title: From triple cysteine mutants to the cysteine-less glucose transporter GLUT1: a functional analysis.
abstract: Two triple cysteine mutants containing Cys-less N- or C-terminal halves and the Cys-less GLUT1 were generated by site-directed mutagenesis. Following expression in Xenopus oocytes, the intrinsic transport activities of the multiple cysteine mutants were slightly decreased when either the cysteine residues of the C-terminal half or all six residues were changed; substitution of serine for cysteine residues located at the N-terminal half was without consequence for the catalytic activity. The exofacial ligand ethylidene glucose inhibited 2-deoxy-D-glucose uptake of wild-type and Cys-less GLUT1-expressing Xenopus oocytes with comparable half-saturation constants (11.5 and 13.2 mM). However, each of the multiple cysteine mutants exhibited an increase in affinity for the endofacial inhibitor cytochalasin B, with the greatest effect being observed for the Cys-less construct (decrease in Ki by the factor 5-6).
authors.affiliation: Institut für Pharmakologie der Freien Universität Berlin, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Wellner
authors.forename: I
authors.initials: I
authors.lastname: Monden
authors.forename: K
authors.initials: K
authors.lastname: Keller
mesh_terms: D000818:Animals; D001483:Base Sequence; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D003062:Codon; D003545:Cysteine; D003571:Cytochalasin B / Q000494:pharmacology; D003847:Deoxyglucose / Q000378:metabolism; D005260:Female; D005947:Glucose / Q000031:analogs & derivatives / Q000494:pharmacology; D051272:Glucose Transporter Type 1; D007700:Kinetics; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D017354:Point Mutation; D017384:Sequence Deletion; D014982:Xenopus laevis
chemical_list: D003062:Codon; D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; C013984:ethylidene glucose; D003571:Cytochalasin B; D003847:Deoxyglucose; D005947:Glucose; D003545:Cysteine