Evidence for the involvement of Ala 166 in coupling Na(+) to sugar transport through the human Na(+)/glucose cotransporter.

TY - JOUR

T1 - Evidence for the involvement of Ala 166 in coupling Na(+) to sugar transport through the human Na(+)/glucose cotransporter.

AU - Meinild, A K

AU - Loo, D D

AU - Hirayama, B A

AU - Gallardo, E

AU - Wright, E M

N1 - Keywords: Alanine; Animals; Glucose; Humans; Kinetics; Membrane Glycoproteins; Monosaccharide Transport Proteins; Sodium; Sodium-Glucose Transporter 1; Xenopus laevis

PY - 2001

Y1 - 2001

N2 - We mutated residue 166, located in the putative Na(+) transport pathway between transmembrane segments 4 and 5 of human Na(+)/glucose cotransporter (hSGLT1), from alanine to cysteine (A166C). A166C was expressed in Xenopus laevis oocytes, and electrophysiological methods were used to assay function. The affinity for Na(+) was unchanged compared to that of hSGLT1, whereas the sugar affinity was reduced and sugar specificity was altered. There was a reduction in the turnover rate of the transporter, and in contrast to that of hSGLT1, the turnover rate depended on the sugar molecule. Exposure of A166C to MTSEA and MTSET, but not MTSES, abolished sugar transport. Accessibility of A166C to alkylating reagents was independent of protein conformation, indicating that the residue is always accessible from the extracellular surface. Sugar and phlorizin did not protect the residue from being alkylated, suggesting that residue 166 is not located in the sugar pathway. MTSEA, MTSET, and MTSES all changed the pre-steady-state kinetics of A166C, independent of pH, and sugars altered these kinetics. The inability of MTSEA-labeled A166C to transport sugar was reversed (with no major change in Na(+) and sugar affinity) if the positive charge on MTSEA was neutralized by increasing the external pH to 9.0. These studies suggest that the residue at position 166 is involved in the interaction between the Na(+) and sugar transport pathways.

AB - We mutated residue 166, located in the putative Na(+) transport pathway between transmembrane segments 4 and 5 of human Na(+)/glucose cotransporter (hSGLT1), from alanine to cysteine (A166C). A166C was expressed in Xenopus laevis oocytes, and electrophysiological methods were used to assay function. The affinity for Na(+) was unchanged compared to that of hSGLT1, whereas the sugar affinity was reduced and sugar specificity was altered. There was a reduction in the turnover rate of the transporter, and in contrast to that of hSGLT1, the turnover rate depended on the sugar molecule. Exposure of A166C to MTSEA and MTSET, but not MTSES, abolished sugar transport. Accessibility of A166C to alkylating reagents was independent of protein conformation, indicating that the residue is always accessible from the extracellular surface. Sugar and phlorizin did not protect the residue from being alkylated, suggesting that residue 166 is not located in the sugar pathway. MTSEA, MTSET, and MTSES all changed the pre-steady-state kinetics of A166C, independent of pH, and sugars altered these kinetics. The inability of MTSEA-labeled A166C to transport sugar was reversed (with no major change in Na(+) and sugar affinity) if the positive charge on MTSEA was neutralized by increasing the external pH to 9.0. These studies suggest that the residue at position 166 is involved in the interaction between the Na(+) and sugar transport pathways.

U2 - 10.1021/bi011026l

DO - 10.1021/bi011026l

M3 - Journal article

C2 - 11570890

SN - 0006-2960

VL - 40

SP - 11897

EP - 11904

JO - Biochemistry

JF - Biochemistry

IS - 39

ER -