Abstract
Antagonists that are sufficiently selective to preferentially block GluN2A-containing N-methyl-d-aspartate receptors (NMDARs) over GluN2B-containing NMDARs are few in number. In this study we describe a pharmacological characterization of 3-chloro-4-fluoro-N-[4-[[2-(phenylcarbonyl)hydrazino]carbonyl]benzyl]benzenesulphonamide (TCN 201), a sulphonamide derivative, that was recently identified from a high-throughput screen as a potential GluN2A-selective antagonist. Using two-electrode voltage-clamp (TEVC) recordings of NMDAR currents from Xenopus laevis oocytes expressing either GluN1/GluN2A or GluN1/GluN2B NMDARs we demonstrate the selective antagonism by TCN 201 of GluN2A-containing NMDARs. The degree of inhibition produced by TCN 201 is dependent on the concentration of the GluN1-site co-agonist, glycine (or d-serine), and is independent of the glutamate concentration. This GluN1 agonist-dependency is similar to that observed for a related GluN2A-selective antagonist, N-(cyclohexylmethyl)-2-[{5-[(phenylmethyl)amino]-1,3,4-thiadiazol-2-yl}thio]acetamide (TCN 213). Schild analysis of TCN 201 antagonism indicates that it acts in a non-competitive manner but its equilibrium constant at GluN1/GluN2A NMDARs indicates TCN 201 is around 30-times more potent than TCN 213. In cortical neurones TCN 201 shows only modest antagonism of NMDAR-mediated currents recorded from young (DIV 9–10) neurones where GluN2B expression predominates. In older cultures (DIV 15–18) or in cultures where GluN2A subunits have been over-expressed TCN 201 gives a strong block that is negatively correlated with the degree of block produced by the GluN2B-selective antagonist, ifenprodil. Nevertheless, while TCN 201 is a potent antagonist it must be borne in mind that its ability to block GluN2A-containing NMDARs is dependent on the GluN1-agonist concentration and is limited by its low solubility.
Highlights
N-methyl-D-aspartate receptors (NMDARs) are a subtype of the ionotropic glutamate receptor family and are tetrameric assemblies, the majority of which contain two GluN1 subunits and two GluN2 subunits (Collingridge et al, 2009; Traynelis et al, 2010)
Inhibition curves for TCN 201 acting at GluN1/GluN2A NMDARs activated by glutamate and glycine or D-serine
We quantified the potency of TCN 201 by determining its IC50 value at GluN1/GluN2A NMDARs under conditions where either glycine or D-serine was used as the GluN1-site agonist
Summary
NMDARs are a subtype of the ionotropic glutamate receptor family and are tetrameric assemblies, the majority of which contain two GluN1 subunits and two GluN2 subunits (Collingridge et al, 2009; Traynelis et al, 2010). Alternative splicing of exons 5, 21 and 22 gives rise to eight GluN1 subunit isoforms (Sugihara et al, 1992) while 4 separate genes encode GluN2A-D subunits. These GluN2 subunits are spatially and temporally regulated Antagonism of NMDARs can be achieved by blocking agonist function at either GluN1 or GluN2 LBDs
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