SUMMARY Structural rearrangements underlying functional transitions of pentameric ligand-gated ion channels (pLGICs) are not fully understood. Using 19F NMR and ESR spectroscopy, we found that ELIC, a pLGIC from Erwinia chrysanthemi, expanded the extracellular end and contracted the intracellular end of its pore during transition from the resting to an apparent desensitized state. Importantly, the contraction at the intracellular end of the pore likely forms a gate to restrict ion transport in the desensitized state. This gate differs from the hydrophobic gate present in the resting state. Conformational changes of the TM2-TM3 loop were limited to the N-terminal end. The TM4 helices and the TM3-TM4 loop appeared relatively insensitive to agonist-mediated structural rearrangement. These results indicate that conformational changes accompanying functional transitions are not uniform among different ELIC regions. This work also revealed the co-existence of multiple conformations for a given state and suggested asymmetric conformational arrangements in a homomeric pLGIC.

Voltage-gated sodium channels (Na V ) play an important role in general anesthesia. Electrophysiology measurements suggest that volatile anesthetics such as isoflurane inhibit Na V by stabilizing the inactivated state or altering the inactivation kinetics. Recent computational studies suggested the existence of multiple isoflurane binding sites in Na V , but experimental binding data are lacking. Here we use site-directed placement of 19 F probes in NMR experiments to quantify isoflurane binding to the bacterial voltage-gated sodium channel NaChBac. 19 F probes were introduced individually to S129 and L150 near the S4-S5 linker, L179 and S208 at the extracellular surface, T189 in the ion selectivity filter, and all phenylalanine residues. Quantitative analyses of 19 F NMR saturation transfer difference (STD) spectroscopy showed a strong interaction of isoflurane with S129, T189, and S208; relatively weakly with L150; and almost undetectable with L179 and phenylalanine residues. An orientation preference was observed for isoflurane bound to T189 and S208, but not to S129 and L150. We conclude that isoflurane inhibits NaChBac by two distinct mechanisms: (i) as a channel blocker at the base of the selectivity filter, and (ii) as a modulator to restrict the pivot motion at the S4-S5 linker and at a critical hinge that controls the gating and inactivation motion of S6.general anesthetics | drug-protein interaction | voltage-gated sodium channel | nuclear magnetic resonance | molecular dynamics simulation

Measurements of distances in cells by pulsed ESR spectroscopy afford tremendous opportunities to study proteins in native environments that are irreproducible in vitro. However, the in-cell environment is harsh towards the typical nitroxide radicals used in double electron-electron resonance (DEER) experiments. A systematic examination is performed on the loss of the DEER signal, including contributions from nitroxide decay and nitroxide side-chain cleavage. In addition, the possibility of extending the lifetime of the nitroxide radical by use of an oxidizing agent is investigated. Using this oxidizing agent, DEER distance measurements are performed on doubly nitroxide-labeled GB1, the immunoglobulin-binding domain of protein G, at varying incubation times in the cellular environment. It is found that, by comparison of the loss of DEER signal to the loss of the CW spectrum, cleavage of the nitroxide side chain contributes to the loss of DEER signal, which is significantly greater in cells than in cell extracts. Finally, local spin concentrations are monitored at varying incubation times to show the time required for molecular diffusion of a small globular protein within the cellular milieu.

Pentameric ligand-gated ion channels (pLGICs) are targets of general anesthetics, but molecular mechanisms underlying anesthetic action remain debatable. We found that ELIC, a pLGIC from Erwinia chrysanthemi, can be functionally inhibited by isoflurane and other anesthetics. Structures of ELIC co-crystallized with isoflurane in the absence or presence of an agonist revealed double isoflurane occupancies inside the pore near T237(6′) and A244(13′). A pore-radius contraction near the extracellular entrance was observed upon isoflurane binding. Electrophysiology measurements with a single-point mutation at position 6′ or 13′ support the notion that binding at these sites renders isoflurane inhibition. Molecular dynamics simulations suggested that isoflurane binding was more stable in the resting than in a desensitized pore conformation. This study presents compelling evidence for a direct pore-binding mechanism of isoflurane inhibition, which has a general implication for inhibitory action of general anesthetics on pLGICs.General anesthetics are administered to approximately 256 million people each year 1 . In addition to their primary action to render analgesia, amnesia, immobility, and unconsciousness, these drugs are also implicated in unwanted side effects, including post-operative cognitive decline 2,3 and neurotoxicity in pediatric and elderly populations [4][5][6] . At the molecular level, how general anesthetics exert their on-target and off-target actions remains poorly understood.Functional measurements suggest that a superfamily of pentameric ligand-gated ion channels (pLGICs) plays a central role in anesthetic action 7,8 . At clinically relevant concentrations, general anesthetics inhibit agonist-elicited currents of cation-conducting channels, such as nicotinic acetylcholine receptors (nAChRs), and potentiate currents of anion-conducting channels, such as GABA A and glycine receptors 7,8 . These channels regulate a myriad of sensory processes and their malfunctions are directly linked to several neurological disorders. A structural insight into the mode of action, particularly the molecular details of anesthetic binding sites, is essential to understand the functional modulation of pLGICs by general anesthetics.Discrete anesthetic binding sites have been proposed on the basis of various experimental and computational studies. In the extracellular domain (ECD), which contains the orthosteric agonist-binding site, crystal structures of prokaryotic pLGICs from Gloeobacter violaceus (GLIC) and Erwinia chrysanthemi (ELIC) have revealed binding sites for anesthetics: ketamine at the interface of two subunits in GLIC 9 and