Glutamate concentration tunes AMPA receptor function through conductance-state occupancy

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) mediate excitatory synaptic transmission across the brain and exhibit distinct modes of opening depending on how much glutamate is bound. Despite extensive work on AMPAR conductance states and subunit composition, whether changes in glutamate levels at and around synapses regulate AMPAR function is unknown. Here we show that glutamate concentration ([glutamate]) at mouse interneuron synapses governs key biophysical features of AMPARs that are commonly used to infer subunit composition. Lower [glutamate] reduces hallmark AMPAR properties, including current-voltage rectification, polyamine block and Ca²⁺ permeability, highlighting a strong dependence of receptor function on synaptic [glutamate]. Recordings from isolated AMPARs combined with numerical simulations reveal differential spermine affinity across distinct conductance states and establish [glutamate], rather than solely subunit composition, as a key determinant of receptor behavior. These findings uncover a previously unrecognized mechanism through which the synaptic glutamate landscape dynamically shapes AMPAR signaling, broadening the framework for how excitatory input is encoded within neural circuits.