Measurements of the timescale and conformational space of AMPA receptor desensitization

Abstract

8 Ionotropic glutamate receptors (iGluRs) are ligand gated ion channels that mediate excitatory 9 synaptic transmission in the central nervous system (CNS). Desensitization of the AMPA-10 subtype following glutamate binding appears critical for brain function, and involves 11 rearrangement of the ligand binding domains (LBDs). Recently, several full-length structures 12 of iGluRs in putative desensitized states were published. These structures indicate movements 13 of the LBDs that might be trapped by cysteine crosslinks and metal bridges. We found that 14 cysteine mutants at the interface between subunits A and C, and lateral zinc bridges (between 15 subunits C & D or A & B) can trap freely-desensitizing receptors in a spectrum of states with 16 different stabilities. Consistent with close approach of subunits during desensitization 17 processes, introduction of bulky amino acids at the A-C interface produced a receptor with slow 18 recovery from desensitization. Further, in wild-type GluA2 receptors, we detected population 19 of stable desensitized state with a lifetime around 1 second. Using mutations that progressively 20 stabilise deep desensitize states (E713T & Y768R), we were able to selectively protect receptors 21 from crosslinks at both the diagonal and lateral interfaces. Ultrafast perfusion enabled us to 22 perform chemical modification in less than 10 ms, reporting movements associated to 23 desensitization on this timescale within LBD dimers in resting receptors. These observations 24 suggest small disruptions of quaternary structure are sufficient for fast desensitization, and 25 that substantial rearrangements likely correspond to stable desensitized states that are adopted 26 relatively slowly, on a timescale much longer than physiological receptor activation. 28Significance statement 29 iGluRs are central components of fast synaptic transmission in the brain. iGluR desensitization occurs as a 30 natural consequence of receptor activation and can reduce the response of an excitatory synapse. AMPA receptor 31 desensitization also appears necessary for proper brain development. Molecular structures of iGluRs in putative 32 desensitized states predict a range of movements during desensitization. In the present study, we performed a 33 series of crosslinking experiments on mutant receptors that we subjected to similar desensitizing conditions 34 over time periods from milliseconds to minutes. These experiments allowed us to count desensitized 35 configurations and rank them according to their stabilities. These data show that large-scale rearrangements 36 occur during long glutamate exposures that are probably not seen in healthy brain tissue, whereas smaller 37 changes in structure probably suffice for desensitization at synapses. 39crosslinked very stably between A & C subunits by the A665C disulfide bond (Lau et al., 2013). Using 79 a fast perfusion system, we used several approaches on mutant and wild-type receptors to count 80 conformational states attained during desensitization. Comparing pot...