AMPA receptors (AMPARs) are present throughout the CNS and mediate the majority of fast excitatory neurotransmission. AMPARs are implicated in CNS diseases such as Alzheimer's, Parkinson's, and epilepsy. Recently, the first AMPAR-selective drug was approved in the form of perampanel for treatment of epilepsy. Perampanel is a negative allosteric modulators (NAMs), inhibiting AMPAR activity via a poorly understood mechanism.Little is known about the location and structure of binding sites for perampanel or other prototypical AMPAR NAMs such as GYKI-53,655 and CP-465,022. Identifying NAM binding sites and binding modes is prerequisite to understanding the molecular mechanism that underlies negative allosteric modulation of AMPAR function and is important for future development of other NAMs. This project aims to characterize the binding sites and binding modes of perampanel and other NAMs. AMPARs are tetrameric assemblies of subunits with a highly modular structure comprising two large extracellular domains -the amino-terminal domain and the ligand-binding domain -connected to the transmembrane domain via three flexible linkers (denoted S1-M1, S2-M3, and S2-M4). Previous work has identified positions within these linkers to control NAM potency (Balannik et al., 2005, Neuron). To identify additional residues that participate in NAM interaction, mutational scanning was performed of the S1-M1 and S2-M4 linker regions. 48 point mutants were functionally characterized using an intracellular calcium imaging assay. For the 37 mutants with intact receptor function, IC 50 was determined for four prototypical NAMs, including perampanel, to identify mutant effects on NAM potency. Several mutants were found having >5-fold change in IC 50 of one or more NAMs and further characterized by two-electrode voltage clamp electrophysiology. The mutational dataset will be used to guide molecular modeling studies of NAM binding modes to the receptor.