Article Figures & Data
Figures
- 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 Po 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 Po (p < 0.05).
Tables
- Table 1.
Rate constants for entry and recovery from desensitization based on single-channel recordings and macroscopic recovery rates
Total events Entry rate, s−1 Recovery rate, s−1 Macroscopic recovery rate, s 1/recovery rate, s−1 GluN1/GluN2A 1,995,992 0.07 ± 0.01 0.48 ± 0.08 0.92 ± 0.07 1.08 ± 0.08 (16) (5) GluN1 (F540L)/ 812,088 0.31 ± 0.09* 0.49 ± 0.08 1.02 ± 0.06 0.98 ± 0.06 GluN2A (4) (3) GluN1/ 25,475 2.68 ± 0.81* 0.13 ± 0.04* 4.02 ± 0.22* 0.25 ± 0.01* GluN2A (F534L) (3) (8) Mean values (±SEM) for the entry and recovery rates derived from the reciprocal of the mean cluster and intercluster durations, respectively, from single-channel recordings (Fig. 3). The number of patches is in parentheses below the total number of events. Macroscopic recovery rates from desensitization and their reciprocal (mean ± SEM) are shown in the right two columns. The number of recordings is shown in parentheses. Significance is shown relative to GluN1/GluN2A (*p < 0.05, Student's t test).
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