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

Liu, Tiemin; Lo, Bryan; Speight, Pam; Silverman, Melvin

doi:10.1152/ajpcell.90602.2007

Cited by 6 publications

(4 citation statements)

References 22 publications

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“…6A) to the cfnt2-K155C mutant transporter. A similar effect was noted for MTSEA modification of K157C in the sodium-glucose cotransporter SGLT1 (44). Although mutation of Lys 155 of CfNT2 to a variety of non-cationic residues resulted in reduced inosine uptake and/or broadened ligand selectivity (Table 1) but correct cell surface localization (Fig.…”

Section: Discussionsupporting

confidence: 64%

Role of Transmembrane Domain 4 in Ligand Permeation by Crithidia fasciculata Equilibrative Nucleoside Transporter 2 (CfNT2)

Arendt

Ullman

2010

Journal of Biological Chemistry

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Equilibrative nucleoside transporters play essential roles in nutrient uptake, cardiovascular and renal function, and purine analog drug chemotherapies. Limited structural information is available for this family of transporters; however, residues in transmembrane domains 1, 2, 4, and 5 appear to be important for ligand and inhibitor binding. In order to identify regions of the transporter that are important for ligand specificity, a genetic selection for mutants of the inosine-guanosine-specific Crithidia fasciculata nucleoside transporter 2 (CfNT2) that had gained the ability to transport adenosine was carried out in the yeast Saccharomyces cerevisiae. Nearly all positive clones from the genetic selection carried mutations at lysine 155 in transmembrane domain 4, highlighting lysine 155 as a pivotal residue governing the ligand specificity of CfNT2. Mutation of lysine 155 to asparagine conferred affinity for adenosine on the mutant transporter at the expense of inosine and guanosine affinity due to weakened contacts to the purine ring of the ligand. Following systematic cysteine-scanning mutagenesis, thiol-specific modification of several positions within transmembrane domain 4 was found to interfere with inosine transport capability, indicating that this helix lines the water-filled ligand translocation channel. Additionally, the pattern of modification of transmembrane domain 4 suggested that it may deviate from helicity in the vicinity of residue 155. Position 155 was also protected from modification in the presence of ligand, suggesting that lysine 155 is in or near the ligand binding site. Transmembrane domain 4 and particularly lysine 155 appear to play key roles in ligand discrimination and translocation by CfNT2.Both hydrophilic nutrients and nutrient analog drugs gain access to target cells via membrane-spanning protein transporters. One family of such proteins, the equilibrative nucleoside transporters (ENTs 2 ; SLC29), transports purine and pyrimidine nucleobases and nucleosides into eukaryotic cells (1).In parasitic protozoa, such as Leishmania, Plasmodium, and trypanosomes, ENTs serve an essential function, because purines cannot be synthesized de novo by these organisms and must be obtained from the environment (2). Mammalian ENTs are of particular interest in renal and cardiovascular function (adenosine transport (3, 4)) and in the pharmacogenomics of nucleoside analog drug uptake in antiparasitic, antiviral, and anticancer chemotherapies (5). Although many ENT genes and cDNAs from diverse organisms have been cloned and biochemically characterized in recent years, and insight into the structure of the ENT family is emerging from recent threading (6, 7) and ab initio (8) structural models, a detailed understanding of how ENTs recognize their ligands has remained elusive. All ENTs studied to date are predicted to be composed of 11 transmembrane-spanning segments (TMs), with a large loop between TM6 and -7 that, like the N terminus, is intracellular, whereas the C terminus projects extracellularly ...

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Section: Discussionsupporting

confidence: 64%

Role of Transmembrane Domain 4 in Ligand Permeation by Crithidia fasciculata Equilibrative Nucleoside Transporter 2 (CfNT2)

Arendt

Ullman

2010

Journal of Biological Chemistry

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“…(See Refs. 3,12,14,15,(19)(20)(21)30,31,34,35,39; Sala-Rabanal M, Martin M, and Wright EM, unpublished observations; and Jiang X, Hirayama BA, Loo DDF, and Wright EM, unpublished observations.) translate these differences into structural motion, they will provide constraints on how the transitions occur.…”

Section: Alternating Accessmentioning

confidence: 88%

Bridging the gap between structure and kinetics of human SGLT1

Sala-Rabanal

Hirayama

Loo

et al. 2012

American Journal of Physiology-Cell Physiology

126

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The Na(+)-glucose cotransporter hSGLT1 is a member of a class of membrane proteins that harness Na(+) electrochemical gradients to drive uphill solute transport. Although hSGLT1 belongs to one gene family (SLC5), recent structural studies of bacterial Na(+) cotransporters have shown that Na(+) transporters in different gene families have the same structural fold. We have constructed homology models of hSGLT1 in two conformations, the inward-facing occluded (based on vSGLT) and the outward open conformations (based on Mhp1), mutated in turn each of the conserved gates and ligand binding residues, expressed the SGLT1 mutants in Xenopus oocytes, and determined the functional consequences using biophysical and biochemical assays. The results establish that mutating the ligand binding residues produces profound changes in the ligand affinity (the half-saturation concentration, K(0.5)); e.g., mutating sugar binding residues increases the glucose K(0.5) by up to three orders of magnitude. Mutation of the external gate residues increases the Na(+) to sugar transport stoichiometry, demonstrating that these residues are critical for efficient cotransport. The changes in phlorizin inhibition constant (K(i)) are proportional to the changes in sugar K(0.5), except in the case of F101C, where phlorizin K(i) increases by orders of magnitude without a change in glucose K(0.5). We conclude that glucose and phlorizin occupy the same binding site and that F101 is involved in binding to the phloretin group of the inhibitor. Substituted-cysteine accessibility methods show that the cysteine residues at the position of the gates and sugar binding site are largely accessible only to external hydrophilic methanethiosulfonate reagents in the presence of external Na(+), demonstrating that the external sugar (and phlorizin) binding vestibule is opened by the presence of external Na(+) and closes after the binding of sugar and phlorizin. Overall, the present results provide a bridge between kinetics and structural studies of cotransporters.

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“…The phloridzin affinity was determined as described previously (20). Transient current measurements were made for an array of voltage steps ranging from À150 to 70 mV in 10 mV steps for mutants.…”

Section: Phloridzin Affinity Measurementsmentioning

confidence: 99%

“…The specific inhibitor, phloridzin, binds to SGLT1 and blocks sugar translocation (24). As described in the Materials and Methods section, the ability of phloridzin to reduce transient charge movement of SGLT1 can been used to measure its apparent affinity for the cotransporter (20,(23)(24)(25). As shown in Fig.…”

Section: Phloridzin Affinitymentioning

confidence: 99%

Effects on Conformational States of the Rabbit Sodium/Glucose Cotransporter through Modulation of Polarity and Charge at Glutamine 457

Liu

Krofchick

Silverman

2009

Biophysical Journal

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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 after 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 wild-type SGLT1, Q457C exhibits approximately 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-MTSET and Q457R with Q457C and wild-type, 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.

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

Cited by 6 publications

References 22 publications

Role of Transmembrane Domain 4 in Ligand Permeation by Crithidia fasciculata Equilibrative Nucleoside Transporter 2 (CfNT2)

Role of Transmembrane Domain 4 in Ligand Permeation by Crithidia fasciculata Equilibrative Nucleoside Transporter 2 (CfNT2)

Bridging the gap between structure and kinetics of human SGLT1

Effects on Conformational States of the Rabbit Sodium/Glucose Cotransporter through Modulation of Polarity and Charge at Glutamine 457

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