Measurements of the timescale and conformational space of AMPA receptor desensitization

Abstract

Ionotropic glutamate receptors (iGluRs) are ligand gated ion channels that mediate excitatory synaptic transmission in the central nervous system (CNS). Desensitization of the AMPA-subtype following glutamate binding appears critical for brain function, and involves rearrangement of the ligand binding domains (LBDs). Recently, several full-length structures of iGluRs in putative desensitized states were published. These structures indicate movements of the LBDs that might be trapped by cysteine crosslinks and metal bridges. We found that cysteine mutants at the interface between subunits A and C, and lateral zinc bridges (between subunits C & D or A & B) can trap freely-desensitizing receptors in a spectrum of states with different stabilities. Consistent with close approach of subunits during desensitization processes, introduction of bulky amino acids at the A-C interface produced a receptor with slow recovery from desensitization. Further, in wild-type GluA2 receptors, we detected population of stable desensitized state with a lifetime around 1 second. Using mutations that progressively stabilise deep desensitize states (E713T & Y768R), we were able to selectively protect receptors from crosslinks at both the diagonal and lateral interfaces. Ultrafast perfusion enabled us to perform chemical modification in less than 10 ms, reporting movements associated to desensitization on this timescale within LBD dimers in resting receptors. These observations suggest small disruptions of quaternary structure are sufficient for fast desensitization, and that substantial rearrangements likely correspond to stable desensitized states that are adopted relatively slowly, on a timescale much longer than physiological receptor activation. Significance statementiGluRs are central components of fast synaptic transmission in the brain. iGluR desensitization occurs as a natural consequence of receptor activation and can reduce the response of an excitatory synapse. AMPA receptor desensitization also appears necessary for proper brain development. Molecular structures of iGluRs in putative desensitized states predict a range of movements during desensitization. In the present study, we performed a series of crosslinking experiments on mutant receptors that we subjected to similar desensitizing conditions over time periods from milliseconds to minutes. These experiments allowed us to count desensitized configurations and rank them according to their stabilities. These data show that large-scale rearrangements occur during long glutamate exposures that are probably not seen in healthy brain tissue, whereas smaller changes in structure probably suffice for desensitization at synapses.