Abstract
Ionotropic glutamate receptors (iGluRs) mediate signal transmission in the brain and are important drug targets. Structural studies show snapshots of iGluRs, which provide a mechanistic understanding of gating, yet the rapid motions driving the receptor machinery are largely elusive. Here we detect kinetics of conformational change of isolated clamshell-shaped ligand-binding domains (LBDs) from the three major iGluR sub-types, which initiate gating upon binding of agonists. We design fluorescence probes to measure domain motions through nanosecond fluorescence correlation spectroscopy. We observe a broad kinetic spectrum of LBD dynamics that underlie activation of iGluRs. Microsecond clamshell motions slow upon dimerization and freeze upon binding of full and partial agonists. We uncover allosteric coupling within NMDA LBD hetero-dimers, where binding of L-glutamate to the GluN2A LBD stalls clamshell motions of the glycine-binding GluN1 LBD. Our results reveal rapid LBD dynamics across iGluRs and suggest a mechanism of negative allosteric cooperativity in NMDA receptors.
Highlights
Ionotropic glutamate receptors mediate signal transmission in the brain and are important drug targets
Signal transmission at excitatory synapses is mediated by ionotropic glutamate receptors that are ubiquitously expressed in the central nervous system1,2. Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that play key roles in brain development and higher-order cognitive functions including learning and memory
Rapid conformational changes that are accompanied by formation and disruption of fluorophore/Trp interaction result in photoinduced electron transfer (PET) fluorescence fluctuations that can be detected by fluorescence correlation spectroscopy (FCS) (PET-FCS)[29,31]
Summary
Ionotropic glutamate receptors (iGluRs) mediate signal transmission in the brain and are important drug targets. We detect kinetics of conformational change of isolated clamshellshaped ligand-binding domains (LBDs) from the three major iGluR sub-types, which initiate gating upon binding of agonists. We observe a broad kinetic spectrum of LBD dynamics that underlie activation of iGluRs. Microsecond clamshell motions slow upon dimerization and freeze upon binding of full and partial agonists. A single subunit consists of the extracellular N-terminal- and ligand-binding domains, the trans-membrane ion channel, and the intracellular C-terminal domain (NTD, LBD, TMD, and CTD, respectively)[4]. IGluR-mediated signal transduction is rooted in a complex network of conformational motions of individual domains, which are elusive to experimental observation. A particular focus is on the LBD, the “muscle” of the receptor, with domain motions providing the trigger that initiates channel gating[23]. Molecular dynamics simulations suggests that the LBD populates a more extended ensemble of conformations than observed experimentally[25]
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