Residues at the tip of the pore loop of NR3B-containing NMDA receptors determine Ca2+ permeability and Mg2+ block.

BACKGROUND: Members of the complex N-methyl-D-aspartate receptor (NMDAR) subfamily of ionotropic glutamate receptors (iGluRs) conventionally assemble from NR1 and NR2 subunits, the composition of which determines receptor properties. Hallmark features of conventional NMDARs include the requirement for a coagonist, voltage-dependent block by Mg2+, and high permeability for Ca2+. Both Mg2+ sensitivity and Ca2+ permeability are critically dependent on the amino acids at the N and N+1 positions of NR1 and NR2. The recently discovered NR3 subunits feature an unprecedented glycine-arginine combination at those critical sites within the pore. Diheteromers assembled from NR1 and NR3 are not blocked by Mg2+ and are not permeable for Ca2+. RESULTS: Employing site-directed mutagenesis of receptor subunits, electrophysiological characterization of mutants in a heterologous expression system, and molecular modeling of the NMDAR pore region, we have investigated the contribution of the unusual NR3 N and N+1 site residues to the unique functional characteristics of receptors containing these subunits. Contrary to previous studies, we provide evidence that both the NR3 N and N+1 site amino acids are critically involved in mediating the unique pore properties. Ca2+ permeability could be rescued by mutating the NR3 N site glycine to the NR1-like asparagine. Voltage-dependent Mg2+ block could be established by providing an Mg2+ coordination site at either the NR3 N or N+1 positions. Conversely, "conventional" receptors assembled from NR1 and NR2 could be made Mg2+ insensitive and Ca2+ impermeable by equipping either subunit with the NR3-like glycine at their N positions, with a stronger contribution of the NR1 subunit. CONCLUSIONS: This study sheds light on the structure-function relationship of the least characterized member of the NMDAR subfamily. Contrary to previous reports, we provide evidence for a critical functional involvement of the NR3 N and N+1 site amino acids, and propose them to be the essential determinants for the unique pore properties mediated by this subunit.

Revisiting the postulated "unitary glutamate receptor": electrophysiological and pharmacological analysis in two heterologous expression systems fails to detect evidence for its existence.

Several years ago evidence for a so-called "unitary glutamate receptor" was published. This unique type of glutamate receptor was reported to be activated by the traditional agonists of all three major glutamate receptor subfamilies [i.e., alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), kainate, and N-methyl-d-aspartate (NMDA)] in a glycine-dependent as well as magnesium-blockable manner and was reported to consist of an NR1 subunit coexpressed with the kainate binding protein (KBP) from Xenopus laevis, XenU1. To re-examine the existence of such a receptor, we cloned two splice variants of the X. laevis NMDA receptor subunit NR1, XenNR1-4a and XenNR1-4b, and expressed them in X. laevis oocytes as well as in human embryonic kidney (HEK) 293 cells, either alone or with the X. laevis KBP subunit XenU1. In addition, we coexpressed XenU1 separately with all eight splice variants of the rat NR1 subunit. In no case did we see evidence of a unitary glutamate receptor pharmacology. In HEK293 cells, we did not get receptor response unless an NR2 subunit was coexpressed. In X. laevis oocytes, we did observe responses to glutamate/glycine as well as small responses to glycine alone, but these were independent of coexpressed XenU1. Neither AMPA nor kainate ever elicited significant responses. Because we verified that XenU1 is expressed and inserted into the plasma membrane of HEK293 cells, we conclude that XenU1 and NR1 do not form the postulated unitary glutamate receptor. Furthermore, successful amplification of a fragment of a X. laevis NR2 subunit indicates that X. laevis uses NR2 subunits and not XenU1 to form heteromeric complexes with NR1.