AMPA receptor ligand binding domain mobility revealed by functional cross linking.
Glutamate receptors mediate the majority of excitatory synaptic transmission in the CNS. The AMPA-subtype has rapid kinetics, with activation, deactivation and desensitization proceeding on the millisecond timescale or faster. Crystallographic, biochemical, and functional studies suggest that GluR2 Cys mutants which form intermolecular disulfide cross-links between the lower D2 lobes of the ligand binding cores can be trapped in a conformation that represents the desensitized state. We used multi-channel rapid perfusion techniques to examine the state dependence of cross-linking in these mutants. Under reducing conditions, both wild-type GluR2 and the G725C and S729C mutants have normal activation and desensitization kinetics, but the Cys mutants can be efficiently trapped in nonconducting states when oxidized. In contrast the I664C mutant is only partially inactivated under oxidizing conditions. For S729C, disulfide cross-links form rapidly when receptors are desensitized in the presence of glutamate, but receptors also become trapped at rest, in the absence of agonist. We assessed such spontaneous trapping in various conditions, including CNQX, a competitive antagonist; kainate, a weak partial agonist; or when desensitization was blocked by the L483Y mutation that stabilizes the D1 dimer interface. These experiments suggest that trapping in the absence of glutamate is due to two motions: Spontaneous breaking of the D1 dimer interface and hyperextension of the lower lobes of the ligand binding core. These data show that the glutamate binding domains are surprisingly mobile in the absence of ligand, which could influence receptor activity in the brain.
Laboratory of Cellular and Molecular Neurophysiology, Porter Neuroscience Research Center, National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA.
Andrew J R
AJ
Plested
Mark L
ML
Mayer
D018750:6-Cyano-7-nitroquinoxaline-2,3-dione / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D007608:Kainic Acid / Q000494:pharmacology; D007700:Kinetics; D008564:Membrane Potentials / Q000235:genetics / Q000502:physiology; D008958:Models, Molecular; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D018408:Patch-Clamp Techniques; D055503:Protein Multimerization; D051381:Rats; D018091:Receptors, AMPA / Q000235:genetics / Q000378:metabolism*
D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; D018091:Receptors, AMPA; D018698:Glutamic Acid; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; C104722:glutamate receptor ionotropic, AMPA 2; D007608:Kainic Acid