Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC181

Poster Communications

Selective inhibition of GluN2A-containing NMDARs by an antagonist acting at the GluN1 subunit.

P. A. Butters1, N. Griffiths1, E. Thubron1, S. McKay1, G. E. Hardingham1, D. J. Wyllie1

1. Centre for Integrative Physiology, University of Edinburgh, Edinburgh, United Kingdom.


The ability to discriminate between subtypes of N-methyl-D-aspartate receptors (NMDARs) is highly desirable in order to determine their various contributions to physiological and pathophysiological processes in the CNS. In the forebrain GluN2A and GluN2B are the most abundant GluN2 NMDAR subunits but a selective antagonist that inhibits GluN2A-containing while sparing GluN2B-containing NMDARs is lacking (Frizelle et al. 2006). A recent report by Bettini et al. (2010) indicated that N-(cyclohexylmethyl)-2-({5-[(phenylmethyl)amino]-1,3,4-thiadiazol-2-yl}thio)acetamide (referred to as Compound 13 in their study and abbreviated here to N-CPTTA) showed selective antagonism of recombinant GluN1/GluN2A NMDARs while having little action at GluN1/GluN2B NMDARs. We have characterized of the antagonist action of N-CPTTA in both recombinant and native NMDARs. Our data indicate that N-CPTTA is able, under certain conditions, to selectively block GluN2A-containing NMDARs while displaying no antagonism at GluN2B-containing NMDARs but does so by acting at the GluN1 NMDAR subunit. Two-electrode voltage-clamp recordings were made from Xenopus laevis oocytes expressing either GluN1/GluN2A or GluN1/GluN2B rat recombinant NMDARs. For GluN1/GluN2A NMDARs antagonism by N-CPTTA was dependent on the glycine concentration used in our external recording solution and was independent of the glutamate concentration. Concentrations of N-CPTTA that produced 50% inhibition of glutamate (100 μM) evoked currents when the external glycine concentration was 150 nM, 1.5 μM and 15 μM were 0.55 ± 0.05, 3.5 ± 0.3 and 39.7 ± 3.4 μM respectively (n = 7, 7, 6). Antagonism by N-CPTTA was surmountable and competitive with Schild analysis giving an estimated KB value of 2 μM (n = 5). For GluN1/GluN2B NMDARs N-CPTTA (10 μM) only produced modest antagonism (5.5 ± 3.4%; n = 6) of agonist evoked currents even when the glycine concentration was low (75 nM) i.e. 10% of its EC50 value at GluN2B-containing NMDARs (Chen et al. 2008). The prototypical competitive glycine-site antagonist, 5,7 dichlorokynurenic acid, did not show selective antagonism and gave comparable IC50 values at GluN2A- and GluN2B-containing NMDARs when equi-potent glycine concentrations were used. Additionally we examined the effects of N-CPTTA acting at NMDARs expressed in rat cultured cortical neurones at a stage where NMDARs predominantly contain GluN2B subunits. Our data indicate that N-CPTTA produces little block of NMDA-evoked currents whereas ifenprodil, a selective blocker of GluN2B-containing NMDARs produced antagonism of around 80%. The fact that N-CPTTA gives selective block of GluN2A-containing NMDARs is being used in on-going investigations to determine the NMDAR subunit dependence of NMDA-induced excitotoxic cell death of rat cultured cortical neurones.

Where applicable, experiments conform with Society ethical requirements