title: 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.
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 transport catalysis by this isoform. We have also examined the role of proline residues in transport catalysis mediated by GLUT3. Substitution of Pro-203 (helix VI), Pro-206, Pro-209 (cytoplasmic loop between helices VI and VII), Pro-381, Pro-383 and Pro-385 (helix X), Pro-399 (intracellular loop between helices X and XI), or Pro-451 (in the carboxy terminus, close to the end of helix XII) with alanine did not change the Km for deoxyglucose transport for any mutant. However, both Pro-381 and Pro-385 when mutated to alanine exhibited a reduction in the Ki for cytochalasin B. In addition, the Ki for maltose inhibition of deoxyglucose transport was increased for mutants Pro206Ala, Pro381Ala, Pro383Ala, and Pro451Ala. These results will be discussed in terms of proposed structural models for the transporters.
authors.affiliation: Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Scotland.
authors.forename: M J
authors.initials: MJ
authors.lastname: Seatter
authors.forename: S
authors.initials: S
authors.lastname: Kane
authors.forename: L M
authors.initials: LM
authors.lastname: Porter
authors.forename: M I
authors.initials: MI
authors.lastname: Arbuckle
authors.forename: D R
authors.initials: DR
authors.lastname: Melvin
authors.forename: G W
authors.initials: GW
authors.lastname: Gould
mesh_terms: D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry; D005260:Female; D005947:Glucose / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D051274:Glucose Transporter Type 3; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D011392:Proline / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
chemical_list: D051274:Glucose Transporter Type 3; D009004:Monosaccharide Transport Proteins; D009419:Nerve Tissue Proteins; D011392:Proline; D005947:Glucose; D000409:Alanine