Disruption Of S2-M4 Linker Coupling Reveals Novel Subunit-specific ...

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Abstract

The N-methyl-d-aspartate (NMDA) receptor is a glutamatergic ion channel and is a known site of ethanol action. Evidence suggests that ethanol inhibits NMDA receptor activity by reducing channel open probability and mean open time potentially via interaction with specific residues within the transmembrane (M) domains 3 and 4 of GluN subunits. Recent models of NMDAR function demonstrate that extracellular residues near the M domains are key regulators of gating, suggesting that they may contribute to ethanol sensitivity. To test this, we substituted cysteines at key positions in GluN1 and GluN2 M3-S2 and S2-M4 regions previously shown to affect channel open probability and mean open time similar to ethanol treatment. Although crosslinking of these domains was predicted to restrict linker domain movement and occlude ethanol inhibition, only intra-GluN1 M1:M4 linker-crosslinked receptors showed a decrease in ethanol sensitivity. For the converse experiment, we expressed NMDARs with glycine substitutions in the S2-M4 domain of GluN subunits to enhance M4 flexibility and recorded currents in response to ethanol. Glycine substitution in the GluN1 S2-M4 region significantly decreased glutamate potency of GluN1(A804G)/GluN2A receptors, while GluN1(A804G)/GluN2B receptors exhibited no change in glutamate sensitivity. In contrast, GluN1/GluN2B(S811G) receptors showed a 10-fold increase in glutamate potency while GluN1/GluN2A(S810G) receptors showed no change. Surprisingly, while S2-M4 glycine substitutions modulated ethanol sensitivity, this was observed only in receptors that did not display a change in agonist potency. Overall, these results suggest that S2-M4 linkers strongly influence receptor function and modestly impact ethanol efficacy in a subunit- and receptor subtype-dependent manner.

Keywords: Channel function; Electrophysiology; Ethanol; N-methyl-D-aspartate receptors; Recombinant expression.

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Figures

Figure 1

Figure 1

Sequence of extracellular linkers and…

Figure 1

Sequence of extracellular linkers and structural topology of GluN subunits. A , Amino…

Figure 1 Sequence of extracellular linkers and structural topology of GluN subunits. A, Amino acid sequence of S1-M1 and S2-M4 linker domains of GluN1 and GluN2 subunits. B, Cartoon depicts the broad structural topology of GluN subunits, highlighting the locations of the ligand binding domain-forming S1/S2 domains, transmembrane (TM) domains, and the S1-M1/S2-M4 linker domains. C–D, Structural models showing the relative locations of cysteine-substituted residues of inter- and intra-subunit crosslinked GluN2B- (C) and GluN2A-containing (D) receptors. MacPymol (The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC) was used to render structures of GluN1/GluN2B (PDB ID: 4PE5; Karakas and Furukawa, 2014) and a homology model of the GluN1/GluN2A receptor described in a previous study (Xu et al., 2012) that was based on the GluA2 structure (PDB ID: 3KG2; Sobolevsky et al., 2009).
Figure 2

Figure 2

Concentration-dependent inhibition of crosslinked receptors…

Figure 2

Concentration-dependent inhibition of crosslinked receptors by ethanol. A , Intra-GluN1 crosslinked GluN2A receptors…

Figure 2 Concentration-dependent inhibition of crosslinked receptors by ethanol. A, Intra-GluN1 crosslinked GluN2A receptors showed a significant reduction in ethanol sensitivity compared to GluN1/GluN2A wild type receptors. Bar graph shows mean ± S.E.M. inhibition of agonist-evoked currents by 30, 100, and 300 mM doses of ethanol (* p < 0.05; two-way ANOVA with Dunnett’s test; effect of mutation, F3,87 = 5.002; N = 6-10 cells). B, Representative trace showing current inhibition by 300 mM ethanol of a GluN1/GluN2A wild type NMDAR. C, Similar to results observed in GluN1/GluN2A receptors, only intra-GluN1 crosslinked GluN2B receptors showed a significant reduction in ethanol sensitivity compared to GluN1/GluN2B wild type receptors. Bar graph shows mean ± S.E.M. inhibition of agonist-evoked currents by 30, 100, and 300 mM doses of ethanol (* p < 0.05; two-way ANOVA with Dunnett’s test; effect of mutation, F3,93 = 7.232; N = 7-10 cells). D, Representative trace showing current inhibition by 300 mM ethanol of a GluN1/GluN2B wild type NMDAR.
Figure 3

Figure 3

Crosslinking M1 and M4 linker…

Figure 3

Crosslinking M1 and M4 linker domains of GluN1/2A receptors alters receptor function. A, …

Figure 3 Crosslinking M1 and M4 linker domains of GluN1/2A receptors alters receptor function. A, Mean current amplitude of GluN1/GluN2A wild type, intra-, and inter-subunit crosslinked receptors in response to 10 uM glutamate/glycine. Bar graph shows mean amplitude ± S.E.M. of WT and mutant receptors from 6–10 cells(** p < 0.001, *** p < 0.0005; one-way ANOVA with Dunnett’s test). B, DTT treatment enhances steady state current amplitude of GluN1/GluN2A crosslinked receptors. Bar graph shows mean percent potentiation of current amplitude by DTT (10 mM; 20 s) ± S.E.M. from 6–10 cells (# p < 0.05; one-sample t-test: **** p < 0.0001; one-way ANOVA with Dunnett’s test). C, Schematic diagram showing location of cysteine-substituted residues in intra- and inter-subunit M1:M4 linker crosslinked GluN1/GluN2A receptors. D, Representative trace demonstrating potentiation of intra-GluN2A crosslinked receptors by DTT treatment.
Figure 4

Figure 4

Crosslinking M1 and M4 linker…

Figure 4

Crosslinking M1 and M4 linker domains of GluN1/2B receptors alters receptor function. A, …

Figure 4 Crosslinking M1 and M4 linker domains of GluN1/2B receptors alters receptor function. A, Bar graph shows mean amplitude ± S.E.M. of wild type and mutant GluN1/GluN2B receptors from 7–10 cells (*** p < 0.0005; one-way ANOVA with Dunnett’s test). B, Effects of DTT treatment on intra- and inter-subunit crosslinked GluN1/GluN2B receptors. Bar graph shows mean ± S.E.M. percent potentiation of current amplitude by DTT (10 mM; 20 s) from 7–10 cells (# p < 0.05; one-sample t-test: **** p < 0.0001; one-way ANOVA with Dunnett’s test). C, Schematic diagram showing location of cysteine-substituted residues in intra- and inter-subunit M1:M4 linker crosslinked GluN1/GluN2B receptors. D, Representative trace demonstrating potentiation of intra-GluN2B crosslinked receptors by DTT.
Figure 5

Figure 5

Pre-TM4 glycine mutations in GluN1/GluN2A…

Figure 5

Pre-TM4 glycine mutations in GluN1/GluN2A and GluN1/GluN2B receptors alter glutamate potency. A, Representative…

Figure 5 Pre-TM4 glycine mutations in GluN1/GluN2A and GluN1/GluN2B receptors alter glutamate potency. A, Representative traces from a GluN1/GluN2A-expressing cell showing response to 0.1 μM, 3 μM, and 30 μM concentrations of glutamate. B/C, Concentration-response curves (B) and summary graph (C) of glutamate EC50 values for GluN1/GluN2A wild type and glycine-substituted mutants. Curves shown are best fits to the equation given in Materials and methods. Bar graph shows mean ± S.E.M. EC50 values for glutamate-activated currents in wild type and mutant GluN1/GluN2A receptors. Data for GluN2A receptors are from 7–9 cells (* p < 0.05; one-way ANOVA with Dunnett’s test). D, Representative traces from a GluN1/GluN2B-expressing cell showing response to 0.03 μM, 1 μM, and 10 μM concentrations of glutamate. E/F, Concentration-response curves (E) and summary graph (F) of glutamate EC50 values for GluN1/GluN2B wild type and glycine-substituted mutants. Curves shown are best fits to the equation given in Materials and methods. Bar graph shows mean ± S.E.M. EC50 values for glutamate-activated currents in wild type and mutant GluN1/GluN2B receptors. Data for GluN2B receptors are from 7–9 cells (* p < 0.05, **** p < 0.0001; one-way ANOVA with Dunnett’s test).
Figure 6

Figure 6

Changes in glutamate potency in…

Figure 6

Changes in glutamate potency in response to mutation of Pre-TM4 GluN2B residues. A, …

Figure 6 Changes in glutamate potency in response to mutation of Pre-TM4 GluN2B residues. A, Substitution of serine (S) 811 of GluN2B with an alanine (A) or aspartate (D) did not significantly change glutamate potency, while mutation of the preceding S810 residue to a glycine produced a significant leftward shift in glutamate potency. For comparison, the effect of GluN2B (S811G) on glutamate potency (data from Figure 5) is represented by the dashed line. Data shown are mean EC50 values ± S.E.M. derived from individual curve fits of 5–10 cells using the equation given in Materials and Methods (*** p < 0.001; one-way ANOVA and Dunnett’s test). B, Substitution of glycine at analogous positions in GluN1 did not alter glutamate potency, while glutamate EC50 of GluN2B (S810G) mutant was not affected by also substituting a glycine at position 811. Data shown are mean ± S.E.M. EC50 values derived from individual curve fits of 5–10 cells using the equation given in Materials and Methods (* p < 0.05; one-way ANOVA and Dunnett’s test).
Figure 7

Figure 7

Concentration-dependent inhibition of glycine-substituted receptors…

Figure 7

Concentration-dependent inhibition of glycine-substituted receptors by ethanol. A, GluN2A (S810G) mutant showed a…

Figure 7 Concentration-dependent inhibition of glycine-substituted receptors by ethanol. A, GluN2A (S810G) mutant showed a significant reduction in ethanol sensitivity compared to wild type GluN2A. Data shown are mean ± S.E.M. inhibition of agonist-evoked currents across three ethanol doses (* p < 0.05; two-way ANOVA with Dunnett’s test, effect of mutation, F2,63 = 5.91, N = 7-10 per group). B, In contrast to GluN1/GluN2A receptors, GluN1 (A804G)/GluN2B significantly increased ethanol sensitivity, with no change observed in GluN2B (S811G) mutants. Data shown are mean ± S.E.M. inhibition of agonist-evoked currents across three ethanol doses (* p < 0.05; two-way ANOVA with Dunnett’s test, effect of mutation, F2,90 = 7.517, N = 7-18 per group).
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References

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