Molecular Basis of NMDA Receptor Allosteric Regulation by New Subunit-selective Modulators Open Access

Ogden, Kevin (2013)

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NMDA receptors are glutamate-gated ion channels that mediate excitatory synaptic transmission in the central nervous system and are critical for learning, cognition, and neuronal development. Dysfunction of NMDA receptors has been implicated in neurological and psychiatric disorders ranging from stroke to schizophrenia. NMDA receptors are tetrameric ion channels comprising GluN1, GluN2, and GluN3 subunits. The four GluN2 subunits, GluN2A, GluN2B, GluN2C, and GluN2D, substantially contribute to functional diversity of NMDA receptors and have distinct expression patterns in the CNS. The promise that subunit-selective allosteric modulators differentially targeting the GluN2 subunit could provide an opportunity to modify the function of select groups of neurons for therapeutic gain has resulted in a handful of new compounds that appear to act at novel sites. In this dissertation, I present data that define the mechanism and site of action of two new classes of NMDA receptor allosteric modulators. I show that a class of tetrahydroisoquinolines, which selectively potentiates GluN2C- and GluN2D-containing NMDA receptors and is exemplified by the molecule CIQ, does not act at previously recognized modulatory sites. Rather, I identified critical determinants of CIQ modulation in the region near the first transmembrane helix of GluN2D, including in a putative pre-M1 cuff helix that may influence channel gating. Further, I investigated the mechanism and molecular determinants of selectivity for a new class of GluN2A-selective antagonists represented by the compound TCN-201. I found that TCN-201 inhibits GluN1/GluN2A receptors by decreasing the potency of the GluN1 agonist in an allosteric manner. Mutagenesis and chimeric data coupled with Schild analysis suggest that TCN-201 binds to a novel allosteric site located at the dimer interface between GluN1 and GluN2 agonist binding domains. Lastly, I show that the pre-M1 region of GluN2A affects desensitization and is critical for normal gating of NMDA receptors. Overall, the data presented here demonstrate new modulatory sites on the NMDA receptor and should facilitate development of novel tools and therapeutics with advantageous mechanisms of action and subunit-selectivity.

Table of Contents

Chapter 1: Introduction Molecular Composition of NMDA Receptors Architecture of NMDA Receptors Carboxy-terminal region Amino-terminal domain Agonist binding in NMDA receptors ABD cleft closure may contribute to NMDA receptor gating Quaternary structure and subunit arrangement Ion channel pore NMDA Receptor Gating NMDA Receptor Function NMDA Receptors in Neurological and Psychiatric Disorders Pharmacology of NMDA Receptors

Recently identified positive modulators improve upon known endogenous potentiators

Non-competitive antagonists target unique modulatory sites with useful subunit-selectivity

Competitive antagonists and channel blockers target highly conserved regions with little selectivity across NMDA receptor subtypes

Conclusion Chapter 2: Materials and Methods DNA Constructs General Molecular Biology Procedures Quikchange Reactions ATD Deletion Constructs DNA Ligations cRNA Synthesis cRNA Injection of Xenopus laevis Oocytes Two-electrode Voltage-clamp Recordings HEK Cell Culture Transfection Whole-cell Patch-clamp Recordings Single-channel Recordings Data Analysis Single-channel analysis Simulations

Chapter 3: Contribution of the M1 transmembrane helix and pre-M1 region to positive allosteric modulation and gating of N-methyl-D-aspartate receptors

Abstract Introduction Results CIQ Does Not Act at Known Modulatory Sites Residues in the M1 helix affect CIQ potentiation Pre-M1 residues control channel open probability CIQ cannot reach its modulatory site by diffusion through the membrane Discussion

Structural determinants of CIQ potentiation reside in the transmembrane region

Role of pre-M1 region in gating

Chapter 4: Subunit-Selective Allosteric Inhibition of Glycine Binding to NMDA Receptors1

Abstract Introduction Results Binding of TCN-201 reduces potency of glycine at the GluN1 subunit TCN-201 is not a competitive antagonist at the GluN1 subunit

Inhibition by TCN-201 is controlled by the agonist binding domain interface

Residue Val783 in GluN2A influences binding of TCN-201 TCN-201 inhibition is mediated by residues from both GluN1 and GluN2A TCN-201 inhibition is mediated by a multi-step mechanism

TCN-201 binding is differentially modulated by glutamate and glycine binding

TCN-201 binding accelerates glycine deactivation TCN-201 is a negative allosteric modulator of glycine binding Discussion

Chapter 5: The pre-M1 region is a critical gating element in NMDA receptors

Abstract Introduction Results Single-channel activity of GluN1/GluN2A NMDA Receptors Effects of pre-M1 Mutations

Gating Impairments in the GluN2 pre-M1 Region Disrupt the Slow Gating Isomerization of NMDA Receptors

Discussion Chapter 6: Discussion TCN-201: a new non-competitive GluN2A-selective antagonist GluN2A-Selective Antagonism TCN-201 Site of Action - ABD Dimer Interface Effects of TCN-201 on Triheteromeric NMDA Receptors Does TCN-201 Change the Gating Equilibrium? Clinical Utility of a GluN2A-Selective Antagonist CIQ: a novel GluN2C- and GluN2D-selective positive allosteric modulator Effects of CIQ on Triheteromeric NMDA Receptors Effect of CIQ on Glutamate Potency Effects of CIQ on fear/emotional conditioned learning Conclusion Chapter 7: References

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