Characterizing the Ion-Conductive State of the α7-Nicotinic Acetylcholine Receptor via Single-Channel Measurements...

The α7-nicotinic acetylcholine receptor (α7-nAChR) is a cation-selective Cys-loop receptor involved in diverse physiological processes and is an important therapeutic target. Multiple cryo-EM structures of putative open states are now available, and their functional relevance is under active investigation. Here, we combined single-channel patch-clamp recordings with atomistic molecular dynamics (MD) simulations to assess the conductive properties of several α7-nAChR structures solved with different ligands. Simulations restrained to the respective cryo-EM structures produced only modest ion flux for all models, inconsistent with experiment, whereas fully unrestrained simulations revealed marked differences in their ability to relax into physiologically conductive ensembles. Two structures, 7KOX and ligand-bound 8V82, consistently stabilized into conductive states whose permeation properties agreed with our measured inward single-channel conductance. The conduction of 8V82 nearly stopped upon removing the ligands resolved in the PDB structures. 8V80 showed only intermittent conduction with ligands, and remained nonconductive without them. 7EKT collapsed into a nonconductive conformation upon relaxation, irrespective of whether the modeled ligands were retained or removed. 9LH5, despite having a transmembrane pore nearly identical to 7KOX's, exhibited approximately 2-fold higher conductance, likely due to a widened extracellular vestibule. Across models, permeation events followed Poissonian statistics with a characteristic entry lag captured by a double-Poisson model. Simulations of outward currents consistently overestimated the conductance compared to experiments, perhaps reflecting the absence of the full intracellular domain in available structural models and/or the presence of current-blocking concentrations of cytosolic Mg²⁺ in patch-clamp cell-attached recordings. These results identify the conformations most compatible with the physiological open state and underscore the importance of unrestrained MD, ligand stabilization, and extracellular-vestibule geometry in shaping α7-nAChR conduction.