Functional Roles of a Conserved Tryptophan at Subunit-Subunit Interfaces in NMDA Receptor Membrane Regions

Abstract

NMDA receptors (NMDARs) are ionotropic glutamate receptors (iGluRs) characterized by channel block by extracellular magnesium (Mg2+o) and slow deactivation kinetics. NMDARs are typically composed of two GluN1 and two GluN2 (GluN2A-GluN2D) subunits. The transmembrane domain (TMD) of each subunit contains three membrane-spanning regions (M1, M3, and M4) and a pore lining re-entrant loop (M2). The M2 region of each NMDAR subunit contains a tryptophan (W) that is highly conserved: nearly all known mammalian iGluR subunits, several potassium channels, and iGluR-like proteins in phylogenetically distant organisms contain W at the homologous site. This highly conserved W is located in NMDARs at the interface between the M2 region and the adjacent M3 region. Previous work demonstrated that mutation of this highly-conserved W in the GluN2B subunit (W607) drastically decreased block by Mg2+o, while mutation of the homologous residue in the GluN2A subunit (W606) had a relatively minor effect (Williams et al., 1998 Mol Pharmacol 53, 933). Interestingly, mutating the homologous residue in GluN1 subunits (W608) also has little effect on Mg2+o block (Siegler Retchless et al., 2012 Nat Neurosci 15, 406). To further explore the function of the conserved W at subunit-subunit interfaces we used whole-cell recordings from recombinant receptors expressed in tsA201 cells. We examined GluN2A(W606) and GluN2B(W607) mutants expressed with wild-type GluN1 subunits or GluN1 subunits with mutations of M3 residues at the M2-M3 interface. We found that receptors containing mutations at both GluN2A(W606) and a residue in the adjacent interface-lining GluN1 M3 region dramatically decreased block by Mg2+o. We also examined GluN1(W608) mutants and observed profoundly slowed deactivation kinetics. Overall, these results suggest that the conserved W is broadly involved in channel properties and gating of NMDARs.