Abstract
The N-methyl-D-aspartate (NMDA) glutamate receptor is a major target of ethanol in the brain. Previous studies have identified positions in the third and fourth membrane-associated (M) domains of the NMDA receptor GluN1 and GluN2A subunits that influence alcohol sensitivity. The predicted structure of the NMDA receptor, based on that of the related GluA2 subunit, indicates a close apposition of the alcohol-sensitive positions in M3 and M4 between the two subunit types. We tested the hypothesis that these positions interact to regulate receptor kinetics and ethanol sensitivity by using dual substitution mutants. In single-substitution mutants, we found that a position in both subunits adjacent to one previously identified, GluN1(Gly-638) and GluN2A(Phe-636), can strongly regulate ethanol sensitivity. Significant interactions affecting ethanol inhibition and receptor deactivation were observed at four pairs of positions in GluN1/GluN2A: Gly-638/Met-823, Phe-639/Leu-824, Met-818/Phe-636, and Leu-819/Phe-637; the latter pair also interacted with respect to desensitization. Two interactions involved a position in M4 of both subunits, GluN1(Met-818) and GluN2A(Leu-824), that does not by itself alter ethanol sensitivity, whereas a previously identified ethanol-sensitive position, GluN2A(Ala-825), did not unequivocally interact with any other position tested. These results also indicate a shift by one position of the predicted alignment of the GluN1 M4 domain. These findings have allowed for the refinement of the NMDA receptor M domain structure, demonstrate that this region can influence apparent agonist affinity, and support the existence of four sites of alcohol action on the NMDA receptor, each consisting of five amino acids at the M3-M4 domain intersubunit interfaces.
Highlights
The NMDA receptor is a major target of alcohol action in the brain
Two interactions involved a position in M4 of both subunits, GluN1(Met818) and GluN2A(Leu-824), that does not by itself alter ethanol sensitivity, whereas a previously identified ethanol-sensitive position, GluN2A(Ala-825), did not unequivocally interact with any other position tested. These results indicate a shift by one position of the predicted alignment of the GluN1 M4 domain. These findings have allowed for the refinement of the NMDA receptor M domain structure, demonstrate that this region can influence apparent agonist affinity, and support the existence of four sites of alcohol action on the NMDA receptor, each consisting of five amino acids at the M3-M4 domain intersubunit interfaces
Single Mutations in the M3 and M4 Domains Alter Ethanol Inhibition of NMDA Receptors—Based on the reported structure of the GluA2 glutamate receptor M domains [37] and previous findings from this and another laboratory, we predicted that sites of alcohol action in the NMDA receptor would be formed by groups of 4 – 6 residues clustered in small regions at the M3-M4 intersubunit interfaces (Fig. 1)
Summary
The NMDA receptor is a major target of alcohol action in the brain. Results: Four pairs of residues at the M3-M4 domain intersubunit interfaces interact to regulate gating and alcohol sensitivity. Ronald et al [32] initially demonstrated that mutations at Phe-639 in the M3 domain of the GluN1 subunit could influence ethanol inhibition of the NMDA receptor and that the characteristics of this position were consistent with a site of alcohol action Studies from this laboratory in the GluN2A subunit found that the cognate position, Phe-637, modulates ethanol inhibition [31] and identified two positions in the M4 domain, Met-823 and Ala825, that regulate ethanol sensitivity [29, 30]. In a previous study we found that these positions functionally interact with respect to ethanol sensitivity but that they do not appear to form a unitary site of alcohol action [34] This finding is consistent with a structural model of the NMDA receptor (supplemental information in Ref. 37) based upon that of the related GluA2 receptor [37]. We propose the existence of four sites of ethanol action on NMDA receptors located at the M3-M4 domain interfaces
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
