A Molecular Determinant of Subtype-Specific Desensitization in Ionotropic Glutamate Receptors

Abstract

AMPA and NMDA receptors are glutamate-gated ion channels that mediate fast excitatory synaptic transmission throughout the nervous system. In the continual presence of glutamate, AMPA and NMDA receptors containing the GluN2A or GluN2B subunit enter into a nonconducting, desensitized state that can impact synaptic responses and glutamate-mediated excitotoxicity. The process of desensitization is dramatically different between subtypes, but the basis for these differences is unknown. We generated an extensive sequence alignment of ionotropic glutamate receptors (iGluRs) from diverse animal phyla and identified a highly conserved motif, which we termed the "hydrophobic box," located at the extracellular interface of transmembrane helices. A single position in the hydrophobic box differed between mammalian AMPA and NMDA receptors. Surprisingly, we find that an NMDAR-to-AMPAR exchange mutation at this position in the rat GluN2A or GluN2B subunit had a dramatic and highly specific effect on NMDAR desensitization, making it AMPAR-like. In contrast, a reverse exchange mutation in AMPARs had minimal effects on desensitization. These experiments highlight differences in desensitization between iGluR subtypes and the highly specific contribution of the GluN2 subunit to this process.

Significance statement: Rapid communication between cells in the nervous system depends on ion channels that are directly activated by neurotransmitter molecules. Here, we studied ionotropic glutamate receptors (iGluRs), which are ion channels activated by the neurotransmitter glutamate. By comparing the sequences of a vast number of iGluR proteins from diverse animal species, assisted by available structural information, we identified a highly conserved motif. We showed that a single amino acid difference in this motif between mammalian iGluR subtypes has dramatic effects on receptor function. These results have implications in both the evolution of synaptic function, as well as the role of iGluRs in health and disease.

Keywords: AMPA receptors; NMDA receptors; gating; sequence alignments; structures.

Figure 1.

MSA across iGluR family proteins. A, Segments of the MSA of a subset of 1047 animal iGluR subunits around the M1 and M3 transmembrane helices (see Materials and Methods). Some of the sequences were identified as specific iGluR subtypes, whereas as others are predicted or hypothetical. The most highly conserved position (99.7%) was a tryptophan (W) in the extracellular end of the M1 helix. We used this as a reference (“W”) for other positions in pre-M1. For positions in M3, we used as a reference the “S” of the highly conserved “SYTANLAAF” motif (Jones et al., 2002). B, Positions around the highly conserved W (green) in the GluA2 structure (3KG2; Sobolevsky et al., 2009). Chances of specific amino acids appearing at each position in the MSA are indicated. The variable position (W-5) was subject to exchange mutagenesis. We used the GluA2 structure (3KG2) for illustration because this region in the NMDAR structures is less resolved. C, Amino acid sequences of Rattus norvegicus iGluRs (NMDAR and AMPAR subunits). Position W-5 is an aromatic residue [phenylalanine (F) or tyrosine (Y)] in NMDAR subunits and L in AMPAR subunits.

Figure 2.

Membrane currents through wild-type and mutant AMPA and NMDA receptors containing exchange mutations at position W-5. A, Outside-out patch recording of membrane currents in HEK 293 cells transfected with wild-type GluA1 (top) or GluA1 containing an exchange mutation at position W-5 (L517F) (bottom). Glutamate (6 mm) was rapidly applied during the shaded box (see Materials and Methods) for 100 ms. Holding potential: −70 mV. B, Whole-cell recording of currents through wild-type GluN1/GluN2A. Glutamate (1 mm; shaded box) was applied for 2.5 s. Cell was continuously bathed in glycine (0.1 mm). Holding potential: −70 mV. C–E, Whole-cell recordings from NMDARs containing exchange mutations at position W-5 for either GluN1 (F540L) (C), GluN2A (F536L) (D), or both subunits (E). Currents recorded as in B. Gray current trace is from wild-type GluN1/GluN2A (B). F, G, Mean values (±SEM) for percentage desensitization (%des; F) and rate of desensitization (τ, ms; G). Significance is shown either relative to their respective wild-type construct (*) or for GluN1 (F540L)/GluN2A (F534L), also to (∧) GluN1/GluN2A (F534L) (p < 0.05, Student's t test). nq, Not quantified.

Figure 3.

Single-channel recordings of wild-type NMDARs and NMDARs with exchange mutations at position W-5. A–C, Example single-channel recordings of GluN1/GluN2A (A), GluN1 (F540L)/GluN2A (B), and GluN1/GluN2A (F534L) (C). Recordings were performed in the cell-attached configuration with a pipette potential of +100 mV. Downward deflections reflect inward currents. For each trace, the top half shows a low-resolution example (filtered at 1 kHz) and the bottom half shows a higher resolution portion of the same record (filtered at 3 kHz). Scale bar for all records is shown in C. D, Open P_o at equilibrium (solid symbols) or during clusters (open symbols) for GluN1/GluN2A (circles), GluN1 (F540L)/GluN2A (squares), and GluN1/GluN2A (F536L) (triangles). *Indicates significant difference from wild-type. #Indicates significant difference between equilibrium and cluster P_o (p < 0.05).