title: A charge-inverting mutation in the "linker" region of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors alters agonist binding and gating kinetics independently of allosteric modulators.
abstract: AMPA receptors are gated through binding of glutamate to a solvent-accessible ligand-binding domain. Upon glutamate binding, these receptors undergo a series of conformational rearrangements regulating channel function. Allosteric modulators can bind within a pocket adjacent to the ligand-binding domain to stabilize specific conformations and prevent desensitization. Yelshansky et al. (Yelshansky, M. V., Sobolevsky, A. I., Jatzke, C., and Wollmuth, L. P. (2004) J. Neurosci. 24, 4728-4736) described a model of an electrostatic interaction between the ligand-binding domain and linker region to the pore that regulated channel desensitization. To test this hypothesis, we have conducted a series of experiments focusing on the R628E mutation. Using ultrafast perfusion with voltage clamp, we applied glutamate to outside-out patches pulled from transiently transfected HEK 293 cells expressing wild type or R628E mutant GluA2. In response to a brief pulse of glutamate (1 ms), mutant receptors deactivated with significantly slower kinetics than wild type receptors. In addition, R628E receptors showed significantly more steady-state current in response to a prolonged (500-ms) glutamate application. These changes in receptor kinetics occur through a pathway that is independent of that of allosteric modulators, which show an additive effect on R628E receptors. In addition, ligand binding assays revealed the R628E mutation to have increased affinity for agonist. Finally, we reconciled experimental data with computer simulations that explicitly model mutant and modulator interactions. Our data suggest that R628E stabilizes the receptor closed cleft conformation by reducing agonist dissociation and the transition to the desensitized state. These results suggest that the AMPA receptor external vestibule is a viable target for new positive allosteric modulators.
authors.affiliation: Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523-1617.
authors.forename: Jonathan E
authors.initials: JE
authors.lastname: Harms
authors.affiliation: Neuroscience Institute, Morehouse School of Medicine, Atlanta, Georgia 30310-1495.
authors.forename: Morris
authors.initials: M
authors.lastname: Benveniste
authors.affiliation: Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, Illinois 62794-9629.
authors.forename: Markus
authors.initials: M
authors.lastname: Kessler
authors.affiliation: Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523-1617.
authors.forename: Leslie M
authors.initials: LM
authors.lastname: Stone
authors.affiliation: Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, Illinois 62794-9629.
authors.forename: Amy C
authors.initials: AC
authors.lastname: Arai
authors.affiliation: Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523-1617. Electronic address: Kathy.partin@colostate.edu.
authors.forename: Kathryn M
authors.initials: KM
authors.lastname: Partin
mesh_terms: D000495:Allosteric Site; D000818:Animals; D001665:Binding Sites; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008962:Models, Theoretical; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D018091:Receptors, AMPA / Q000737:chemistry* / Q000235:genetics*; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000737:chemistry*
chemical_list: D008024:Ligands; D018091:Receptors, AMPA; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; C104722:glutamate receptor ionotropic, AMPA 2
keywords: Computer Modeling; Electrophysiology; Gating; Glutamate Receptors, Ionotropic (AMPA, NMDA); Glutamate Receptors, Metabotropic; Structural Biology