title: Transmembrane domains 1 and 3 of the glycine transporter GLYT1 contain structural determinants of N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)-propyl]sarcosine specificity.
abstract: The neurotransmitter glycine is removed from the synaptic cleft by two Na(+)-and Cl(-)-dependent transporters: GLYT1 and GLYT2. GLYT1, expressed in glial processes of glycinergic areas and in glia and neurons of glutamatergic pathways that contain N-methyl-d-aspartate (NMDA) receptors, is essential for regulating glycine levels both at glycinergic and NMDA-containing synapses. GLYT2 is the transporter present in glycinergic neurons and provides cytosolic glycine for vesicular release from glycinergic terminals. GLYT1 is selectively inhibited by the sarcosine derivative N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)-propyl]sarcosine (NFPS). In the present report, GLYT1-GLYT2 chimeric transporters have been generated and their inhibition by NFPS has been studied. The introduction of GLYT2 transmembrane domains (TMs) 1 or 3, but not 2, on GLYT1 structure reduced the inhibition potency of NFPS and sarcosine. Binding studies and kinetic analysis of NFPS inhibition indicate lower affinity and smaller sensitivity of the chimeras to the compound. Opposite chimeras containing TM1 or TM3 of GLYT1 on GLYT2 structure became sensitive to NFPS. Individual substitution mutants of GLYT2 TM1 residues on GLYT1 and opposite GLYT1 TM1 residues on GLYT2 indicate that the more N-terminal portion of GLYT1 including residue E40 contributes to NFPS specificity. Our results demonstrate that TM1 and TM3, but not TM2, contain residues involved in the specific action of NFPS on GLYT1.
authors.affiliation: Centro de Biología Molecular "Severo Ochoa", Facultad de Ciencias, Universidad Autónoma de Madrid, Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain.
authors.forename: Enrique
authors.initials: E
authors.lastname: Núñez
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Martínez-Maza
authors.forename: Arjan
authors.initials: A
authors.lastname: Geerlings
authors.forename: Carmen
authors.initials: C
authors.lastname: Aragón
authors.forename: Beatriz
authors.initials: B
authors.lastname: López-Corcuera
mesh_terms: D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D005786:Gene Expression Regulation / Q000187:drug effects*; D005998:Glycine / Q000378:metabolism* / Q000494:pharmacology; D050487:Glycine Plasma Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D016296:Mutagenesis / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000502:physiology; D012521:Sarcosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors; D014316:Tritium / Q000378:metabolism
chemical_list: D050487:Glycine Plasma Membrane Transport Proteins; C435025:N-(3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)-3-(4'-phenylphenoxy)propyl)sarcosine; D014316:Tritium; D005998:Glycine; D012521:Sarcosine