Extracellular loops 2 and 4 of GLYT2 are required for N-arachidonylglycine inhibition of glycine transport.
Concentrations of extracellular glycine in the central nervous system are regulated by Na(+)/Cl(-)-dependent glycine transporters, GLYT1 and GLYT2. N-Arachidonylglycine (NAGly) is an endogenous inhibitor of GLYT2 with little or no effect on GLYT1 and is analgesic in rat models of neuropathic and inflammatory pain. Understanding the molecular basis of NAGly interactions with GLYT2 may allow for the development of novel therapeutics. In this study, chimeric transporters were used to determine the structural basis for differences in NAGly sensitivity between GLYT1 and GLYT2 and also the actions of a series of related N-arachidonyl amino acids. Extracellular loops 2 and 4 of GLYT2 are important in the selective inhibition of GLYT2 by NAGly and by the related compounds N-arachidonyl-gamma-aminobutyric acid and N-arachidonyl-d-alanine, whereas only the extracellular loop 4 of GLYT2 is required for N-arachidonyl-l-alanine inhibition of transport. These observations suggest that the structure of the head group of these compounds is important in determining how they interact with extracellular loops 2 and 4 of GLYT2. Site-directed mutagenesis of GLYT2 EL4 residues was used to identify the key residues Arg(531), Lys(532), and Ile(545) that contribute to the differences in NAGly sensitivity.
Edington
Amelia R
AR
Transporter Biology Group, Discipline of Pharmacology, School of Medical Sciences, Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia.
McKinzie
Audra A
AA
Transporter Biology Group, Discipline of Pharmacology, School of Medical Sciences, Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia.
Reynolds
Aaron J
AJ
Brain and Mind Research Institute, 100 Mallett Street, Camperdown, New South Wales 2050, Australia; School of Chemistry, University of Sydney, Sydney, New South Wales 2006, Australia.
Kassiou
Michael
M
Brain and Mind Research Institute, 100 Mallett Street, Camperdown, New South Wales 2050, Australia; School of Chemistry, University of Sydney, Sydney, New South Wales 2006, Australia; Discipline of Medical Radiation Sciences, University of Sydney, Sydney, New South Wales 2006, Australia.
Ryan
Renae M
RM
Transporter Biology Group, Discipline of Pharmacology, School of Medical Sciences, Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia.
Vandenberg
Robert J
RJ
Transporter Biology Group, Discipline of Pharmacology, School of Medical Sciences, Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia. Electronic address: rvan9242@mail.usyd.edu.au.
D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects / Q000235:genetics; D005998:Glycine / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D050487:Glycine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010146:Pain / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
D001095:Arachidonic Acids; D050487:Glycine Plasma Membrane Transport Proteins; C465623:N-arachidonylglycine; D011993:Recombinant Fusion Proteins; C493269:Slc6a5 protein, rat; C493266:Slc6a9 protein, rat; D005998:Glycine