Molecular Determinants of Proton Sensitivity and Gating in NMDA Receptors
Murthy, Swetha E.
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NMDA receptors are glutamate-activated ion-channels and are required for fast excitatory transmission and synaptic plasticity in mammalian brain. Over-activation of these receptors mediates neurotoxic effects implicated in neurodegenerative disorders. NMDA receptor activity is attenuated by physiologic concentrations of extracellular protons (IC 50 = pH 7.3) and by acidification during periods of intense glutamate signaling. Inhibition of NMDA receptor activity by protons plays a neuroprotective role during regular biological processes and during pathological conditions. NMDA receptors are tetramers of two GluN1 and two GluN2 subunits. The GluN1 subunit is encoded by a single gene, which undergoes alternative splicing of three exons: 5, 21 and 22, resulting in 8 splice variants. The GluN2 subunit is encoded by four genes, GluN2A-D. NMDA receptor isoforms differ in regional and developmental expression, as well as in kinetics and pharmacology, including sensitivity to protons. GluN1-1a/2A receptors (-exon 5) are more sensitive to protons compared to GluN1-1b/2A receptors (+exon 5). In NMDA receptors, the proton binding site is unresolved; however, certain regions in the N-terminal domain and transmembrane domain have been implicated. In my thesis, I investigated the mechanism by which molecular determinants of NMDA receptors in NTD (specifically exon 5) and TMD (residues in the Lurcher motif) alter the sensitivity to protons. My results show that exon 5 does not alter gating kinetics of NMDA receptors when the proton sensor is unprotonated or protonated. Proton inhibition mechanism is similar between the two splice variants, which is to stabilize the closed conformation and destabilize the open state of the receptor. Exon 5 reduces sensitivity of the receptor to protons by reducing the affinity to protons. Additionally, I propose that exon 5 can interact with the proton binding site therefore hindering the interaction with protons. In NMDA receptors, the M3 helix of the TMD contains a highly conserved 9 amino acid motif, Lurcher (SYTANLAAF). Previous studies have shown that an alanine to tyrosine substitution at position 7 of this motif reduces sensitivity of the receptor to protons. I tested the effect of this substitution (A7Y) on NMDA receptor gating and proton sensitivity. The results obtained showed that the A7Y substitution alters gating kinetics by stabilizing the receptor in the open conformation resulting in a high channel MOT. The reduced sensitivity to protons arises from the altered gating kinetics conferred by the mutation. Further, my results are consistent with the hypothesis that A7 forms the agonist controlled gate and tightly couples ligand binding to channel opening. Additional substitutions made at A7 and A8 positions of the motif showed that these residues contribute in regulating gating of NMDA receptors in a subunit specific fashion. Based on these results I propose that in the reaction mechanism of NMDA receptors, the two pre-open gating steps are controlled by the movement of the M3 helix in the GluN2 subunit followed by the GluN1 subunit, after which the channel opens. Taken together, the results obtained in this thesis further our understanding of the complex nature of NMDA receptor gating. The detailed analysis of the mechanism by which specific sites of NMDA receptors alter sensitivity to protons helps present these sites as potential drug targets to control NMDA receptor activity during pathological conditions. Taken together, the results obtained in this thesis further our understanding of the complex nature of NMDA receptor gating. The detailed analysis of the mechanism by which specific sites of NMDA receptors alter sensitivity to protons helps present these sites as potential drug targets to control NMDA receptor activity during pathological conditions.