Abstract
AMPA subtype ionotropic glutamate receptors mediate fast excitatory neurotransmission and are implicated in numerous neurological diseases. Ionic currents through AMPA receptor channels can be allosterically regulated via different sites on the receptor protein. We used site-directed mutagenesis and patch-clamp recordings to probe the ion channel extracellular collar, the binding region for noncompetitive allosteric inhibitors. We found position and substitution-dependent effects for introduced mutations at this region on AMPA receptor gating. The results of mutagenesis suggested that the transmembrane domains M1, M3 and M4, which contribute to the ion channel extracellular collar, undergo significant relative displacement during gating. We used molecular dynamics simulations to predict an AMPA receptor open state structure and rationalize the results of mutagenesis. We conclude that the ion channel extracellular collar plays a distinct role in gating and represents a hub for powerful allosteric modulation of AMPA receptor function that can be used for developing novel therapeutics.
Highlights
Structural studies of isolated LBDs that have been crystallized in complex with numerous ligands and uncovered a diverse ensemble of gating conformations[5,6,7,8,9], greatly facilitating our understanding of the molecular basis of gating initiation
Supporting this idea, desensitization in the highly homologous and structurally similar NMDA receptors was greatly affected by mutations in a “hydrophobic box”[36], a region that in AMPA receptors is adjacent to the noncompetitive inhibitor binding sites
Comparing the modelled open state and experimental apo state structures, we rationalized the results of our mutagenesis experiments and predicted gating-related conformational rearrangements in the TMD, including relative displacement of the pre-M1, M3 and M4 segments that contribute to the noncompetitive inhibitor binding sites
Summary
Structural studies of isolated LBDs that have been crystallized in complex with numerous ligands and uncovered a diverse ensemble of gating conformations[5,6,7,8,9], greatly facilitating our understanding of the molecular basis of gating initiation. Our recent study of the allosteric mechanism of AMPA receptor noncompetitive inhibition by antiepileptic drugs pyridone perampanel (PMP)[40,41,42], GYKI 53655 (GYKI)[43, 44] and CP 465022 (CP)[44,45,46] identified novel antagonist binding sites in the ion channel extracellular collar, at the interface between TMD and LBD-TMD linkers[47] We hypothesized that these inhibitors stabilize the AMPA receptor in the closed state and act as wedges between transmembrane segments, thereby preventing gating rearrangements necessary for ion channel opening. Using the mutations that promote ion channel opening or inhibit receptor desensitization, we performed targeted molecular dynamics (MD)[48] simulations of the TMD and LBD-TMD linkers in lipid membrane and water (full atomistic model) environments to predict an AMPA receptor open state structure. Comparing the modelled open state and experimental apo state structures, we rationalized the results of our mutagenesis experiments and predicted gating-related conformational rearrangements in the TMD, including relative displacement of the pre-M1, M3 and M4 segments that contribute to the noncompetitive inhibitor binding sites
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