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

Tiemin Liu, Bryan Lo, Pam Speight, Mel Silverman

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Investigation of the structure/function relationships of the sodium-glucose transporter (SGLT1) is crucial to understanding the cotransporter mechanism. In the present study, we used cysteine-scanning mutagenesis and chemical modification by methanethiosulfonate (MTS) derivatives to test whether predicted transmembrane IV participates in sugar binding. Five charged and polar residues (K139, Q142, T156, K157, and D161) and two glucose/galactose malabsorption missense mutations (I147 and S159) were replaced with cysteine. Mutants I147C, T156C, and K157C exhibited sufficient expression to be studied in detail using the two-electrode voltage-clamp method in Xenopus laevis oocytes and COS-7 cells. I147C was similar in function to wild-type and was not studied further. Mutation of lysine-157 to cysteine (K157C) causes loss of phloridzin and α-methyl-D-glucopyranoside (αMG) binding. These functions are restored by chemical modification with positively charged (2-aminoethyl) methanethiosulfonate hydrobromide (MTSEA). Mutation of threonine-156 to cysteine (T156C) reduces the affinity of αMG and phloridzin for T156C by ∼5-fold and ∼20-fold, respectively. In addition, phloridzin protects cysteine-156 in T156C from alkylation by MTSEA. Therefore, the presence of a positive charge or a polar residue at 157 and 156, respectively, affects sugar binding and sugar-induced Na+ currents.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume295
Issue number1
DOIs
StatePublished - Jul 2008

Fingerprint

Sodium-Glucose Transport Proteins
Cysteine
Threonine
Phlorhizin
Mutation
COS Cells
Xenopus laevis
Alkylation
Missense Mutation
Mutagenesis
Lysine
Oocytes
Electrodes

Keywords

  • Chemical modification by methanethiosulfonate reagents
  • Cysteine scanning mutagenesis

ASJC Scopus subject areas

  • Cell Biology
  • Physiology

Cite this

Transmembrane IV of the high-affinity sodium-glucose cotransporter participates in sugar binding. / Liu, Tiemin; Lo, Bryan; Speight, Pam; Silverman, Mel.

In: American Journal of Physiology - Cell Physiology, Vol. 295, No. 1, 07.2008.

Research output: Contribution to journalArticle

@article{64c78c8e05714637beb5b85488aafda5,
title = "Transmembrane IV of the high-affinity sodium-glucose cotransporter participates in sugar binding",
abstract = "Investigation of the structure/function relationships of the sodium-glucose transporter (SGLT1) is crucial to understanding the cotransporter mechanism. In the present study, we used cysteine-scanning mutagenesis and chemical modification by methanethiosulfonate (MTS) derivatives to test whether predicted transmembrane IV participates in sugar binding. Five charged and polar residues (K139, Q142, T156, K157, and D161) and two glucose/galactose malabsorption missense mutations (I147 and S159) were replaced with cysteine. Mutants I147C, T156C, and K157C exhibited sufficient expression to be studied in detail using the two-electrode voltage-clamp method in Xenopus laevis oocytes and COS-7 cells. I147C was similar in function to wild-type and was not studied further. Mutation of lysine-157 to cysteine (K157C) causes loss of phloridzin and α-methyl-D-glucopyranoside (αMG) binding. These functions are restored by chemical modification with positively charged (2-aminoethyl) methanethiosulfonate hydrobromide (MTSEA). Mutation of threonine-156 to cysteine (T156C) reduces the affinity of αMG and phloridzin for T156C by ∼5-fold and ∼20-fold, respectively. In addition, phloridzin protects cysteine-156 in T156C from alkylation by MTSEA. Therefore, the presence of a positive charge or a polar residue at 157 and 156, respectively, affects sugar binding and sugar-induced Na+ currents.",
keywords = "Chemical modification by methanethiosulfonate reagents, Cysteine scanning mutagenesis",
author = "Tiemin Liu and Bryan Lo and Pam Speight and Mel Silverman",
year = "2008",
month = "7",
doi = "10.1152/ajpcell.90602.2007",
language = "English (US)",
volume = "295",
journal = "American Journal of Physiology - Heart and Circulatory Physiology",
issn = "0363-6135",
publisher = "American Physiological Society",
number = "1",

}

TY - JOUR

T1 - Transmembrane IV of the high-affinity sodium-glucose cotransporter participates in sugar binding

AU - Liu, Tiemin

AU - Lo, Bryan

AU - Speight, Pam

AU - Silverman, Mel

PY - 2008/7

Y1 - 2008/7

N2 - Investigation of the structure/function relationships of the sodium-glucose transporter (SGLT1) is crucial to understanding the cotransporter mechanism. In the present study, we used cysteine-scanning mutagenesis and chemical modification by methanethiosulfonate (MTS) derivatives to test whether predicted transmembrane IV participates in sugar binding. Five charged and polar residues (K139, Q142, T156, K157, and D161) and two glucose/galactose malabsorption missense mutations (I147 and S159) were replaced with cysteine. Mutants I147C, T156C, and K157C exhibited sufficient expression to be studied in detail using the two-electrode voltage-clamp method in Xenopus laevis oocytes and COS-7 cells. I147C was similar in function to wild-type and was not studied further. Mutation of lysine-157 to cysteine (K157C) causes loss of phloridzin and α-methyl-D-glucopyranoside (αMG) binding. These functions are restored by chemical modification with positively charged (2-aminoethyl) methanethiosulfonate hydrobromide (MTSEA). Mutation of threonine-156 to cysteine (T156C) reduces the affinity of αMG and phloridzin for T156C by ∼5-fold and ∼20-fold, respectively. In addition, phloridzin protects cysteine-156 in T156C from alkylation by MTSEA. Therefore, the presence of a positive charge or a polar residue at 157 and 156, respectively, affects sugar binding and sugar-induced Na+ currents.

AB - Investigation of the structure/function relationships of the sodium-glucose transporter (SGLT1) is crucial to understanding the cotransporter mechanism. In the present study, we used cysteine-scanning mutagenesis and chemical modification by methanethiosulfonate (MTS) derivatives to test whether predicted transmembrane IV participates in sugar binding. Five charged and polar residues (K139, Q142, T156, K157, and D161) and two glucose/galactose malabsorption missense mutations (I147 and S159) were replaced with cysteine. Mutants I147C, T156C, and K157C exhibited sufficient expression to be studied in detail using the two-electrode voltage-clamp method in Xenopus laevis oocytes and COS-7 cells. I147C was similar in function to wild-type and was not studied further. Mutation of lysine-157 to cysteine (K157C) causes loss of phloridzin and α-methyl-D-glucopyranoside (αMG) binding. These functions are restored by chemical modification with positively charged (2-aminoethyl) methanethiosulfonate hydrobromide (MTSEA). Mutation of threonine-156 to cysteine (T156C) reduces the affinity of αMG and phloridzin for T156C by ∼5-fold and ∼20-fold, respectively. In addition, phloridzin protects cysteine-156 in T156C from alkylation by MTSEA. Therefore, the presence of a positive charge or a polar residue at 157 and 156, respectively, affects sugar binding and sugar-induced Na+ currents.

KW - Chemical modification by methanethiosulfonate reagents

KW - Cysteine scanning mutagenesis

UR - http://www.scopus.com/inward/record.url?scp=52749095703&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=52749095703&partnerID=8YFLogxK

U2 - 10.1152/ajpcell.90602.2007

DO - 10.1152/ajpcell.90602.2007

M3 - Article

C2 - 18448629

AN - SCOPUS:52749095703

VL - 295

JO - American Journal of Physiology - Heart and Circulatory Physiology

JF - American Journal of Physiology - Heart and Circulatory Physiology

SN - 0363-6135

IS - 1

ER -