The earliest metazoan ancestors of humans include the ctenophore Mnemiopsis leidyi. The genome of this comb jelly encodes homologs of vertebrate ionotropic glutamate receptors (iGluRs) that are distantly related to glycine-activated NMDA receptors and that bind glycine with unusually high affinity. Using ligandbinding domain (LBD) mutants for electrophysiological analysis, we demonstrate that perturbing a ctenophore-specific interdomain Arg-Glu salt bridge that is notably absent from vertebrate AMPA, kainate, and NMDA iGluRs greatly increases the rate of recovery from desensitization, while biochemical analysis reveals a large decrease in affinity for glycine. X-ray crystallographic analysis details rearrangements in the binding pocket stemming from the mutations, and molecular dynamics simulations suggest that the interdomain salt bridge acts as a steric barrier regulating ligand binding and that the free energy required to access open conformations in the glycine-bound LBD is largely responsible for differences in ligand affinity among the LBD variants.glutamate receptors | X-ray crystallography | electrophysiology | molecular dynamics simulations | free energy calculations

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a novel functional assessment of the GAS belt in ASIC1a by incorporating unnatural amino acids (UAAs) into the channel to modify the main chain atoms implicated in ion conduction. We also engineered channels containing either one, two or three mutant subunits (concatemers) to assess the contribution of individual subunits. Together with molecular dynamics simulations of ion permeation through ASIC1, this revealed that the GAS belt does not determine sodium selectivity. We therefore screened the channel via conventional mutagenesis for other determinants of selectivity, identifying a ring of glutamate residues at the lower end of the pore. Results from UAA incorporation, concatemeric channels, and energetics of ion binding to this part of the pore suggest that these carboxylate side chains directly determine selective sodium conduction. These results suggest that the sodium selectivity filter of ASIC1a resides at the lower end of the channel pore.