Mechanisms underlying TARP modulation of the GluA1/2-γ8 AMPA receptor

Abstract

AMPA-type glutamate receptors (AMPARs) mediate rapid signal transmission at excitatory synapses in the brain. Glutamate binding to the receptor’s ligand-binding domains (LBDs) leads to ion channel activation and desensitization. Gating kinetics shape synaptic transmission and are strongly modulated by transmembrane AMPAR regulatory proteins (TARPs) through currently incompletely resolved mechanisms. Here, electron cryo-microscopy structures of the GluA1/2 TARP-γ8 complex, in both open and desensitized states (at 3.5 Å), reveal state-selective engagement of the LBDs by the large TARP-γ8 loop (‘β1’), elucidating how this TARP stabilizes specific gating states. We further show how TARPs alter channel rectification, by interacting with the pore helix of the selectivity filter. Lastly, we reveal that the Q/R-editing site couples the channel constriction at the filter entrance to the gate, and forms the major cation binding site in the conduction path. Our results provide a mechanistic framework of how TARPs modulate AMPAR gating and conductance.

Asymmetry and ion selectivity properties of bacterial channel NaK mutants mimicking ionotropic glutamate receptors

“…In our ion binding analysis, we found distinct binding profiles of larger alkali ions (K + , Rb + and Cs + ) in the TTV part of the SF, while the larger vestibule at the upper part of the SF in NaK-CDI plays a much weaker role in binding and selectivity of ions. In line with this finding, our previous simulations 6 and recent cryo-EM data 36 of AMPA receptors revealed that the QQ part constitutes the major binding sites for monovalent ions, while the CDI part is wide and less important for coordination of monovalent ions with scarcely any metastable sites where ions reside. The present patch-clamp electrophysiology data showed that mutation of the CDI sequence in GluA2 by the DGNF of NaK had no obvious influence on channel activity, demonstrating again that the QQ filter in GluA2 is the most essential part of the SF for selection of monovalent ions.…”

Modulatory mechanisms of TARP γ8-selective AMPA receptor therapeutics

“…Positive modulation is facilitated by F527A and by mutants in the lower part of M1, including V534A, F537A, R541A and F542A. Interestingly, this pattern correlates with M1-TARP interaction strength – the upper part of M1 is loosely coupled to γ8, while the region below Phe527 is coupled more tightly 38 . Moreover, this enhanced TARP modulation is not immune to NAM action, as positions distal from the binding pocket are also robustly blunted by all three NAMs, as shown for R541A and F542A ( Fig.…”

“…Resolutions of the ion channel/TARP sector at ∼ 3.0 Å, with the LY-481 structure reaching 2.6 Å, enabled an unprecedented view of the modulator binding site with associated lipids and putative water molecules (Fig. In addition, we subjected these structures to large scale MD simulations in an explicit lipid membrane together with water and ions, alongside previously reported apostate complexes (open and resting; PDB: 7QHB and 7OCD, respectively) 22,38 . Three sets of simulations for each receptor, totaling 1.25-1.5 μs of sampling for each system, offered additional insight into ligand behavior in their binding pocket, as well as enabling a comprehensive comparison of local and global receptor dynamics in response to NAM binding (Supplemental Movie 2).…”

“…5c ) directly contact the M2 pore loop of the selectivity filter and will thereby influence the lower half of the conduction path ( Fig. 5d; right panel), while Arg541 targets the acidic M1/2 cytoplasmic loop, implicated in AMPAR regulation 38,47 . In summary, contacts in the AMPAR-TARP transmembrane sector appear to be finely tuned for transmission via the M1/M4 periphery (‘transmitter’) to the ‘effector’ (the M3 gate and M2 selectivity filter) ( Fig.…”

“…Four ligand -bound, and two apo structures of the LBD-TMD-TARP complex described in this and previous studies from the lab 38,65 , were used to set up simulations. Missing residues and loops were added using MODELLER 66 , with restraints applied to the 1-loop to extend its conformation.…”