A novel NMDA receptor positive allosteric modulator that acts via the transmembrane domain

Abstract

Ionotropic glutamate receptors (iGluRs) mediate fast excitatory neurotransmission and are key nervous system drug targets. While diverse pharmacological tools have yielded insight into iGluR extracellular domain function, less is known about molecular mechanisms underlying the ion conduction gating process within the transmembrane domain (TMD). We have discovered a novel NMDAR positive allosteric modulator (PAM), GNE-9278, with a unique binding site on the extracellular surface of the TMD. Mutation of a single residue near the Lurcher motif on GluN1 M3 can convert GNE-9278 modulation from positive to negative, and replacing three AMPAR pre-M1 residues with corresponding NMDAR residues can confer GNE-9278 sensitivity to AMPARs. Modulation by GNE-9278 is state-dependent and significantly alters extracellular domain pharmacology. The unique properties and structural determinants of GNE-9278 reveal new modulatory potential of the iGluR TMD.

An NMDAR positive and negative allosteric modulator series share a binding site and are interconverted by methyl groups

“…Most PAMs were also capable of potentiating GluN1/GluN2B ( Table 1 , Figure 2A ). As seen with other series of NMDAR PAMs ( Malayev et al, 2002 ; Horak et al, 2004 ; Horak et al, 2006 ; Hackos et al, 2016 ; Wang et al, 2017 ), allosteric modulation can be dependent on agonist concentrations. Thus, we assessed the ability of EU1794-27 to modulate NMDAR responses activated by sub-saturating co-agonist concentrations, clearly exemplified by modulation of GluN1/GluN2C ( Figure 2B , Table 2 ).…”

“…Given that other NMDAR modulators have displayed agonist-dependence ( Petrovic et al, 2005 ; Acker et al, 2011 ; Hansen and Traynelis, 2011 ; Borovska et al, 2012 ; Vyklicky et al, 2015 ; Wang et al, 2017 ), we performed rapid solution exchange experiments to examine if these modulators had different affinities at agonist-bound and apo receptors ( Figure 5A,B ). The instantaneous current response to a rapid step into glutamate plus glycine (from glycine alone) gives an estimate of whether a modulator was pre-bound to receptors with only glycine (but not glutamate) bound.…”

“…We therefore examined a GluA2 AMPA receptor structure bound to CP-465,022, which shares a core scaffold with QNZ modulators ( Yelshanskaya et al, 2016 ). Residues identified as being important for CP-465,022 binding in Yelshanskaya et al, 2016 were aligned to the GluN1 and GluN2D subunits to map this modulatory site onto the NMDAR subunits, in addition to critical residues for GNE-9278 in this same region ( Figure 6A , Wang et al, 2017 ). This broader range of residues were located on the pre-M1, M3, and pre-M4 regions of both GluN1 and GluN2D, which have previously been suggested to cooperate to control gating ( Ogden and Traynelis, 2013 ; Alsaloum et al, 2016 ; Chen et al, 2017 ; Ogden et al, 2017 ; Yelshanskaya et al, 2017 ).…”

“… ( A ) Ribbon representation ( left ) of a GluN1/GluN2D model based on published crystal structures of GluN1/GluN2B and GluA2 ( Karakas and Furukawa, 2014 ; Lee et al, 2014 ; Yelshanskaya et al, 2016 ). The GluN1 subunits are grey and the GluN2D are purple; surface shell indicate residues of interest in GluN1 and GluN2D, including residues that are homologous to those identified in recent studies investigating transmembrane domain interacting modulators of NMDAR and AMPAR ( Yelshanskaya et al, 2016 ; Wang et al, 2017 ). These GluN1 and GluN2D residues were changed to alanine (or tyrosine in two cases, as previously reported) and tested for effects on the NMDAR sensitivity to EU1794-2 and EU1794-27 .…”

“…An increasing number of NMDAR and AMPAR modulators have been identified with structural determinants of action that reside in transmembrane linker regions and extracellular portions of the transmembrane domain ( Mullasseril et al, 2010 ; Acker et al, 2011 ; Hansen and Traynelis, 2011 ; Ogden and Traynelis, 2013 ; Yelshanskaya et al, 2016 ; Swanger et al, 2018 ; Wang et al, 2017 ). Other cell surface receptor families have bi-directional modulator pockets, including multiple GPCRs as well as the benzodiazepine binding site in GABA-A receptors ( Barnard et al, 1998 ; Rudolph and Knoflach, 2011 ; Wootten et al, 2013 ).…”