STRUCTURE-FUNCTION STUDIES OF THE BRAIN-TYPE GLUCOSE-TRANSPORTER, GLUT3 - ALANINE-SCANNING MUTAGENESIS OF PUTATIVE TRANSMEMBRANE HELIX-VIIIAND AN INVESTIGATION OF THE ROLE OF PROLINE RESIDUES IN TRANSPORT CATALYSIS

The brain-type glucose transporter (GLUT3) is a high-affinity transpor ter for D-glucose and D-galactose and is a member of a family of mamma lian sugar transporters, each of which are proposed to adopt a seconda ry structure containing 12 transmembrane helices. In an effort to unde rstand structure-function relationships within such transporters, we h ave employed alanine-scanning mutagenesis to examine the functional im portance of each residue within putative transmembrane helix VIII of t he human GLUT3 isoform. Each residue in this helix was replaced indivi dually 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 K-m for deoxygluc ose transport and the K-i for maltose. In contrast, Asn-315 --> Ala-31 5 exhibited a significant increase in the K-m for deoxyglucose indepen dently of any effect on the K-i 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 cat alysis by this isoform. We have also examined the role of proline resi dues 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, c lose to the end of helix XII) with alanine did not change the K-m for deoxyglucose transport for any mutant. However, both Pro-381 and Pro-3 85 when mutated to alanine exhibited a reduction in the K-i for cytoch alasin B. In addition, the K-i for maltose inhibition of deoxyglucose transport was increased for mutants Pro206Ala, Pro381Ala, Pro383Ala, a nd Pro451Ala. These results will be discussed in terms of proposed str uctural models for the transporters.