title: A novel dualistic profile of an allosteric AMPA receptor modulator identified through studies on recombinant receptors, mouse hippocampal synapses and crystal structures.
abstract: Positive allosteric modulators (PAMs) of 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors receive increasing interest as therapeutic drugs and have long served as important experimental tools in the study of the molecular mechanisms underlying glutamate-mediated neurotransmission. The aim of this study was to investigate functional and structural aspects of a novel analog of the AMPA receptor PAM cyclothiazide (CTZ) on recombinant and native glutamate receptors. We expressed rat GluA4flip and flop in Xenopus oocytes and characterized NS1376 and CTZ under two-electrode voltage-clamp. The dose-response analyses revealed dual effects of NS1376. The modulator induced 30-fold and 42-fold reductions in glutamate potency and increased the glutamate efficacy by 3.2-fold and 5.3-fold at GluA4flip and GluA4flop, respectively. Rapid application of glutamate to excised outside-out patches showed that NS1376 markedly attenuated desensitization, supporting the increased efficacy observed in the oocytes. Furthermore, when applied to acutely isolated mouse brain slices, NS1376 reduced the field excitatory postsynaptic potentials (fEPSPs) in the hippocampus to 51.6 ± 4.3% of baseline, likely as a consequence of reduced glutamate potency. However, the modulator displayed no effects on a sub-maximal long-term potentiation (LTP) protocol. We confirmed that CTZ increases presynaptic transmitter release, a property which was not shared by NS1376. Finally, we obtained detailed molecular information through X-ray structures, docking and molecular dynamics, which revealed that NS1376 interacts at the dimer interface of the ligand-binding domain in a manner overall similar to CTZ. NS1376 reveals that minor structural changes in CTZ can result in an altered modulatory profile, both enhancing agonist efficacy while markedly reducing agonist potency. These unique properties add new aspects to the complexity of allosteric modulations in neuronal systems.
authors.affiliation: Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark.
authors.forename: G B
authors.initials: GB
authors.lastname: Christiansen
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Jagtvej 162,, DK-2100 Copenhagen, Denmark.
authors.forename: B
authors.initials: B
authors.lastname: Harbak
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark; NeuroSearch A/S, Pederstrupvej 93, DK-2750 Ballerup, Denmark.
authors.forename: S E
authors.initials: SE
authors.lastname: Hede
authors.affiliation: NeuroSearch A/S, Pederstrupvej 93, DK-2750 Ballerup, Denmark.
authors.forename: A H
authors.initials: AH
authors.lastname: Gouliaev
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Jagtvej 162,, DK-2100 Copenhagen, Denmark.
authors.forename: L
authors.initials: L
authors.lastname: Olsen
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Jagtvej 162,, DK-2100 Copenhagen, Denmark.
authors.forename: K
authors.initials: K
authors.lastname: Frydenvang
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark; H. Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, Denmark.
authors.forename: J
authors.initials: J
authors.lastname: Egebjerg
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Jagtvej 162,, DK-2100 Copenhagen, Denmark. Electronic address: jsk@sund.ku.dk.
authors.forename: J S
authors.initials: JS
authors.lastname: Kastrup
authors.affiliation: Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark; Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark. Electronic address: mmh@biomed.au.dk.
authors.forename: M M
authors.initials: MM
authors.lastname: Holm
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects*; D000818:Animals; D001581:Benzothiadiazines / Q000737:chemistry / Q000494:pharmacology*; D019706:Excitatory Postsynaptic Potentials / Q000187:drug effects; D018698:Glutamic Acid / Q000494:pharmacology; D006624:Hippocampus / Q000187:drug effects / Q000502:physiology*; D017774:Long-Term Potentiation / Q000187:drug effects; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018091:Receptors, AMPA / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects; D013569:Synapses / Q000187:drug effects / Q000502:physiology*; D014982:Xenopus laevis
chemical_list: D001581:Benzothiadiazines; D018091:Receptors, AMPA; D011994:Recombinant Proteins; D018698:Glutamic Acid; C004639:cyclothiazide
keywords: AMPA receptor; allosteric modulator; crystal structure; hippocampus; outside-out patch; synaptic plasticity