Neuron NMDA receptors

Structure-function relationship and mechanisms N-methyl-D-aspartate receptor (NMDAR) antagonism

One of the research programs at the Keck Center is towards understanding the underlying mechanism of aberrant activation of NMDAR during ischemic stroke. Currently, the only FDA approved treatment for ischemic stroke is tissue plasminogen activator (tPA) that is effective only when administered with 3 hours of stroke onset. Our working hypothesis is that insights in understanding the NMDAR-driven pathological changes accompanying ischemia will provide new channels for developing therapeutic treatment.

The NMDAR are cation permeable gated by the neurotransmitter glutamate and co-agonist glycine. The receptors are hetero-oligomeric proteins consisting of the mandatory GluN1 (a-h) subunit and a GluN2 (A-D) subunit that confers upon the functional ion channels its unique electrophysiological, biochemical, and pharmacological properties. Our laboratory has characterized the NMDAR using specific antagonists, the conantokins and its effect on NMDA/glutamate-induced neurotoxicity. Conantokins, though originally found as peptides in the venom of marine snails are synthesized at the Keck Center, and contain the unusual post-transitionally modified amino acid, Υ-carboxyglutamate. Electrophysiological assays and real-time imaging data have shown that conantokins inhibited NMDA-induced excitoxicity, as well as have effects on downstream signaling events and neuronal integrity in dissociated neurons. Conantokins are also employed as pharmacological tools to assess changes in intrinsic electrophysiological properties in genetically altered mice that lack a specific GluN subunit and in which stroke has been induced. This is achieved by performing intracellular recordings of neurons by whole cell patch-clamp electrophysiology in ex vivo brain slices of mice.

Studying structure-function relationship of NMDAR inhibition by conantokins involves designing strategic variants of these peptides and quantifying their inhibitory potency towards recombinant NMDARs or in neurons lacking a particular GluN subunit. This helps to identify conantokin residues involved in NMDAR-specific antagonism. Biophysical approaches involving NMR spectroscopy elucidates cation-binding interactions with conantokins in terms of structural stability and its biological efficacy. Additionally, modular components of the extracellular regions of the GluN1 and GluN2 subunits are being expressed to structurally characterize the binding to glutamate/NMDA and conantokins. Thus, biochemical studies on the structural requirements of conantokins for NMDAR antagonism are beneficial for the development of improved treatment of neuropathologies.

Representation of the NMDAR spanning the neuronal membrane

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Links: Ischemic Strokes; NMDA Receptors; Stroke Treatments

References 

Yuan Y, Balsara RD, Zajicek J, Kunda S, Castellino FJ. Discerning the Role of the Hydroxyproline Residue in the Structure of Conantokin Rl-B and Its Role in GluN2B Subunit-Selective Antagonistic Activity toward N-Methyl-d-Aspartate Receptors. Biochemistry. 2016 Dec 27;55(51):7112-7122. doi: 10.1021/acs.biochem.6b00962. Epub 2016 Dec 16. PMID: 27981829

Cheriyan J, Balsara RD, Hansen KB, Castellino FJ. Pharmacology of triheteromeric N-Methyl-D-Aspartate Receptors. Neurosci Lett. 2016 Mar 23;617:240-6. doi: 10.1016/j.neulet.2016.02.032. Epub 2016 Feb 23. PMID: 26917100

Kunda S, Yuan Y, Balsara RD, Zaijcek J, Castellino FJ. Hydroxyproline-induced Helical Disruption in Conantokin RI-B Affects Subunit-selective Antagonistic Activities Toward Ion Channels of NMDA Receptors. J Biol Chem. 2015 Jun 5. pii: jbc.M115.650341. [Epub ahead of print] PMID: 26048991

Balsara R, Dang A, Donahue DL, Snow T, Castellino FJ. Conanntokin-G Attenuates Detrimental Effects of NMDAR Hyperactivity in an Ischemic Rat Model of Stroke. PLoS One. 2015 Mar 30;10(3):e0122840.doi: 10.1371/journal.pone.0122840. eColletion 2015. PMID: 25822337

Cheriyan J, Mezes C, Zhou N, Balsara RD, Castellino FJ. Heteromerization of ligand binding domains of N-methyl-D-aspartate receptor requires both coagonists, L-glutamate and glycine. Biochemistry. 2015 Jan 27;54(3):787-94.doi: 10.1021/bi501437s. Epub 2015 Jan 8. PMID: 25544544

Balsara RD, Chapman SE, Sander IM, Donahue DL, Liepert L, Catellino FJ, Leevy WM. Non-invasivie imaging and analysis of cerebral ischemia in living rats using positron emission tomography with 18F-FDG.J Vis Exp.2014 Dec 28;(94).doi: 10.3791/51495.PMID: 25590998

Balsara RD, Ferreira AN, Donahue DL, Castellino FJ, Sheets PL. Probing NMDA receptor GluN2A and GluN2B subunit expression and distribution in cortical neurons. Neuropharmacology. 2014 Apr;79:542-9. doi: 10.1016/j.neuropharm.2014.01.005. Epub 2014 Jan 15. PMID:24440368

Kunda S, Cheriyan J, Hur M, Balsara RD, Castellino FJ (2013). Antagonist properties of Conus parius peptides on N-methyl-D-aspartate receptors and their effects on CREB signaling. PLoS One. Nov 18;8(11):e81405. doi: 10.1371/journal.pone.0081405. eCollection 2013. PMID:24260577

Balsara RD, Li N, Weber-Adrian D, Huang L, Castellino FJ. (2012). Opposing action of conantokin-G on synaptically and extrasynaptically-activated NMDA receptors. Neuropharmacology 62(7): 2226-2237. Jun;62(7):2227-38. doi: 10.1016/j.neuropharm.2012.01.018. Epub 2012 Jan 27. PMID:22306487.

Cheriyan J, Balsara RD, Hansen KB, Castellino FJ. Pharmacology of triheteromeric N-Methyl-d-Aspartate Receptors. Neurosci Lett. 2016 Mar 23;617:240-6. doi: 10.1016/j.neulet.2016.02.032. Epub 2016 Feb 23.  PMID: 26917100 [PubMed - in process]