Kainate induces various domain closures in AMPA and kainate receptors Raminta Venskutonyte ˙ a , Karla Frydenvang a , Helle Hald a,b , Anna Ceravalls de Rabassa a , Michael Gajhede a , Philip K. Ahring b , Jette Sandholm Kastrup a,⇑ a Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark b NeuroSearch A/S, Drug Discovery, Pederstrupvej 93, DK-2750 Ballerup, Denmark article info Article history: Available online 7 March 2012 Keywords: Ionotropic glutamate receptors Crystal structures Domain closure Binding site interactions Electrophysiology abstract Ionotropic glutamate receptors are key players in fast excitatory synaptic transmission within the central nervous system. These receptors have been divided into three subfamilies: the N-methyl-d-aspartic acid (NMDA), 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) and kainate receptors. Kainate has previously been crystallized with the ligand binding domain (LBD) of AMPA receptors (GluA2 and GluA4) and kainate receptors (GluK1 and GluK2). Here, we report the structures of the kainate recep- tor GluK3 LBD in complex with kainate and GluK1 LBD in complex with kainate in the absence of glycerol. Kainate introduces a conformational change in GluK3 LBD comparable to that of GluK2, but different from the conformational changes induced in GluA2 and GluK1. Compared to their domain closures in a gluta- mate bound state, GluA2 and GluK1 become more open and kainate induces a domain closure of 60% and 62%, respectively, relative to glutamate (100%). In GluK2 and GluK3 with kainate, the domain closure is 88% and 83%, respectively. In previously determined structures of GluK1 LBD in complex with kainate, glycerol is present in the binding site where it bridges interlobe residues and thus, might contribute to the large domain opening. However, the structure of GluK1 LBD with kainate in the absence of glycerol confirms that the observed domain closure is not an artifact of crystallization conditions. Comparison of the LBD structures with glutamate and kainate reveals that contacts are lost upon binding of kainate in the three kainate receptors, which is in contrast to the AMPA receptors where similar contacts are seen. It was revealed by patch clamp electrophysiology studies that kainate is a partial agonist at GluK1 with 36% efficacy compared to glutamate, which is in between the published efficacies of kainate at GluK2 and AMPA receptors. The ranking of efficacies seems to correlate with LBD domain closures. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Kainate was originally isolated from the seaweed Digenea simplex in 1953 (Moloney, 1998). The compound acts as a potent neuroexcitatory amino acid on the central nervous system and has been used for the induction of seizures in experimental ani- mals (Tanaka et al., 1992). Kainate targets an important class of excitatory ion channel receptors in the central nervous system, i.e. the ionotropic glutamate receptors (iGluRs). These receptors allow cations to pass the channel pore, triggered by conformational rearrangement and channel opening upon binding of glutamate (Traynelis et al., 2010). Binding of the excitatory neurotransmitter glutamate to iGluRs is a key step in the mechanism of fast excit- atory synaptic transmission among nerve cells within the central nervous system. Present in more than 80% of all excitatory syn- apses, iGluRs are critically important for normal brain function. The iGluRs are tetrameric membrane bound receptors. They have been divided into three subfamilies based on their pharma- cology and sequence similarity: 2-amino-3-(3-hydroxy-5-methyl- 4-isoxazolyl)propionic acid (AMPA) receptors comprising subunits GluA1–4, N-methyl-d-aspartic acid (NMDA) receptors comprising subunits GluN1, GluN2A–D and GluN3A, B and kainate receptors comprising GluK1–5 (Traynelis et al., 2010). NMDA receptors differ from other iGluRs as in addition to glutamate they also require glycine for activation. One additional subfamily of iGluRs was de- scribed later on, the so-called delta receptors comprising subunits GluD1 and GluD2. Whereas AMPA receptors bind kainate with micromolar affini- ties, nanomolar affinities are seen at the kainate receptors (Sagot et al., 2008; Keinänen et al., 1990). GluK4–5 subunits cannot form 0197-0186/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2012.02.016 Abbreviations: AMPA, 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid; ATD, amino-terminal domain; ATPA, 2-amino-3-(5-tert-butyl-3-hydroxy-4- isoxazolyl)propanoic acid; CHO, Chinese hamster ovary; CTD, intracellular carboxy- terminal domain; iGluRs, ionotropic glutamate receptors; LBD, ligand binding domain; NMDA, N-methyl-d-aspartic acid; TMD, transmembrane domain. ⇑ Corresponding author. Tel.: +45 3533 6000; fax: +45 3533 6001. E-mail address: jsk@farma.ku.dk (J.S. Kastrup). Neurochemistry International 61 (2012) 536–545 Contents lists available at SciVerse ScienceDirect Neurochemistry International journal homepage: www.elsevier.com/locate/nci