Ionotropic glutamate receptors are postsynaptic tetrameric ligand-gated channels whose activity mediates fast excitatory transmission. Glutamate binding to clamshell-shaped ligand binding domains (LBDs) triggers opening of the integral ion channel, but how the four LBDs orchestrate receptor activation is unknown. Here, we present a high-resolution x-ray crystal structure displaying two tetrameric LBD arrangements fully bound to glutamate. Using a series of engineered metal ion trapping mutants, we showed that the more compact of the two assemblies corresponds to an arrangement populated during activation of full-length receptors. State-dependent cross-linking of the mutants identified zinc bridges between the canonical active LBD dimers that formed when the tetramer was either fully or partially bound by glutamate. These bridges also stabilized the resting state, consistent with the recently published full-length apo structure. Our results provide insight into the activation mechanism of glutamate receptors and the complex conformational space that the LBD layer can sample.
SummarySignal transduction at vertebrate excitatory synapses involves the activity of ionotropic glutamate receptors, including the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptor. Technical advances in cryo-electron microscopy have brought a slew of full-length structures of AMPA receptors, on their own and in combination with auxiliary subunits. These structures illustrate a wide range of conformations, indicating that individual domains might undergo substantial lateral motions during gating, resulting in an open, “relaxed” extracellular layer. Here, we used bifunctional methanethiosulfonate cross-linkers to calibrate the conformations found in functional AMPA receptors both in the presence and absence of the auxiliary subunit Stargazin. Our data indicate that AMPA receptors have considerable conformational freedom and can get trapped in stable, relaxed conformations, especially upon long exposures to glutamate. In contrast, Stargazin limits this conformational flexibility. Thus, under synaptic conditions, where brief glutamate exposures and the presence of Stargazin dominate, AMPA receptors are unlikely to adopt very relaxed conformations during gating.
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