SUMMARY Excitatory neurotransmission plays a key role in epileptogenesis. Correspondingly, AMPA-subtype ionotropic glutamate receptors, which mediate the majority of excitatory neurotransmission and contribute to seizure generation and spread, have emerged as promising targets for epilepsy therapy. The most potent and well-tolerated AMPA receptor inhibitors act via a noncompetitive mechanism, but many of them produce adverse side effects. The design of better drugs is hampered by the lack of a structural understanding of noncompetitive inhibition. Here, we report crystal structures of the rat AMPA-subtype GluA2 receptor in complex with three noncompetitive inhibitors. The inhibitors bind to a novel binding site, completely conserved between rat and human, at the interface between the ion channel and linkers connecting it to the ligand-binding domains. We propose that the inhibitors stabilize the AMPA receptor closed state by acting as wedges between the transmembrane segments, thereby preventing gating rearrangements that are necessary for ion channel opening.

The transient receptor potential channel TRPM7 is a master regulator of the organismal balance of divalent cations that plays an essential role in embryonic development, immune responses, cell mobility, proliferation, and differentiation. TRPM7 is implicated in neuronal and cardiovascular disorders, tumor progression and has emerged as a new drug target. Here we use cryo-EM, functional analysis, and molecular dynamics simulations to uncover two distinct structural mechanisms of TRPM7 activation by a gain-of-function mutation and by the agonist naltriben, which show different conformational dynamics and domain involvement. We identify a binding site for highly potent and selective inhibitors and show that they act by stabilizing the TRPM7 closed state. The discovered structural mechanisms provide foundations for understanding the molecular basis of TRPM7 channelopathies and drug development.

Zn2+, Mg2+ and Ca2+ are essential divalent cations implicated in many metabolic processes and signalling pathways. An emerging new paradigm is that the organismal balance of these cations predominantly depends on a common gatekeeper, the channel-kinase TRPM7. Despite extensive electrophysiological studies and recent cryo-EM analysis, an open question is how the channel activity of TRPM7 is activated. Here, we performed site-directed mutagenesis of mouse TRPM7 in conjunction with patch-clamp assessment of whole-cell and single-channel activity and molecular dynamics (MD) simulations to show that the side chains of conserved N1097 form an inter-subunit Mg2+ regulatory site located in the lower channel gate of TRPM7. Our results suggest that intracellular Mg2+ binds to this site and stabilizes the TRPM7 channel in the closed state, whereas the removal of Mg2+ favours the opening of TRPM7. Hence, our study identifies the structural underpinnings through which the TRPM7 channel is controlled by cytosolic Mg2+, representing a new structure–function relationship not yet explored among TRPM channels.

Noncompetitive inhibitors of AMPA receptors have attracted interest in recent years as antiepileptic drugs. However, their development is hindered by a lack of detailed understanding of the protein–inhibitor interaction mechanisms. Recently, structures of AMPA receptor complexes with the structurally dissimilar, noncompetitive, small-molecule inhibitors pyridone perampanel (PMP), GYKI 53655 (GYKI), and CP 465022 (CP) were resolved, revealing that all three share a common binding site. However, due to the low resolution of the ligands, their exact binding modes and protein–ligand interactions remain ambiguous and insufficiently detailed. We carried out molecular dynamics (MD) simulations on X-ray-resolved and docked AMPA receptor complexes, including thermodynamic integration (TI) to compute ligand binding constants, in order to investigate the inhibitor binding modes in detail and identify key protein–ligand interaction mechanisms. Our analysis and simulations show that the ligand binding pocket at the interface of the receptor’s transmembrane domain exhibits features also found in the binding pockets of the multidrug-resistance proteins. The inhibitors bind to such promiscuous pockets by forming multiple weak contacts, while the large, flexible pocket undergoes adjustments to accommodate structurally different ligands in different orientations. TI was able to identify a specific more favorable binding mode for GYKI, while PMP, which has a symmetric ring structure, produced several comparable poses indicating that it may bind in several orientations.

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