Disruption of S2-M4 linker coupling reveals novel subunit-specific contributions to N-methyl-d-aspartate receptor function and ethanol sensitivity

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.