title: Cysteine 144 in the third transmembrane domain of the creatine transporter is located close to a substrate-binding site.
abstract: All creatine transporters contain a cysteine residue (Cys(144)) in the third transmembrane domain that is not present in other members of the Na+,Cl(-)-dependent family of neurotransmitter transporters. Site-directed mutagenesis and reaction with methane thiosulfonates were used to investigate the importance of Cys(144) for transporter function. Replacement of Cys(144) with Ser did not significantly affect the kinetics or activity of the transporter, whereas a C144A mutant had a higher K(m) (0.33 compared with 0.18 mm). Substitution of Cys(144) with Leu gave a mutant with a 5-fold higher K(m) and a reduced specificity for substrate. Low concentrations of 2-aminoethyl methanethiosulfonate (MTSEA) resulted in rapid inactivation of the creatine transporter. The C144S mutant was resistant to inactivation, indicating that modification of Cys(144) was responsible for the loss of transport activity. Creatine and analogues that function as substrates of the creatine transporter were able to protect from MTSEA inactivation. Na+ and Cl(-) ions were not necessary for MTSEA inactivation, but Na+ was found to be important for creatine protection from inactivation. Our results indicate that cysteine 144 is close to the binding site or part of a permeation channel for creatine.
authors.affiliation: Protein Structure and Function Research Group, School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand.
authors.forename: J R
authors.initials: JR
authors.lastname: Dodd
authors.forename: D L
authors.initials: DL
authors.lastname: Christie
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D019921:Biotinylation; D002417:Cattle; D002460:Cell Line; D002713:Chlorine / Q000378:metabolism; D003401:Creatine / Q000378:metabolism / Q000493:pharmacokinetics; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D007477:Ions; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D008698:Mesylates / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011817:Rabbits; D051381:Rats; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D012964:Sodium / Q000378:metabolism; D014162:Transfection
chemical_list: D007202:Indicators and Reagents; D007477:Ions; D026901:Membrane Transport Proteins; D008698:Mesylates; C080679:creatine transporter; C093171:methanethiosulfonate ethylammonium; C074502:methanethiosulfonate; D012694:Serine; D002713:Chlorine; D005020:Ethyl Methanesulfonate; D012964:Sodium; D003545:Cysteine; D003401:Creatine