Bridging the gap between structural models of nicotinic-receptor superfamily ion channels and their corresponding functional states

摘要

Aromatic–aromatic interactions are a most prominent feature of the crystal structure of ELIC (PDB code 2VL0), a bacterial member of the nicotinic-receptor superfamily of ion channels where five pore-facing phenylalanines come together to form a structure akin to a narrow iris that occludes the transmembrane pore. To identify the functional state of the channel this structure represents, we engineered phenylalanines at various pore-facing positions of the muscle acetylcholine receptor (one position at a time), including the position that aligns with the native phenylalanine 246 of ELIC, and assessed the consequences of such mutations using electrophysiological and toxin-binding assays. From our experiments, we conclude that the interaction among the side chains of pore-facing phenylalanines, rather than the accumulation of their independent effects, leads to the formation of a non-conductive conformation that is unresponsive to the application of acetylcholine and is highly stable even in the absence of ligand. Moreover, electrophysiological recordings from a GLIC channel (another bacterial member of the superfamily) engineered to have a ring of phenylalanines at the corresponding pore-facing position suggest that this novel refractory state is distinct from the well-known desensitized state. It seems reasonable to propose, then, that it is in this peculiar non-conductive conformation that the ELIC channel was crystallized. It seems also reasonable to propose that, in the absence of rings of pore-facing aromatic side chains, such stable conformation may never be attained by the acetylcholine receptor. Incidentally, we also noticed that the response of the proton-gated, wild-type GLIC channel to a fast change in pH from 7.4 to 4.5 (on the extracellular side) is only transient, with the evoked current fading completely in a matter of seconds. This raises the possibility that the crystal structures of GLIC obtained at pH 4.0 (PDB code 3EHZ) and 4.6 (PDB code 3EAM) correspond to the to the (well-known) desensitized state.