Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 403, Issue 5, Pages 693-705Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2010.09.026
Keywords
Cys-loop receptors; aromatic-aromatic interactions; desensitization; electrophysiology; patch clamp
Categories
Funding
- US National Institute of Neurological Disorders and Stroke [R01-NS042169]
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Aromatic aromatic interactions are a prominent feature of the crystal structure of ELIC [Protein Data Bank (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 ins that occludes the transmembrane pore To identify the functional state of the channel that this structure represents, we engineered phenylalanines at various pore-facing positions of the muscle acetylcholine (ACh) 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 nonconductive conformation that is unresponsive to the application of ACh 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 nonconductive 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 ACh receptor Incidentally, we also noticed that the response of the proton-gated wild-type GLIC channel to a fast change in pH from pH 7 4 to pH 4 5 (on the extracellular side) is only transient, with the evoked current fading completely m a matter of seconds This raises the possibility that the crystal structures of GLIC obtained at pH 4 0 (PDB code 3EHZ) and pH 4 6 (PDB code 3EAM) correspond to the to the (well-known) desensitized state (C) 2010 Elsevier Ltd All rights reserved
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