Subunit-Selective Allosteric Inhibition of Glycine Binding to NMDA Receptors

Abstract

NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission in the brain, and are involved in numerous neuropathological conditions. NMDA receptors are activated upon simultaneous binding of co-agonists glycine and glutamate to the GluN1 and GluN2 subunits, respectively. Subunit-selective modulation of NMDA receptor function by ligand binding to modulatory sites distinct from the agonist binding sites could allow pharmacological intervention with therapeutically beneficial mechanisms. Here, we show the mechanism of action for TCN-201, a new GluN1/GluN2A-selective NMDA receptor antagonist whose inhibition can be surmounted by glycine. Electrophysiological recordings from chimeric and mutant rat NMDA receptors suggest that TCN-201 binds to a novel allosteric site located at the dimer interface between the GluN1 and GluN2 agonist binding domains. Furthermore, we demonstrate that occupancy of this site by TCN-201 inhibits NMDA receptor function by reducing glycine potency. TCN-201 is therefore a negative allosteric modulator of glycine binding.

Contribution of the M1 Transmembrane Helix and Pre-M1 Region to Positive Allosteric Modulation and Gating of N-Methyl-d-Aspartate Receptors

“…1) and is critical for glycine-dependent inhibition by and N-(cyclohexylmethyl)-2-({5-[(phenylmethyl)amino]-1,3,4-thiadiazol-2-yl}thio)acetamide (TCN-213) (Hansen et al, 2012;McKay et al, 2012). Given the role of the ABD dimer interface in mediating allosteric coupling between the ATD and the ion channel gate (Gielen et al, 2008(Gielen et al, , 2009, and the importance of the ABD dimer interface of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors for the activity of positive modulators such as cyclothiazide (Sun et al, 2002), we asked whether the ABD dimer interface of NMDA receptors might contribute to potentiation by CIQ.…”

“…Given the role of the ABD dimer interface in mediating allosteric coupling between the ATD and the ion channel gate (Gielen et al, 2008(Gielen et al, , 2009, and the importance of the ABD dimer interface of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors for the activity of positive modulators such as cyclothiazide (Sun et al, 2002), we asked whether the ABD dimer interface of NMDA receptors might contribute to potentiation by CIQ. Two residues in GluN1 contributing to the dimer interface, Phe754 and Arg755, were critical for inhibition of GluN1/GluN2A receptors by TCN-201 (Hansen et al, 2012), and mutation of these residues to alanine altered the binding of TCN-201. By contrast, CIQ potentiation was…”

“…3E). The S2 segment of the GluN2 ABD also contributes residues to the ABD dimer interface and introducing this region of GluN2A into GluN2D conferred inhibition by TCN-201 to GluN2D (Hansen et al, 2012). Yet, CIQ potentiation was not diminished in GluN2D chimeric receptors containing the entire ABD from GluN2A [i.e., 2D(2A S1S2)] or just the S2 segment, as in the chimera 2D(2A S2) (Fig.…”

“…The transmembrane domain consists of three transmembrane helices-M1, M3, and M4-and a re-entrant pore loop, called M2. NMDA receptors have several allosteric sites, including the side-to-side GluN1/ GluN2 dimer interface of the ATD (Karakas et al, 2011;Mony et al, 2011), the back-to-back GluN1/GluN2 dimer interface of the ABD (Gielen et al, 2008;Hansen et al, 2012) and the ion channel pore (Antonov and Johnson, 1996;Kashiwagi et al, 2002;Blanpied et al, 2005).…”

Sensitivity of N-Methyl-d-Aspartate Receptor–Mediated Excitatory Postsynaptic Potentials and Synaptic Plasticity to TCN 201 and TCN 213 in Rat Hippocampal Slices

“…Because TCN 201 is reported to be a negative allostereic modulator of glycine binding in GluN1/2A receptors (Edman et al, 2012;Hansen et al, 2012), we examined it in the presence of bitopertin, a glycine uptake inhibitor. It has been reported that 100 nM, but not 300 nM, bitopertin facilitates LTP induction in rat slices (Alberati et al, 2012).…”