Received for publication June 4, 2008.
Revised September 2, 2008.
Accepted for publication September 23, 2008.

Inhibitors of GlyT1 affect glycine transport via discrete binding sites

Abstract

In the forebrain, synaptic glycine concentrations are regulated through the glycine transporter GlyT1. Since glycine is a co-agonist of the NMDA receptor (NMDAR), which has been implicated in schizophrenia, inhibition of GlyT1 is thought to provide an option for the treatment of schizophrenia. In support of this hypothesis, GlyT1 inhibitors facilitate in vivo NMDAR function and demonstrate antipsychotic-like effects in animal models. Among the specific GlyT1 inhibitors, substituted N-methyl-glycine (sarcosine) derivatives (e.g. NFPS, (R)-NPTS and Org24589), and non-sarcosine containing inhibitors, such as SSR504734, have been described. In the present study, we analyzed the mode of interaction of these compounds with GlyT1 by using electrophysiological measurements in Xenopus oocytes, and with two binding assays, using [³H]-(R)-NPTS or [³H]-N-methyl-SSR504734 as radioligands. Inhibition of electrogenic glycine transport by sarcosine-based compounds was apparently irreversible and independent of glycine concentration. The latter indicates a non-competitive mode of action. In contrast, both SSR504734 and N-methyl-SSR504734 exhibited reversible and competitive inhibition of glycine transport. In GlyT1-expressing membranes, the binding of the novel radioligand[³H]-N-methyl-SSR504734 to a single site on GlyT1 was competitively displaced by glycine and SSR504734, but non-competitively by sarcosine-based compounds. Conversely, [³H]-(R)-NPTS binding was competitively inhibited by sarcosine-based compounds, while glycine, SSR504734 and N-methyl-SSR504734 non-competitively decreased maximal binding. Our data indicate that besides exerting an apparently irreversible or reversible inhibition, GlyT1 inhibitors differ by exhibiting either a non-competitive or competitive mode of inhibition. The divergent modes of inhibition may significantly impact the efficacy and tolerability of these drugs.

Key words: Ion channel regulation, Amino Acid