title: Pharmacology and expression analysis of glycine transporter GlyT1 with [3H]-(N-[3-(4'-fluorophenyl)-3-(4'phenylphenoxy)propyl])sarcosine.
abstract: In the central nervous system, re-uptake of the neurotransmitter glycine is mediated by two different glycine transporters, GlyT1 and GlyT2. GlyT2 is found in brainstem and spinal cord, whereas GlyT1 is expressed in rat forebrain regions where it is responsible for most glycine transport activity. Initially, GlyT1 and GlyT2 were pharmacologically differentiated by sarcosine, a weak selective inhibitor of GlyT1. The recently described selective and potent GlyT1 antagonist, NFPS/ALX-5407 provided an important additional tool to further characterize GlyT1 pharmacology. In the present study, we have radiolabeled the racemic form of NFPS (N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl])sarcosine (also known as ALX-5407) to investigate its interaction with GlyT1, as well as define GlyT1 expression in the rat central nervous system. Kinetic studies indicated that [3H]NFPS binds rapidly to rat forebrain membranes and dissociates with a t(1/2) of 28 +/- 5 min. [3H]NFPS labeled a saturable population of sites in rat forebrain with a Kd of 7.1+/-1.3 nM and a B(max) of 3.14 +/- 0.26 pmol/mg protein. Bound [3H]NFPS was fully and potently displaced by unlabeled NFPS, whereas glycine and sarcosine were weak, Na+-dependent inhibitors with IC50 of 1,008 and 190 microM, respectively. Additional saturation experiments indicated that glycine and sarcosine were non-competitive antagonists of [3H]NFPS binding. Functional studies revealed that NFPS was a non-competitive inhibitor of [3H]glycine uptake and does not interact with Na+ and Cl- binding sites of GlyT1. Overall, this work shows that [3H]NFPS is a valuable tool in studying GlyT1 expression and pharmacology and that NFPS interacts with GlyT1 at a site different from the transporter translocation and ion binding sites.
authors.affiliation: Merck and Co. Inc., Department of Neuroscience, West Point, WP26A-3000, P.O. Box 4, West Point, PA 19486, USA.
authors.forename: Pierre J
authors.initials: PJ
authors.lastname: Mallorga
authors.forename: Jacinta B
authors.initials: JB
authors.lastname: Williams
authors.forename: Marlene
authors.initials: M
authors.lastname: Jacobson
authors.forename: Rosemary
authors.initials: R
authors.lastname: Marques
authors.forename: Ashok
authors.initials: A
authors.lastname: Chaudhary
authors.forename: P Jeffrey
authors.initials: PJ
authors.lastname: Conn
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Pettibone
authors.forename: Cyrille
authors.initials: C
authors.lastname: Sur
mesh_terms: D026921:Amino Acid Transport Systems, Neutral / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000704:Analysis of Variance; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002490:Central Nervous System / Q000187:drug effects* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005998:Glycine / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D050487:Glycine Plasma Membrane Transport Proteins; D006801:Humans; D007700:Kinetics; D018021:Lithium Chloride / Q000494:pharmacology; D011869:Radioligand Assay / Q000379:methods; D051381:Rats; D012521:Sarcosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D019346:Sodium Acetate / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013997:Time Factors; D014316:Tritium / Q000378:metabolism
chemical_list: D026921:Amino Acid Transport Systems, Neutral; D050487:Glycine Plasma Membrane Transport Proteins; C435025:N-(3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)-3-(4'-phenylphenoxy)propyl)sarcosine; C493271:SLC6A5 protein, human; C493268:SLC6A9 protein, human; C493269:Slc6a5 protein, rat; C493266:Slc6a9 protein, rat; C435781:org 24598; D014316:Tritium; D012965:Sodium Chloride; D019346:Sodium Acetate; D018021:Lithium Chloride; D005998:Glycine; D012521:Sarcosine