacitance) and an SGLT1 specific component. To isolate SGLT1 specific currents, the well-known non-transported sugar inhibitor, phloridzin, is used to block SGLT1 activity. Then, recorded currents acquired in the presence of saturating phloridzin (200 µM) are subtracted from currents acquired in the absence of phloridzin, to provide the currents due exclusively to SGLT1.

100

Figure 5.4: The kinetic models for SGLT1 in the presence of sugar or absence of sugar.

(A) A simple six-state model for SGLT1 with sugar in a certain time range (163 ). The

+ shaded region represents the voltage-dependent step: Na binding/dissociation C2↔C1; orientation of empty carrier between inward-facing state and outward-facing state

C1↔C6. (B) A four-state model of SGLT1 for pre-steady state currents in the absence of sugar (157, 158 ), which added an intermediate empty carrier conformational state ( C1a ) between C1 and C6. (C) In the absence of sugar, a four-state model of SGLT1 for charge movement, decay constants ( τ) and rate constants ( k).

101 In the presence of Na + and absence of sugar, using the two-electrode voltage clamp technique and the phloridzin subtraction protocol with sufficient duration of voltage pulse (150 ms), there is now evidence or an additional intermediate empty carrier conformational state ( C1a ) (Fig. 5.4B, C) (157, 158 ). Thus, it is now believed that reorientation of empty carrier from an inward to outward-facing state occurs via two transitions, C6↔C1a , C1a ↔C1. The cotransporter is in state C 2 at large hyperpolarizing

+ voltages, and at C 6 at large depolarizing voltages. In the presence of Na and absence of sugar, the 4 transporter transition states are characterized by three time constants (158 ).

Accordingly, the pre-steady state transient currents of mutants Q457C and

Q457C-MTSET were fitted to obtain first-, second-, and third-order decays. Fig. 5.5 shows the fit to the pre-steady state currents for mutants Q457C (Figs. 5.5A, B) and

Q457C-MTSET (Figs. 5.5D, E), using first- and second- order exponential decays. The nonrandom regions in the first 25 ms (Figs. 5.5B, E) demonstrate inadequacy of the fit with second- order exponential decays. The residuals of the third-order exponential decay

(Figs. 5C, F) exhibit random oscillations about the zero axis, indicating best fit of the data with three exponential decay components for the pre-steady state currents of mutants

Q457C and Q457C-MTSET. These results suggest that the pre-steady state currents of mutants Q457C and Q457C-MTSET can also be represented by three decay constants ( τf, the fast decay constant; τm, the medium decay constant; and τs, the slow decay constant), consistent with those reported for WT rabbit SGLT1 (158 ).

102

Figure 5.5: The fit residuals for mutant rabbit Q457C before or after reaction with

MTSET. First- (A) or (D), second- (B) or (E), and third- (C) or (F) order fit residuals for the –130 mV pre-step, 10 mV post-step potential transient.

103 Figs. 5.6A, B, C show the relationships between decay constants and voltage.

Inspection of Fig. 5.6 reveals that in the presence of 100 mM Na +, the three decay constants for WT (158 ) and mutant Q457C are similar both in magnitude and voltage dependence. The fast decay constant of mutant Q457C ( τf, 0.5–1.1 ms) is relatively voltage-independent with a range between 0.5 ms and 1.1 ms (Fig. 5.6A). The medium decay constant of mutant Q457C ( τm, 1.2–6.5 ms) has a voltage-dependence and increases from ~1.2 ms at hyperpolarizing potentials to ~6.5 ms at depolarizing potentials

(Fig. 5.6B). The slow decay constant of mutant Q457C ( τs, 60–15 ms) has a sigmoid- shaped voltage-dependence and decreases rapidly from ~60 ms at hyperpolarizing potentials to ~15 ms at depolarizing potentials (Fig. 5.6C).

+ + At low Na concentrations (40 mM Na ), both the fast decay constant ( τf, 0.9–1.3 ms) and the medium decay constant ( τm, 1.4–5 ms) are only slightly altered (Figs. 5.6A

and B). But the slow decay constant ( τs, 100–6.5 ms) is significantly slower at potentials more hyperpolarizing than -50 mV and faster at potentials more depolarizing than -50 mV

(Fig. 5.6C).

[Na +] The fast decay The medium decay The slow decay (mM) constant ( τf) constant ( τm) constant ( τs) rWT 100 0.5–1 ms 0.5–4 ms 50–8 ms rQ457C 100 0.5–1 ms 1.2–6.5 ms 60–15 ms 40 0.9–1.3 ms 1.4–5 ms 100–6.5 ms rQ457C-MTSET 100 0.5–1.1 ms 1.2–6 ms ~30 ms rQ457R 100 0.5–1.4 ms 1.2–6 ms ~16 ms

Table 5.2: Summary of the time constants for WT and mutants

104 The decay constants for mutants Q457C-MTSET and Q457R are illustrated in

+ Figs. 5.6D, E, F. In the presence of 100 mM Na , the fast decay constant ( τf, 0.5–1.4 ms) and the medium decay constant ( τm, 1.2–6 ms) for both Q457C-MTSET and Q457R are similar to that of WT and mutant Q457C (Figs. 5.6D and E). However, the slow decay constant ( τs) of Q457C-MTSET and Q457R are substantially changed. Both exhibit voltage-independence from –150 mV to 0 mV, with reduced magnitudes of ~30 ms and

~16 ms, respectively. At depolarizing voltages ( ≥ 20 mV), the slow component exhibits very low amplitude of the transient currents (small signal-to-noise ratio), precluding our obtaining estimates of τs for mutants Q457C-MTSET and Q457R over this voltage range.

The slow decay constants of mutants Q475C-MTSET and Q457R are reduced in magnitude (i.e. faster) at potentials more hyperpolarizing than -50 mV and increased in magnitude (i.e. slower), at potentials more depolarizing than -50 mV, compared to the slow decay constant of mutant Q475C (Compare Fig. 5.6C and Fig. 5.6E). When the Na + concentration is reduced from 100 mM to 40 mM, a condition in which Q457C exists preferentially in an inward facing conformational state, we observe a marked slowing of

τs compared to mutants Q475C-MTSET and Q457R (Fig. 5.6C). Collectively, these results suggest that addition of positive charge at position 457 significantly increases return of empty carrier from inward-facing state to outward-facing state.

Table 5.2 summarizes the decay constants for WT in 100 mM Na +, mutants

Q457C in 100 and 40 mM Na +, Q457C-MTSET in 100 mM Na + and Q457R in 100 mM

Na +.

105

Figure 5.6: Voltage- and Na +-dependence of the three decay constants (n ≥3). For WT in

+ + 100 mM Na () and mutant Q457C in 100mM ( ) and 40 mM Na ( ), ( A) τf, fast decay. ( B) τm, medium decay. ( C) τs, slow decay. For mutants Q457C-MTSET in 100

+ + mM Na ( ) and Q457R in 100 mM Na ( ), (D) τf, fast decay. (E) τm, medium decay.

(F) τs, slow decay.

106 5.3.6 Model Simulations

Computer model simulations were carried out based on a four-state model (Fig.

5.4B) proposed by Chen et al (157 ) and Krofchick et al (158 ), to help evaluate the functional differences between WT and mutant Q457C before and after reaction with

MTSET. The top panels of Fig. 5.7 A and B show the simulated pre-steady state currents and charge transfer using the best-fit parameters in Table 5.3. The bottom panel of Fig.

5.7 A and B show the three decay constants plotted as a function of voltage and compared to the experimentally derived τ values in Fig. 5.6. As shown in the middle panels of Fig. 5.7 A and B, the ON and OFF charges in the experiments are not equal.

While the reason for this phenomena is unknown, similar results have been shown by

Loo et al. (261 ). One possibility is that these currents do not represent pure displacement currents (i.e. currents due to the displacement of charged residues attached to the transporter). Fig. 5.7 reveals good agreement between model simulations and experimental data for mutants Q457C and Q457C-MTSET for pre-steady state ON currents, the medium decay constant, and the slow decay constants. In contrast, Fig. 5.7 reveals a difference between model simulations and experimental data for pre-steady state OFF currents and the fast decay constants. The model proposed with the parameters shown in Table 5.3 represents fairly well the “ON” data for mutants Q457C and Q457C-

MTSET. The reason for the poor fit to the “OFF” data is unknown but it clearly indicates that the real mechanism responsible for the transient current recorded is more complex than the proposed model.

107

108

Figure 5.7: Simulated transient currents, charge transfer and decay constants in 100 mM Na + were predicted by the model solution in Table 3. Predictions of the kinetic model superimposed on the experimental data for mutant Q457C rabbit SGLT1 (A) and mutant Q457C exposed to MTSET rabbit SGLT1 (B). The top panel shows simulated currents (gray line ) at the potentials of –130 mV and +50 mV superimposed on the experimental current ( black line ). The middle panel illustrates the Q-V curves. The bottom panel shows three decay constants ( τf, the fast decay constant; τm, the medium decay constant; and τs, the slow decay constant) as a function of voltage.

109 A summary of representative model parameters for mutants Q475C and Q475C-

MTSET derived from model simulations is shown in Table 5.3. Comparing the parameter values for WT and mutant Q457C (Table 5.3), the ratio for transitions C1a ↔C6 are

+ similar. But the ratio for transition C2 ↔C1, the Na dissociation rate k21 / k 12 , is altered

(0.37 for WT versus 0.94 for mutant Q457C), which correlates with the observed

Na+ decrease in K 0.5 of Q457C. Reacting mutant Q457C with positively charged MTSET,

+ partly restores the Na dissociation rate k21 / k 12 (0.75 for Q457C-MTSET versus 0.94 for

Q457C). Computer simulation also reveals that the rate observed for empty carrier transition from outside to inside, k1 1a / k1a 1 , decreases from 4.2 for WT to 2.2 for mutant

Q457C-MTSET. This suggests that there is a faster rate for reorientation of empty carrier back to the outside facing state and correlates with the increase in Qdep / Q max for the inward/outward-facing distribution and abolishing of sugar translocation.

-1 (s ) k21/ k1 1a / k1a 6 / k k k Z Z Z Z Z Z 12 1a 1 6 1a k21 k12 k1 1a k1a 1 k1a 6 k6 1a 21 12 1 1a 1a 1 1a 6 6 1a WT 66 180 100 24 330 380 0.44 0.69 0 0 0.31 0.2 0.37 4.2 0.87 (158 ) Q457C 150 160 80 24 750 850 0.3 0.7 0.8 0 0.17 0.07 0.94 3.3 0.88 Q457C- 90 120 80 36 750 850 0.3 0.5 0.8 0 0.25 0.2 0.75 2.2 0.88 MTSET Table 5.3: Rate constants of a four-state kinetic model used for the pre-steady state current simulations of WT rabbit SGLT1 and mutants in 100 mM Na +.

110 5.4 DISCUSSION

In human SGLT1, mutant Q457R or mutant Q457C exposed to thiol-reactive reagents (methanethiosulfonates and maleimides), abolishes sugar translocation.

However, under these conditions, transporter still binds Na + and sugar (163 ). The sugar

(αMG) affinities of mutants Q457E (polar to negative) (194 ), Q457C (polar to neutral)

(194 ) and Q457R (polar to positive) (120, 215 ) are reduced by ~4, ~7 and ~10–fold respectively, compared to WT, suggesting that the position 457 is quite tolerant to charge.

Diez-Sampedro et al. (194 ) compared the kinetics of transport of glucose analogues (each modified at one position of the pyranose ring) for hSGLT1, Q457C and Q457E and then proposed that Q457 was essential for binding of sugar through hydrogen bond interactions with O1 and O5 of the pyranose ring.

Because Q457 human SGLT1 is tolerant to charge, and still binds to sugar in the absence of transport, it seemed to us that Q457 is more important for translocation of sugar. To further investigate this issue, mutants Q457C and Q457R in rabbit SGLT1 were characterized using the two-electrode voltage-clamp technique. On the one hand our experimental findings indicate similar functions of rabbit and human isoforms following a glutamine to cysteine mutation (Q457C) with regard to decreased apparent affinity for

αMG and Na +. Also in both instances Q457R or Q457C reaction with MTSET abolishes sugar transport, leaving binding of Na + and sugar intact. But the results of the present study also provide new insights into the functional significance of glutamine 457 in rabbit

SGLT1. Specifically our findings support the hypothesis that loss of sugar transport in the mutants Q457C-MTSET and Q457R of rabbit SGLT1, can be explained by changes in

111 the conformational equilibrium of the transport cycle with accumulation of transporter in an outward-facing state. The evidence supporting this hypothesis is discussed below.

Our data show that most of the transporters remain in a non-transporting outward- facing state as a consequence of reacting Q457C rabbit SGLT1 with MTSET. Therefore, the co-transporter under these conditions is more available for binding of phloridzin, and the substantial alteration in phloridzin affinities of mutants Q457C and Q457C-MTSET

(Fig. 5.3) can be explained on the basis of a changed equilibrium between inward- and outward-facing conformational states. Changes in the equilibrium between inward- and outward-facing conformational states and altered substrate/inhibitor affinity have been observed for other transporters. For example, mutant D176A rabbit SGLT1 exhibits an increased rate of empty carrier transition from outside to inside, which is accompanied by a decreased apparent affinity for phloridzin (227 ). Mutants K264A, Y335A and D345A alter the conformation of the dopamine transporter and result in changed apparent affinities for inhibitors (262 ). Mutant M345H in the γ-aminobutyric acid transporter-1

(that belongs to a large family of Na +/Cl –-coupled neurotransmitter transporters) shifts the transporter toward the outward-facing Na +-bound conformation, resulting in an increased apparent affinity for Na + (263 ). Finally, mutant T349H in the γ-aminobutyric acid transporter-1 shifts the transporter toward the inward-facing empty conformation causing a decrease in the apparent affinity for Na + (263 ).

A common behavior observed for ion-coupled co-transporters is that empty carrier will orientate from internal to external membrane surface during the initial transport step and is associated with charge movement (242, 249, 264 ). For example, Loo et al. (163, 185, 258 ) studied the fluorescence changes of tetramethylrhodamine-6-

112 maleimide-labeled human Q457C (Q457C-TMR6M) under voltage clamp, and suggested the major voltage-dependent step in the SGLT1 transport cycle was the return of the empty carrier from inward-facing to outward-facing states. Also, based on a four-state model for SGLT1, charge movements were associated with conformational transitions of sodium binding/debinding (C1↔C2) and reorientation of the unloaded protein across the membrane ( C1↔C1a ↔C6) (Fig. 5.4B, C) (158 ).

Thus, SGLT1 pre-steady state currents provide direct insight into the conformational changes that accompany rearrangements of charges within the protein. In particular, the slow decay constant reflects the rate limiting transition of the empty carrier

(218 ). However there is some discrepancy in the results for Q457C reacted with MTS and maleimides from earlier investigations. Previous studies recording charge movement reported that the slow decays ( τs) of mutant Q457C-TMR6M human SGLT1 (185 ) is voltage- and Na +- dependent, whereas the results from recording fluorescence changes seem to indicate that the slow decay ( τs) of mutant Q457C-TMR6M human SGLT1 is voltage- and Na +- independent. These differences may be due to the different experimental protocols used and the very low amplitude of the transient currents exhibited by the slow component (small signal-to-noise ratio). The pre-steady state currents for slow component of mutant Q457C-TMR6M human SGLT1 was less than 20 nA between –150 mV and +50 mV, compared to ~200 nA for total components of mutant

Q457C-TMR6M human SGLT1 (185 ). The fluorescence level for the controls, TMR6M- labeled human SGLT1 Q457C expressing oocytes was 187±15 au and 229±25 au, respectively (258 ). The ∆F for slow component of mutant Q457C-TMR6M human

SGLT1 was 0.36 au at –190 mV and 0.38 au at +90 mV (185 ).

113 In the present study, to resolve the experimental difficulty of dealing with small signals for the slow component, we took advantage of the modified OFF protocol (158 ) that yields adequate currents at large voltage jumps, to obtain estimates of τs for mutants

Q457C-MTSET and Q457R of rabbit SGLT1. Our experimental data and the computer simulations based on a four-state model lend further support to the conclusion that addition of positive charge at position 457 significantly slows return of empty carrier from an outward-facing to inward-facing state. It is interesting that similar results have been found for three other SGLT1 mutants- Q170E rabbit SGLT1 (214 ), C255A human

SGLT1 (259 ) and C511A human SGLT1 (259 ). In each case, the major changes identified in computer simulations are that the rate of empty carrier movement from outside to inside ( k1 1a / k1a 1 ) is decreased, resulting in a greater number of transporters in the outward-facing state (214, 217, 259 ).

114

+ Figure 5.8: Occupancy probability ( Ci) in 100 mM Na as a function of time as calculated by the four-state kinetic model for WT rabbit SGLT1 and mutant Q457C before or after exposed to MTSET. (A) Time course of WT occupancy probabilities for a Vm pulse from

+70 mV to –150 mV. (B) Time course of mutant Q457C occupancy probabilities for a Vm pulse from +70 mV to –150 mV. (C) Time course of mutant Q457C exposed to MTSET occupancy probabilities for a Vm pulse from +70 mV to –150 mV. (D) Time course of

WT ( solid line ), Q457C ( dotted line ) and Q457C-MTSET ( dashed line ) C2 occupancy probability for a Vm pulse from +70 mV to –150 mV. (E) Time course of WT ( solid line ),

Q457C ( dotted line ) and Q457C-MTSET ( dashed line ) C1a occupancy probability for a

Vm pulse from +70 mV to –150 mV.

115 The occupancy probability ( Ci) as a function of time as calculated by kinetic model for SGLT1 has been used to evaluate the functional differences between WT and mutants (164, 183, 259 ) of rabbit SGLT1. Based on a four-state model (Fig. 5.4B), computer model simulations for occupancy probability ( Ci) as a function of time as calculated by the four-state kinetic model for WT rabbit SGLT1 and mutant Q457C before or after exposure to MTSET in the presence of 100 mM Na + and absence of αMG, could help us to evaluate the functional differences between WT and mutant Q457C before and after exposure to MTSET. Fig. 5.8 shows the occupancy probabilities for a voltage step from the most depolarizing potential (+70 mV; most of the transporters will stay in the inward-facing states) to the most hyperpolarizing potential (-150 mV; most of the transporters will stay in the outward-facing states). The transition, C1a →C1 is clearly related to the slow component of the experimental transient currents. At depolarizing potential (+70 mV), the starting probabilities for WT and Q457C-MTSET are similar ( C6 is ~80% and C1a is ~20%; Fig. 5.8A and C) and different with the starting probabilities for Q457C ( C6 is ~70% and C1a is ~30%; Fig. 5.8B). At hyperpolarizing potential (-150 mV), the rate of Q457C-MTSET passing through the slow transition ( C1a ; Fig. 5.8E) and reaching the outward-facing state ( C2; Fig. 5.8D), is faster than those of WT and Q457C.

The occupancy probability for WT, Q457C and Q457C-MTSET is consistent with the experimental slow decay constants.

+ Fig. 5.9 shows the variability of the occupancy probabilities ( Ci) in 100 mM Na with voltage. The occupancy probabilities of predominant state ( C2) at the extreme negative voltage (-150 mV) are 97%, 97% and 94% for WT, Q457C and Q457C-MTSET of rabbit SGLT1, respectively. The occupancy probabilities of predominant state ( C6) at

116 the extreme positive voltage (+70 mV) are 76%, 68% and 79% for WT, Q457C and

Q457C-MTSET of rabbit SGLT1, respectively. For WT rabbit SGLT1, the occupancy probabilities of C2, C1, C1a and C6 at –50 mV based on the decay constants in Table 5.3 are 26%, 10%, 43% and 21%, suggesting that 36% of transporters are in an outward- facing free or Na +-bound state. However, based on a five-state kinetic model, the occupancy probabilities of WT human SGLT1 in a free or Na +-bound state at –50 mV are

73% (157, 217, 259 ). The discrepancy between these two sets of observations is due to the different values of V 1/2 for WT human SGLT1 (-46 ± 3 mV) (217 ) and WT rabbit

SGLT1 (-2.5 ± 0.7 mV) (159 ). For mutant Q457C rabbit SGLT1, the occupancy probabilities of C2, C1, C1a and C6 at –50 mV are 26%, 34%, 23% and 17%, suggesting that 60% of transporters are in a free or Na +-bound state. For mutant Q457C rabbit

SGLT1 exposed to MTSET, the occupancy probabilities of C2, C1, C1a and C6 at –50 mV are 28%, 43%, 20% and 9%, suggesting that 71% of transporters are in a free or Na +- bound state. The occupancy probability for WT, Q457C and Q457C-MTSET is consistent with the hypothesis that Q457C-MTSET preferentially occupies an outward– facing state.

Gagnon et al. (217 ) found a disulfide bridge between C255 and C511 of human

SGLT1, and were the first to quantitatively study the dose-dependent effect of αMG on the pre-steady state currents of these mutants as well as for WT human SGLT1 (259 ).

They also proposed a five-state kinetic model to quantitatively explain the effect of αMG on the pre-steady state currents. They found that the reorientation of free transporter was the slowest step for WT human SGLT1 either in the presence or in the absence of αMG.

117

+ Figure 5.9: Simulation predictions on occupancy probabilities ( Ci) in 100 mM Na as a function of voltage as calculated by the four-state kinetic model for WT rabbit SGLT1

(A), mutant Q457C (B) and Q457C exposed to MTSET (C).

In present study, we suggest that modulation of charge and polarity of glutamine

457 in rabbit SGLT1 likely influences sugar translocation by affecting reorientation of the empty carrier from one side of the membrane to the other side. Because no steady state currents could be measured for mutants Q457C-MTSET and Q457R of rabbit

SGLT1, we could not study the mutants’ effect on reorientation of the fully loaded carrier from one side of the membrane to the other side.

Since our results show an outward-facing preference of the empty carrier, together with the findings from Gagnon et al, it seems reasonable to speculate that modulation of

118 charge and polarity of glutamine 457 likely influences sugar translocation by affecting reorientation of the fully loaded carrier from one side of the membrane to the other side.

In summary, the experimental and computer simulation data may provide a better understanding of the relationships for charge/conformational change and translocation at the position 457 in rabbit SGLT1. As an explanation for the altered conformational equilibrium, it is possible that mutants Q457C-MTSET and Q457R may disrupt the intramolecular interactions involved in stabilizing the transporter in the inward facing conformation and that this results in an impaired ability of the transporter to return to this conformation. Therefore, the transporter may accumulate in the outward facing conformation. Taken together, in addition to its involvement in sugar binding (194, 207 ), our results suggest that modulation of charge and polarity of glutamine 457 in rabbit

SGLT1 is likely associated with a minor modification of the orientation of the free carrier and a complete abolition of the translocation of the fully loaded carrier.

119 Chapter 6

Structural implications of structure-function studies in SGLT1

The major goal of structure-function studies is to identify the crucial sites and the common slippage/cotransport pathway for substrates. It would seem reasonable to expect that proper integration of substrate binding and translocation functions would be reflected structurally. Although no definitive structural data of SGLT1 is currently available, functional information is beginning to provide some insights.

1) Our data show several functionally important residues for substrate and cation recognition are located in the amino-terminal half of the transporter. Lysine 157 and threonine 156 in TM 4 (conserved in all members of both SGLT1 and SGLT2) participate in sugar binding (230, 265 ). Mutation of lysine 157 to cysteine (K157C) causes loss of phloridzin and αMG binding ( α-methyl-D-glucopyranoside). These functions are restored by chemical modification with positively charged (2-aminoethyl) methanethiosulphonate hydrobromide (MTSEA). Mutation of threonine 156 to cysteine

(T156C) reduces the affinity of αMG and phloridzin for T156C by ~5–fold and ~20-fold, respectively. Sodium affinity of mutant T156C is modestly reduced at hyperpolarizing potentials, unchanged at the holding potential, and relatively insensitive to voltage.

Na +/D-glucose stoichiometry is unchanged at 2:1. T156 is located more hydrophobically than K157. Neither membrane-impermeant MTSET, nor MTSES, modify T156C function. αMG induced Na + current was abolished after T156C was labeled with

120 membrane-permeant MTSEA. In addition phloridzin protects cysteine 156 in T156C from alkylation by MTSEA. Our group also identified that the reaction of MTS reagents with F163C, A166C, and Q170C in the putative loop joining TM 4 significantly decreased the sugar transport to 20% -50% of its original value (159 ). There is evidence that A166 is important for the interaction between the Na + and sugar pathways and is close to TMs 10-11 (241 ). All of these results suggest that TM 4 is close to the sugar binding-site.

2) Alkylation of Q457C in TM 11 is prevented in the presence of 20 mM aMG, or

100 uM phloridzin. This protection afforded by sugar and phloridzin, indicates that the

Q457 site is at or near the sugar-binding site. Further evidence supporting such a conclusion comes from experiments in which Q457C hSGLT1 is labeled with a fluorophore, and then exposed to sugar. Under these conditions, sugar induces changes in fluorescence (258 ), indicating that residue 457 lies at or near the sugar binding site. The experimental and computer simulation data from our lab suggest that modulation of charge and polarity of glutamine 457 in rabbit SGLT1 is likely associated with a minor modification of the orientation of the free carrier and a complete abolition of the translocation of the fully loaded carrier (266 ).

Collectively these functional data suggest that position 457 and 157 are in close proximity and that TMs 4 and 11 are close to the substrate binding/translocation pathway.

Alkylation of cysteine in T156C (230 ) and Q457C (163 ) profoundly increases the Na + leak, is suggesting that Na + and sugar enter a common permeation pathway.

We also summarize other important residues and domains that play critical roles in the SGLT1 binding/transport mechanism as follows.

121 1) Application of the substituted cysteine accessibility method (SCAM) has shown that the putative external loop joining TM 4-5 (residues 163, 166, 170, and 173) in the N-terminal half of SGLT1 is involved in Na + interaction (2, 214, 235, 236 ).

2) Panayotova-Heiermann et al (227 ) have shown that phloridzin (176) recognition sites are located in the putative external loop joining TMs 4-5 in the N- terminal half of the transporter. They also suggest that K321 in TM 8 play a major role in the Na + and sugar binding during cotransporter activity (210 ).

3) Puntheeranurak et al (182 ) expressed rSGLT1 in COS-7 and G6D3 cells and found that mutants C255 (in the putative external loops joining TMs 6-7) and C608 (the putative external loops joining TMs 13-14) formed a disulfide bridge. These results suggest that the putative external loops joining TM 8-9 are involved in sugar binding.

Gagnon et al (217, 218 ), using the Xenopus laevis oocytes expression system, showed that mutants C255 and C511 form a disulfide bridge, suggesting the putative loop TMs 6-

7 is close to TMs 11-13. Despite the discrepancy between these two sets of observations

(which might be due to the different experimental systems used), both studies suggest that the putative loop TMs 6-7 also participates as part of the extracellular binding pocket for sugar.

4) D454 in TM 11 does not form part of either the sugar and Na + binding sites, but is involved in the coupling reaction (200 ).

5) A chimera made by substituting the putative external loop joining TMs 12-13 of Xenopus SGLT1-like protein (xSGLT1L) with the homologous region of rabbit

SGLT1 suggests that the putative external loop joining TMXII-XIII participates in the sugar transport of SGLT1 (267 ).

122 A

Figure 6.1: (A) A proposed structural model viewed from the extracellular side for substrate binding in SGLT1. The positions of key residues (T156, K157, F163, A166,

Q170, L173, C255, D454, Q457 and C511) are indicated. The highlighted TM 4 and TM

11 are the key domains for substrate binding and translocation as described in the text.

(B) Structure of vSGLT viewed from the intracellular side with the central helices (1).

123 Based on these considerations we propose a limited structural model, depicted in

Fig. 6.1A, 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 common permeation pathway, analogous to all of the known crystal structures of ion-coupled transporters (the Na +/leucine transporter (268 ), Na +/aspartate transporter (269 ) and lactose permease (14 )). The substrate binding sites of those transporters are located in a hydrophilic cavity formed by multiple transmembrane helices. In Fig. 6.1A, TMs 4-7 and

TMs 10-13, form a hydrophilic cavity, which is involved in substrate binding and translocation.

This SGLT1 limited structural model is supported by the 3 Å crystal structure of bacterial SGLT ( Vibrio parahaemolyticus SGLT, vSGLT) that was published recently

(Fig. 6.1B) (1). The 3D structure of vSGLT, a homolog of SGLT1, was recently determined and provides important insights into the structure/function of this bacterial co-transporter. Both SGLT1 and vSGLT belong to the solute sodium symporters family

(145, 270 ), have a sequence similarity of 60%, contain 14 transmembrane domains and share an alternating-access mechanism with tight coupling between sodium and solute transport. Although there are significant functional differences between vSGLT and mammalian SGLT1 (the Na +/galactose stoichiometry is 1:1 and the Na +/glucose stoichiometry is 2:1, respectively), it is informative to explore how much of the information obtained from vSGLT structure can be extrapolated to SGLT1. The crystal structure of vSGLT contains a hydrophilic core structure formed by multiple transmembrane helices from both N-terminus (TMs 2-6) and C-terminus (TMs 7-11). Na + and galactose are bound in the center of the core.

124

Figure 6.2: Amino acid sequence alignment and secondary structure of the transmembrane segments 2-11 in vSGLT, hSGLT1, NIS and PutP. The secondary structure of vSGLT is shown on top. The positions of functionally important residues in SGLT1 (T156,

K157, F163, A166, Q170, L173, C255, D454, and Q457) are indicated as circles.

125 The theme of N- and C-terminal segments participating in formation of a common cavity for Na + and co-transported substrate is also emerging from studies of other ion coupled co-transporters, such as the Na +/leucine transporter (253 ), Na +/aspartate transporter (254 ), Na +/glutamate transporter (255 ), Na +/H + antiporter (256 ) and lactose permease (14 )). For example, crystal studies of lactose permease complexed with lactose demonstrate the involvement of TM I, IV and V in sugar binding (14 ).

In the region (155-173), vSGLT has a sequence similarity of 68% to all members of SGLT1 (Fig. 6.2). Therefore, it is possible that the topologies in this region surrounding predicted TM 4 of vSGLT and SGLT1 are similar. Based on previous cysteine scanning mutagenesis (amino acids 155-173 of rabbit SGLT1) in Xenopus laevis oocytes (159, 162, 213, 214, 230, 265 ) and molecular dynamics (MD) simulations at low dielectric constant (251 ), our results support the conclusion that the published crystal structure of vSGLT can be extended to mammalian SGLT1. Therefore, we speculate to indicate the positions of residues in SGLT1 (156, 157, 163, 166, 170, 173, 321, 454, and

457) with circles and correspond with residues in vSGLT based on the amino acid sequence alignment of SGLT1 and vSGLT (Fig. 6.3).

Taken together our study along with previous biochemical characterization of

SGLT1 (Fig. 6.1A) and crystal structure of vSGLT (Fig. 6.1B), we propose that TM 4 in the N-terminal half of SGLT1 and TM 11 in the C-terminal half of SGLT1 form part of aqueous Na +- and sugar-binding cavity. The functionally important residues in SGLT1

(T156 and K157 in TM 4, D454 and Q457 in TM 11) are close to sugar binding sites.

126

Figure 6.3: Topology of vSGLT. The trapeziums represent the inverted topology of TMs

2 –6 and TMs 7-11. Based on the amino acid sequence alignment of SGLT1 and vSGLT, the positions of residues in SGLT1 (156, 157, 163, 166, 170, 173, 321, 454, and 457) are indicated with circles and correspond with residues in vSGLT.

In summary, for years, it has been accepted that sugar-binding involved only the last three TM segments of SGLT1. My work, however, disproved this by showing that the N-terminal half of SGLT1 is involved in sugar substrate binding. My work, together with previous works, provides some novel structural insights, suggesting that the N- terminus and C-terminus combine together via TM 4 and TM 11, to establish a common substrate permeation pathway for glucose and Na +. Further mutagenesis and cross-linking studies have been initiated to extend our understanding of sugar and Na + binding/translocation, and transmembrane helix packing.

127 Chapter 7

Future directions

7.1 Background overview

The high affinity sodium/glucose co-transporter (SGLT1) belongs to the homologous family of Na +/solute symporters (SLC5), and is a secondary active transporter that utilizes the sodium electrochemical gradient to transport sugar substrates uphill against a concentration gradient (56 ). SGLT1 is expressed most abundantly at the mucosal surface of the small intestine but is also present in the S3 segment of the proximal tubule (74, 232-234 ). SGLT1, located in an apical site of the small intestinal epithelia, serves as the principal uptake pathway for glucose derived from dietary sources. Mutations that result in dysfunctional SGLT1 affect intestinal glucose/galactose absorption (120 ). Recently, SGLT1 has been a target protein for diabetes treatment (Fig.

7.1) (250, 271 ). The transporter functions as a monomer with 14 transmembrane domains and exhibits a stoichiometry of 2 Na + ions : 1 sugar molecule (59 ) (Fig. 1.4).

The low affinity sodium/glucose co-transporter (SGLT2) is expressed in the S1 segment of the proximal tubule and has a sequence identity of 59% with SGLT1 (133 ).

Although SGLT1 is expressed in the S3 segment of the proximal tubule, SGLT2 mediates the majority of renal glucose reabsorption. Blood glucose in the circulation is continuously filtered in the glomeruli of the kidneys and then reabsorbed in the renal proximal tubules via SGLT2 and to a lesser extent via SGLT1. Mutations that result in dysfunctional SGLT2 cause a rare renal tubular disorder (familial renal glucosuria, FRG)

128 (272 ). Type II diabetes (T2D) is currently a worldwide epidemic and an estimated 246 million people world have T2D (273) . Recently, SGLT2 has been chosen as a drug target for the treatment of type II diabetes by a new mechanism of suppressing renal glucose reabsorption and effectively enhancing urinary glucose excretion via SGLT2 inhibition

(Fig. 7.1) (274, 275) . Several SGLT2 inhibitors are in phase II-III clinical trials

[Dapagliflozin from Bristol-Myers Squibb and AstraZeneca (276, 277) ; Sergliflozin from

Kissei Pharmaceutical Co. Ltd (278-280) ; GW-189075, GW-869682 and KGT-1681 from GlaxoSmithKline and Kissei Pharmaceutical Co. Ltd (281) , etc].

Figure 7.1: Blocking glucose absorption into blood by inhibiting SGLT1 or excretion of excessive glucose out of the blood to the urine by inhibiting SGLT2

129 7.2 Biophysical characterization of SGLT1

Investigation of the structure/function relationships of SGLT1 is crucial to understanding co-transporter mechanism.

7.2.1 Sodium interaction domain of SGLT1

213, 214 ). The polar residues at position 176 hydrogen bond to the hydroxyl group on the

β–phenyl ring of phloridzin (237 ). There is also evidence that D454 in the putative external loop joining TM X-XI is involved in the coupling of Na + and sugar in the transport process (238 ).

7.2.2 Sugar interaction domain of SGLT1

The sugar binding domain, on the other hand, has been localized to the C-terminal half of SGLT1 (239 ). Q457, located in the putative external loop joining TM X-XI, appears to be particularly important because sugar transport is abolished by the reaction of MTS reagents and maleimides with Q457C (240 ). However, under these conditions, the transporter still binds Na + and sugar (240 ). These results suggest that although residue

457 is at or near the sugar-translocation site, other unidentified residue(s) must be involved in the interaction with the sugar ligand.

130 In the 34 different missense mutations that were identified for GGM (56 ), four mutations are located in TMIV and the putative loop joining TM IV-V (R135W, L147R,

S159P and A166T). In the region of TM IV, eighteen of twenty-four residues (138-161) are conserved in all members of SGLT1 and SGLT2. Therefore, it is possible that TM IV may be important for SGLT1 function. Mutation of lysine 157 to cysteine (K157C) causes loss of phloridzin and αMG binding ( α-methyl-D-glucopyranoside). These functions are restored by chemical modification with positively charged (2-aminoethyl) methanethiosulphonate hydrobromide (MTSEA). Mutation of threonine 156 to cysteine

(T156C) reduces the affinity of αMG and phloridzin for T156C by ~5–fold and ~20-fold, respectively. In addition, phloridzin protects cysteine 156 in T156C from alkylation by

MTSEA. Detailed investigation of two single cysteine mutants (K157C and T156C, which are conserved across species in SGLT1 and SGLT2) suggests that TM IV participates in sugar interaction with SGLT1 (230 ).

7.3 Biophysical characterization of SGLT2

Human SGLT1 and Human SGLT2 show different ability to handle Na + and substrate. Human SGLT1 exhibits a stoichiometry of 2 Na + ions : 1 glucose molecule.

But, human SGLT2 exhibits a stoichiometry of 1 Na + ion : 1 glucose molecule (70) . The apparent affinities of human SGLT1 and human SGLT1 for αMG using COS-7 cells expression system are 1.8 ± 0.4 mM and 4.8 ± 1.7 mM, respectively (282 ). In contrast to

SGLT1 , human or rat SGLT2 has no affinity for D-galactose (70, 274) .

131 7.4 Rationale of experimental strategy

The recently published crystal structure of Vibrio parahaemolyticus SGLT

(vSGLT) provides important insights into the structure/function of this bacterial cotransporter (1) (Fig. 7.2). The crystal structure of vSGLT contains a hydrophilic core structure formed by multiple transmembrane helices from both N-terminus (TMs II-VI) and C-terminus (TMs VII-XI). Na + and galactose are bound in the center of the core. The theme of N- and C-terminal segments participating in formation of a common cavity for

Na + and co-transported substrate is also emerging from studies of other ion coupled cotransporters, such as the Na +/leucine transporter (253 ), Na +/aspartate transporter (254 ),

Na +/glutamate transporter (255 ), Na +/H + antiporter (256 ) and lactose permease (14 )). For example, crystal studies of lactose permease complexed with lactose demonstrate the involvement of TM I, IV and V in sugar binding (257 ).

132

Figure 7.2: Crystal structure of vSGLT (1). (A) Topology. The structure is colored as a rainbow from the N terminus (red) to the C terminus (purple). The blue and red trapeziums represent the inverted topology of TM2 to TM6 and TM7 to TM11. The gray hexagon with red trim represents the galactose. Residues involved in sugar recognition, gate residues, and a proposed Na + site are shown in cyan, gray, and yellowcircles. (B) Structure viewed in the membrane plane. The coloring scheme and numbering of a helices is the same as in Fig. 2A. Bound galactose is shown as black and red spheres for the C and O atoms. The proposed Na + ion is colored as a blue sphere. (C) Overview of the galactose and proposed Na +-binding site viewed in the membrane plane. (D) Structure of vSGLT viewed from the intracellular side with the central helices colored as in (B) and the peripheral helices in grey.

133 In contrast to the structural data for vSGLT, previous studies involving functional characterization of SGLT1 showed that TMs X-XIII in the C-terminal half of SGLT1 retained sufficient tertiary structure to transport sugar downhill in a stereospecific and selective manner (172, 207, 215 ). However, studies from other groups suggest that the putative external loop joining TM IV-V in the N-terminal half of SGLT1 is involved in the Na + interaction (159, 213 ). In addition, TM IV participates in sugar binding (230 ).

Interestingly, there is evidence that A166 is important for the interaction between the Na + and sugar pathways and that helices TMIV-V are close to TMX-XI (241 ). Although there are differences between vSGLT and mammalian SGLT1 (the Na +/glucose stoichiometry is 1:1 and 2:1, respectively), it is necessary to correlate the available structure/function information for SGLT1 if the functional interpretation of the vSGLT structure is to be extrapolated to mammalian SGLT1.

The properties of SGLT1 have been well characterized (56 ). However, the properties of SGLT2 have not been well characterized because of the low expression in heterologous systems (283) .

SGLT1, SGLT2 and vSGLT belong to the solute sodium symporters family. They have a sequence similarity of 60%, contain 14 transmembrane domains and share an alternating-access mechanism with tight coupling between sodium and solute transport

(270, 283, 284 ). This suggests that they may have similar structure and function.

Therefore, the aim of present study is to investigate TMs II-IV of human SGLT1 and human SGLT2 to explore the structural relationship in SGLT1, SGLT2 and vSGLT (Fig.

7.3).

134

Figure 7.3: Amino acid sequence alignment and secondary structure of the transmembrane segments II-IV in human SGLT1, human

SGLT2 and vSGLT.

135 7.5 Research plan

7.5.1 Investigation of the function/structure of SGLT1

In this first part of the investigative plan we outline experiments to study the Na + ion- and substrate-binding domain with special focus on transmembrane segments II-IV of human SGLT1 and vSGLT.

7.5.1.1 Investigation of the Na +- and substrate-binding domain for transmembrane segments II-IV in human SGLT1 and vSGLT

Previous studies found that human SGLT1 and vSGLT show different abilities of handling Na + and substrate. Human SGLT1 exhibits a stoichiometry of 2 Na + ions : 1 glucose molecule. vSGLT1 exhibits a stoichiometry of 1 Na + ion : 1 galactose molecule.

To identify whether transmembrane segments II-IV are responsible for differences in Na + and substrate recognition, we plan to construct several chimeric transporters between human SGLT1 and vSGLT.

In the presence of Na +, the galactose uptake in control or oocytes expressing vSGLT or SGLT1 is 0.8 ± 0.04 pmol/oocyte/hour, 4.3 ± 0.3 pmol/oocyte/hour and 254 ±

13 pmol/oocyte/hour, respectively (285 ). Since vSGLT has been expressed in Xenopus laevis oocytes expression system, we will also test if vSGLT could be expressed in COS-

7 cells expression system.

In the presence of Na +, the α-methyl-D-glucopyranoside ( αMG) uptake in control or COS-7 cells expressing human SGLT1 is ~1 pmol/well-min and ~50 pmol/well-min, respectively (282 ). In order to obtain a large signal-to-noise ratio, we will only substitute

136 the transmembrane segments II-IV in human SGLT1. The chimeric transporters (2VH;

3VH; 4VH; 2,3VH; 2,4VH; 3,4VH and 2,3,4VH) are designated using the letters H

(human SGLT1), V (vSGLT), and the number of TM contributed by vSGLT at the amino terminus. For example, 2VH contains TMs II from vSGLT1, and TM I, TMs III-XIV from human SGLT1 (Fig. 7.4).

To test the chimeric transporters we design several experiments:

(i) Expression and α-methyl-D-glucopyranoside ( αMG) transport activity of chimeric transporters in COS-7 cells. We believe that the chimeras may express in COS-7 cells because they are primarily SGLT1. Previous research found that human SGLT1 had uptake for αMG (282 ), but vSGLT had no uptake for αMG (285, 286 ). Therefore, we can indicate which TM in the transmembrane segments II-IV responsible for differences in

αMG recognition.

(ii) To determine the selectivity of chimeras for sugar substrate (D-glucose, D- galactose and αMG) by measuring Ratio gal (the proportional affinity for a sugar

sugar gal compared to the K0.5 value for galactose, K0.5 / K0.5 ).

Previous studies found that human SGLT1 and vSGLT show different ability to handle sugar substrate. The selective of human SGLT1 for sugar substrate (D-glucose, D- galactose and αMG) is D-glucose > D-galactose> αMG (194 ). The apparent affinities for

D-glucose, D-galactose and αMG using Xenopus laevis oocytes expression system are

0.5 ± 0.02 mM, 0.6 ± 0.02 mM and 0.7 ± 0.04 mM, respectively. Therefore, the Ratio gal of human SGLT1 for D-glucose and αMG is 0.8 and 1.2, respectively.

137

Figure 7.4: Secondary structure models of chimeric transporters used in this study. The cylinders and lines represent predicted transmembrane helices (TM) and associated loops of human SGLT1 (unfilled cylinders, black lines) and vSGLT (filled cylinders, gray lines). The Y indicates the N-glycosylation site. The chimeric transporters are designated using the letters H (human SGLT1), V (vSGLT), and the number of TM contributed by vSGLT at the amino terminus. 2VH contains TMs II from vSGLT1, and TM I, TMs III- XIV from human SGLT1. 3VH contains TM III from vSGLT1, and TMs I-II, TMs IV- XIV from human SGLT1. 4VH contains TM IV from vSGLT1, and TMs I-III, TMs V- XIV from human SGLT1. 2,3VH contains TMs II-III from vSGLT1, and TM I, TMs IV- XIV from human SGLT1. 2,4VH contains TM II and TM IV from vSGLT1, and TM I, TM III, TMs IV-XIV from human SGLT1. 3,4VH contains TMs III-IV from vSGLT1, and TM I-II, TMs V-XIV from human SGLT. 2,3,4VH contains TMs II-IV from vSGLT1, and TM I, TMs V-XIV from human SGLT1.

138 The selective of vSGLT for sugar substrate (D-glucose, D-galactose and αMG) is

D-galactose > D-glucose >> αMG. The apparent affinities for D-galactose and D-glucose are 40 µM and 420 µM, respectively (287 ). Therefore, the Ratio gal of vSGLT for D- glucose and αMG is 10 and ∞, respectively.

By measuring Ratio gal of chimeras, we can indicate which TM in the transmembrane segments II-IV is responsible for differences in sugar substrate recognition.

7.5.1.2 Investigation of the topology and important residues for

Na +- and substrate-binding in transmembrane segments II-III of the human SGLT1 by scanning cysteine accessibility methods

To investigate the region in TMs II-III, residues will be individually replaced with cysteine and then expressed in COS-7 cells. It has been previously demonstrated that N- terminal myc-tagged WT human SGLT1 is expressed at the cell surface of transiently transfected COS-7 cells or Xenopus laevis oocytes and its activity is similar to the non- myc-tagged WT humanSGLT1 in terms of sugar transport and phloridzin binding (217 ).

The experiments for function/topology include (i) αMG Transport Activity and

Expression of Cysteine-substituted Mutants; (ii) Functional Characteristics of Cysteine

Mutants by measuring the apparent affinities for ions (Na +, Li + and H+), sugar substrates and inhibitors; (iii) To determine the stoichiometry of Na + ions : sugar molecule; (iv)

MTSET Accessibility of Cysteine Mutants; (v) Effects of Cations and Substrate on

MTSET Sensitivity; (vi) Concentration Dependence of MTSET Inhibition; (vii) Effect of

139 Temperature on MTSET Inhibition; (viii) Labeling of Substituted Cysteines with

MTSEA-biotin.

7.5.1.3 Investigation of a negative residue that forms a charged pair with K157 in TM IV for regulating sugar binding

Previously we found that when Lys157 in TM IV was replaced with Cys (230 ) or

Ala (59 ), both binding and active transport were abolished. These results suggest that

K157 may be essential and forms a charged pair with a negative residue for regulating sugar binding. A mechanism involves charged pairs for regulating substrate binding has been proposed by several publications on Na +/leucine transporter (268, 288 ) and Lactose

Permease (14, 289 ). Therefore, the aim of this study is to investigate the negative residue that forms a charged pair with K157 in TM IV for regulating sugar binding.

The conserved negative charge residues in SGLT1 (E102; E130; D161; D204;

E225; D261; D268; D273; D294; E350; E372; D408; E419; E421; D454; E482; E503;

E513; D553; E568; E569; D572; D574; E578; D608; E623; E624; E625; D634; E637) are identified by the amino acid sequence alignment of SGLT1 (Human, Rabbit, Rat,

Mouse, Sheep, Pig). Based on the crystal structure of vSGLT, the conserved negative charge residues, located in TMs 2, 3, 7,8 and 11 of human SGLT1, are E102 (TM 3),

E130 (IL 3), D273 (TM 7E), D294 (Loop joining TM 7E and TM7I), E350 (EL 8a), E372

(EL 8b) and D454 (TM 11). These residues will be individually replaced with cysteine and then expressed in COS-7 cells. If we can find the essential negative charge residues, we will measure the transport activity by double neutral replacements or inversion of the charged residues at these positions.

140

7.5.2 Investigation of the function/structure of SGLT2

In this second part of the investigative plan we outline experiments to study the

Na +- and substrate-binding domain with special focus on transmembrane segments II-IV of human SGLT2.

7.5.2.1 Investigation of the non-conserved charged or polar residues in transmembrane segments II-IV of human SGLT1 and human SGLT2 by scanning cysteine accessibility methods

Previous research found that human SGLT1 and human SGLT2 show different abilities to handle Na + and substrate. Human SGLT1 exhibits a stoichiometry of 2 Na + ions : 1 glucose molecule. Human SGLT2 exhibits a stoichiometry of 1 Na + ion : 1 glucose molecule (70) .

Both SGLT1 and SGLT2 have been expressed in Xenopus laevis oocytes. The apparent affinities of SGLT1 and SGLT2 for Na + are 32 mM (177 ) and 250-300 mM

(133 ), respectively. The apparent affinities of human SGLT1 and human SGLT2 for

αMG are 0.7 mM and 1.6 mM, respectively (282 ). Since the extracellular physiological concentration of Na + (~100 mM, oocytes could not tolerate Na + concentration

Na+ significantly higher above this concentration) is significantly lower than the Km of

sugar SGLT2, the Km of SGLT2 may be overestimated (156 ). In contrast to the high affinity of human SGLT1 for D-galactose (0.6 ± 0.02 mM) (194 ), human or rat SGLT2 has no affinity for D-galactose (70, 274) .

141 To identify whether transmembrane segments II-IV are responsible for differences in Na + and substrate recognition between SGLT1 and SGLT2, we plan to investigate the non-conserved charged or polar residues in transmembrane segments II-IV of human SGLT1 and human SGLT2 by scanning cysteine accessibility methods. Fig. 7.3 shows the multiple sequence alignment of human SGLT1 and human SGLT2 .

In the presence of Na +, the α-methyl-D-glucopyranoside ( αMG) uptake in control or COS-7 cells expressing human SGLT1, human SGLT2 or mouse SGLT2 is ~1 pmol/well-min, ~50 pmol/well-min, ~5 pmol/well-min and ~12 pmol/well-min (282 ).

The apparent affinities of human SGLT1 and human SGLT2 for αMG using COS-7 cells expression system are 1.8 ± 0.4 mM and 4.8 ± 1.7 mM, respectively (282 ). In order to obtain a large signal-to-noise ratio, we will only substitute the residues in human SGLT1

(K122T, E130Q, Q139R, Q142R, I162M, N170Q, and L171Q ).

Human SGLT1 mutants will be individually expressed in COS-7 cells, followed by the measurement of: (i) Expression and αMG and D-galactose transport activity of mutants in COS-7 cells; (ii) To determine the apparent affinities and selective of mutants for ions (Na +, Li + and H+) and sugar substrate (D-glucose, D-galactose and αMG; Ratio gal

sugar gal + = K0.5 / K0.5 ); (iii) To determine the stoichiometry of Na ions : glucose molecule.

7.5.2.2 Investigation of the essential Na +- and substrate-binding residues in human SGLT2

Based on the function/structure studies on SGLT1 (163, 230 ) and vSGLT (1), several residues in human SGLT2 (A73, I76, H80, L84, F97, E99, A102, T153, K154,

142 S287, Y290, K321, S393, F453 and Q457) will be individually replaced with cysteine and then expressed in COS-7 cells.

The experiments for function/topology include (i) Expression and αMG and D- galactose transport activity of Cysteine-substituted mutants in COS-7 cells; (ii)

Functional Characteristics of Cysteine Mutants by measuring the apparent affinities for ions (Na +, Li + and H+), sugar substrates and inhibitors; (iii) Determination of the stoichiometry of Na + ions: sugar molecule; (iv) MTSET Accessibility of Cysteine

Mutants; (v) Effects of Cations and Substrate on MTSET Sensitivity. (vi) Concentration

Dependence of MTSET Inhibition; (vii) Effect of Temperature on MTSET Inhibition;

(viii) Labeling of Substituted Cysteines with MTSEA-biotin.

7.6 Materials and methods

7.6.1 Construction of Chimeric Transporter cDNAs

Construction of Chimeric Transporter cDNAs will be done as described previously (228, 290 ). Chimeric transporter cDNAs containing portions of human SGLT1 and vSGLT cDNAs are constructed by subcloning using endogenous or introduced restriction enzyme sites. The junctions of the chimeras are located in loops outside of putative TMs. The numbering of the junctions is based on the vSGLT amino acid sequence, and the equivalent positions in human SGLT1 are selected to avoid additional mutations in the chimeras. All of the chimeras will be confirmed by sequencing.

143 7.6.2 Transport assay

Transport assays will be carried out 48 h after transfections as described previously (282, 291 ). The sodium buffer contains 140 mM NaCl, 2 mM KCl, 1 mM

MgCl 2, 1 mM CaCl 2, and 10 mM HEPES, pH adjusted to 7.4 with 1 M Tris. Choline buffer contains 140 mM choline chloride in place of NaCl. For the assays, each well is washed twice with 1 ml of choline buffer, then incubated with 0.25 ml of sodium buffer containing [ 14 C] α-methyl-D-glucopyranoside for 10-30 min. Inhibitors are added to the transport solutions from dimethyl sulfoxide stocks, with a final volume of dimethyl sulfoxide less than 1%. Inhibition experiments included a 10-min preincubation with inhibitor, which reduces variability in the results. The plates are rocked in a heated incubator during the incubation period. The uptakes are stopped and extracellular radioactivity removes with four 1-ml washes of choline buffer. After the fourth rinse, the buffer is completely removed from the wells and the cells are solubilazed by adding

500 µl of ATPlite substrate solution (Perkin-Elmer). Then luminescence ATP detection is assessed using a MicroBeta Trilux (Perkin-Elmer). A standard curve is used to determine the amount ofATP as milligram of protein, based on a high correlation ( r = 0 .95) between

ATP detection and protein content determined by Bradford assay (Sigma). Each well is washed with four 1-ml washes of choline buffer. After the last wash is removed, each well of cells is dissolved in 1 ml of scintillation cocktail, OptiPhase Supermix (GE

Healthcare). The plates are sealed with plastic plate covers and counted directly in a

Microbeta Trilux 1450 plate scintillation counter (Perkin-Elmer). For all experiments, uptakes in vector-transfected cells are subtracted from uptakes in SGLT plasmid- transfected cells. There is no difference in the background counts obtained with each

144 vector. For calculation of Ki values from Dixon plots, inhibition of transport activity is measured at multiple inhibitor and substrate concentrations. The data are analyzed by linear regression, and the x-axis value corresponding to the intersection of the lines is

taken as –Ki. To determine IC 50 values (the inhibitor concentration resulting in 50% inhibition), the transport of 100 µM [ 14 C] AMG is measured in the presence of increasing concentrations of inhibitor and the resulting activity (v%, as a percentage of control) is

analyzed by nonlinear regression using v% = IC 50 /([I] + IC 50 ) x 100, where [I] represents the inhibitor concentration. Statistical analysis with Student's t test or with one-way analysis of variance followed by Dunnett's test is done using the Sigma Stat program

(SPSS Inc., Chicago, IL).

7.7 Preliminary data for human SGLT1-Cell

Transfection and Western Blot Detection

Non-transfected COS-7 cells or COS-7 cells transfected with WT-myc human

SGLT1 were grown and maintained in RPMI 1640 medium (Invitrogen Canada,

Burlington, ON). Non-transfected COS-7 cells served as controls. Proteins samples were resolved on 10% SDS-PAGE and transferred to nitrocellulose. The myc-epitope was detected with mouse monoclonal 9E10 (anti-c-myc, 1:1000) antibody (Berkley Antibody

Company), followed by peroxidase conjugated anti-mouse IgG (1:200,000) (Sigma)

Immunoblots were developed by chemiluminescence and area analysis was performed using the public domain NIH Image program (developed at the US National Institutes of

Health). Western blot for β-actin was performed to check equal loading.

145 A B

Figure 7.5: Western blot analysis of WT-myc human SGLT1 [a gift from Dr. Lapointe JY

(292 )], detected with an anti-myc antibody. (A) Non-transfected COS-7 cells. (B) COS-7 cells were transfected with WT-myc human SGLT1.

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