A Regulatory Domain (R1–R2) in the Amino Terminus of theN-Methyl-d-Aspartate Receptor: Effects of Spermine, Protons, and Ifenprodil, and Structural Similarity to Bacterial Leucine/Isoleucine/Valine Binding Protein

Abstract

There are complex interactions between spermine, protons, and ifenprodil at N-methyl-D-aspartate receptors. Spermine stimulation may involve relief of proton inhibition, whereas ifenprodil inhibition may involve an increase in proton inhibition. We studied mutations at acidic residues in the NR1 subunit using voltage-clamp recording of NR1/NR2B receptors expressed in Xenopus oocytes. Mutations at residues near the site of the exon-5 insert, including E181 and E185, reduced spermine stimulation and proton inhibition. Mutation NR1(D130N) reduced sensitivity to ifenprodil by more than 500-fold, but had little effect on sensitivity to spermine and pH. Mutations at six other residues in this region of the NR1 subunit reduced the potency and, in some cases, the maximum effect of ifenprodil. These mutants did not affect sensitivity to pH, glutamate, glycine, or other hallmark properties of N-methyl-D-aspartate channels such as Mg2+ block and Ba2+ permeability. Residues in this region presumably form part of the ifenprodil-binding site. To model this region of NR1 we compared the predicted secondary structure of NR1 (residues 19-400) with the known structures of 1,400 proteins. This region of NR1 is most similar to bacterial leucine/isoleucine/valine binding protein, a globular amino acid binding protein containing two lobes, similar to the downstream S1-S2 region of glutamate receptors. We propose that the tertiary structure of NR1(22-375) is similar to leucine/isoleucine/valine binding protein, containing two "regulatory" domains, which we term R1 and R2. This region, which contains the binding sites for spermine and ifenprodil, may influence the downstream S1 and S2 domains that constitute the glycine binding pocket.

Genetically encoding a light switch in an ionotropic glutamate receptor reveals subunit-specific interfaces

“…S1 A and B). In agreement with previous studies (20), we also observed that mutations at this position affect sensitivities to various allosteric modulators, including proton, zinc, and spermine (SI Appendix, Fig. S1 C-E).…”

Polyamine and Redox Modulation of [3H]MK‐801 Binding to N‐Methyl‐d‐aspartate Receptors in the Spinal Cord and Cerebral Cortex

“…Spermine stimulation of NMDA receptors has been suggested to be due to a relief of the proton inhibition (Traynelis et al 1995;Masuko et al 1999). Our data support this since the reduction in [ 3 H]MK-801 binding at low pH was abolished by spermine.…”

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Channels Lacking the N-terminal Domain

“…Glycine-independent desensitization of NMDA receptors was assigned to two regions in the NR2A subunit, the X domain and an area within the S1 segment (13,17). Moreover, participation of the N-terminal domain in the regulation of NMDA receptor channel function by allosteric modulators, including Zn 2ϩ ions (14,16), ifenprodil (8,15,16), spermine (8), protons (25), and redox agents (26), has been demonstrated. In some cases, this modulation has been shown to involve an direct allosteric interaction between the N-terminal domain and the agonist-binding domain (16).…”

“…In addition, an N-terminal ϳ400-residue segment preceding S1 shows sequence similarity to bacterial binding proteins specific for leucine (LBP) and leucine/isoleucine/valine (LIVBP) and to the N-terminal extracellular Nterminal segments of Class C G-protein-coupled receptors, which include the metabotropic glutamate receptors (3). The three-dimensional structure of this domain has not been determined, but homology models predict an LIVBP-like structure consisting of two lobes surrounding a central cleft (8,9).…”

“…First, this domain is implicated as a determinant in the assembly of oligomeric channels (10 -12). Second, the X domain may mediate the allosteric transitions involved in the channel activation, desensitization, or modulation by ions and drugs as has been observed for NMDA receptors (8,(13)(14)(15)(16)(17). Third, the X domain may provide docking sites for extracellular proteins, which serve to cluster the receptors or stabilize their localization.…”