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Pharmacology and Structural Analysis of Ligand Binding to the Orthosteric Site of Glutamate-Like GluD2 Receptors.

ABSTRACT: The GluD2 receptor is a fundamental component of postsynaptic sites in Purkinje neurons, and is required for normal cerebellar function. GluD2 and the closely related GluD1 are classified as members of the ionotropic glutamate receptor (iGluR) superfamily on the basis of sequence similarity, but do not bind l-glutamate. The amino acid neurotransmitter D-Ser is a GluD2 receptor ligand, and endogenous D-Ser signaling through GluD2 has recently been shown to regulate endocytosis of ?-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type iGluRs during synaptic plasticity in the cerebellum, such as long-term depression. Here, we investigate the pharmacology of the orthosteric binding site in GluD2 by examining the activity of analogs of D-Ser and GluN1 glycine site competitive antagonists at GluD2 receptors containing the lurcher mutation (GluD2(LC)), which promotes spontaneous channel activation. We identify several compounds that modulate GluD2(LC), including a halogenated alanine analog as well as the kynurenic acid analog 7-chloro-4-oxo-1H-quinoline-2-carboxylic acid (7-chlorokynurenic acid; 7-CKA). By correlating thermodynamic and structural data for 7-CKA binding to the isolated GluD2 ligand binding domain (GluD2-LBD), we find that binding 7-CKA to GluD2-LBD differs from D-Ser by inducing an intermediate cleft closure of the clamshell-shaped LBD. The GluD2 ligands identified here can potentially serve as a starting point for development of GluD2-selective ligands useful as tools in studies of the signaling role of the GluD2 receptor in the brain.

SUBMITTER: Kristensen AS

PROVIDER: S-EPMC4746485 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Pharmacology and Structural Analysis of Ligand Binding to the Orthosteric Site of Glutamate-Like GluD2 Receptors.

Kristensen Anders S AS Hansen Kasper B KB Naur Peter P Olsen Lars L Kurtkaya Natalie L NL Dravid Shashank M SM Kvist Trine T Yi Feng F Pøhlsgaard Jacob J Clausen Rasmus P RP Gajhede Michael M Kastrup Jette S JS Traynelis Stephen F SF

Molecular pharmacology 20151210 2

The GluD2 receptor is a fundamental component of postsynaptic sites in Purkinje neurons, and is required for normal cerebellar function. GluD2 and the closely related GluD1 are classified as members of the ionotropic glutamate receptor (iGluR) superfamily on the basis of sequence similarity, but do not bind l-glutamate. The amino acid neurotransmitter D-Ser is a GluD2 receptor ligand, and endogenous D-Ser signaling through GluD2 has recently been shown to regulate endocytosis of α-amino-3-hydrox ...[more]

PMID: 26661043

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Project description:Background and purposeThe binding of transmitter to specialized binding pockets leads to rearrangements in the structure of the receptor eventually resulting in channel opening. We used voltage-clamp fluorometry to investigate the pharmacological basis and biophysical processes that underlie structural changes at the transmitter binding site of the rat ?1?2?2L GABA(A) receptor.Experimental approachSimultaneous electrophysiological and site-specific fluorescence measurements were conducted on receptors expressed in Xenopus oocytes and labelled with an environmentally-sensitive fluorophore, Alexa 546 maleimide, at the ?1L127C site.Key resultsReceptors activated by GABA demonstrate a concentration-dependent increase in fluorescence intensity, indicating that the environment surrounding the fluorophore becomes less polar upon activation. Qualitatively similar responses were observed with other GABA site ligands such as piperidine-4-sulphonic acid, muscimol, ?-alanine and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol. Fluorescence changes were not affected by the direction of current flow. During long applications of GABA significant desensitization developed, which was not accompanied by additional changes in fluorescence. Pentobarbital was an efficacious agonist of the labelled mutant receptor but did not cause changes in fluorescence. Direct activation by etomidate or the steroid allopregnanolone also did not result in fluorescence changes. Functional potentiation of GABA-activated receptors by allopregnanolone or etomidate enhanced both the GABA-elicited functional response and the fluorescence change. In contrast, potentiation by pentobarbital was not accompanied by an enhanced fluorescence response.Conclusions and implicationsThe data indicate that there is no direct correlation between current flow or position of the activation gate and the structural changes as detected by Alexa 546-labelled ?1L127C?2?2L GABA(A) receptors. Channel potentiation by pentobarbital qualitatively differs from potentiation by etomidate or allopregnanolone.

Dataset Information

Pharmacology and Structural Analysis of Ligand Binding to the Orthosteric Site of Glutamate-Like GluD2 Receptors.

Publications

Pharmacology and Structural Analysis of Ligand Binding to the Orthosteric Site of Glutamate-Like GluD2 Receptors.

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