Structure-function studies of the brain-type glucose transporter, GLUT3: alanine-scanning mutagenesis of putative transmembrane helix VIII and an investigation of the role of proline residues in transport catalysis

Biochemistry
M J SeatterG W Gould

Abstract

The brain-type glucose transporter (GLUT3) is a high-affinity transporter for D-glucose and D-galactose and is a member of a family of mammalian sugar transporters, each of which are proposed to adopt a secondary structure containing 12 transmembrane helices. In an effort to understand structure-function relationships within such transporters, we have employed alanine-scanning mutagenesis to examine the functional importance of each residue within putative transmembrane helix VIII of the human GLUT3 isoform. Each residue in this helix was replaced individually with alanine, and the functional properties of the mutants were examined by microinjection of in vitro transcribed mRNA into Xenopus oocytes. We show that substitution of residues 305, 306, 308-314, and 316-325 with alanine had minimal effect on the functional activity of the transporter, as determined by measurement of the Km for deoxyglucose transport and the Ki for maltose. In contrast, Asn-315 > Ala-315 exhibited a significant increase in the Km for deoxyglucose independently of any effect on the Ki for maltose. This data suggests that, despite the strong sequence conservation in this helix among the GLUT family, no individual residue is absolutely required for transp...Continue Reading

Citations

Jan 14, 2017·Frontiers in Plant Science·Peter PrislanKatarina Čufar
Dec 31, 2002·Physiological Reviews·Giovanni E MannLuis Sobrevia
Jun 4, 2002·The Journal of Biological Chemistry·Suzanne K ManningSanjeev Krishna

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