Tuning activation of the AMPA-sensitive GluR2 ion channel by genetic adjustment of agonist-induced conformational changes.
The (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazole) propionic acid (AMPA) receptor discriminates between agonists in terms of binding and channel gating; AMPA is a high-affinity full agonist, whereas kainate is a low-affinity partial agonist. Although there is extensive literature on the functional characterization of partial agonist activity in ion channels, structure-based mechanisms are scarce. Here we investigate the role of Leu-650, a binding cleft residue conserved among AMPA receptors, in maintaining agonist specificity and regulating agonist binding and channel gating by using physiological, x-ray crystallographic, and biochemical techniques. Changing Leu-650 to Thr yields a receptor that responds more potently and efficaciously to kainate and less potently and efficaciously to AMPA relative to the WT receptor. Crystal structures of the Leu-650 to Thr mutant reveal an increase in domain closure in the kainate-bound state and a partially closed and a fully closed conformation in the AMPA-bound form. Our results indicate that agonists can induce a range of conformations in the GluR2 ligand-binding core and that domain closure is directly correlated to channel activation. The partially closed, AMPA-bound conformation of the L650T mutant likely captures the structure of an agonist-bound, inactive state of the receptor. Together with previously solved structures, we have determined a mechanism of agonist binding and subsequent conformational rearrangements.
Department of Biochemistry and Molecular Biophysics and Howard Hughes Medical Institute, Columbia University, New York, NY 10032, USA.
Neali
N
Armstrong
Mark
M
Mayer
Eric
E
Gouaux
D019943:Amino Acid Substitution; D000818:Animals; D018360:Crystallography, X-Ray; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology*; D005260:Female; D007608:Kainic Acid / Q000494:pharmacology*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010053:Ovary / Q000502:physiology; D011487:Protein Conformation / Q000187:drug effects; D017433:Protein Structure, Secondary; D018091:Receptors, AMPA / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D014982:Xenopus laevis; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology*
D018690:Excitatory Amino Acid Agonists; D018091:Receptors, AMPA; D011994:Recombinant Proteins; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; C104722:glutamate receptor ionotropic, AMPA 2; D007608:Kainic Acid