Pentameric ligand-gated ion channels (pLGICs) mediate fast chemical signaling through global allosteric transitions. Despite the existence of several high-resolution structures of pLGICs, their dynamical properties remain elusive. Using the proton-gated channel GLIC, we engineered multiple fluorescent reporters, each incorporating a bimane and a tryptophan/tyrosine, whose close distance causes fluorescence quenching. We show that proton application causes a global compaction of the extracellular subunit interface, coupled to an outward motion of the M2-M3 loop near the channel gate. These movements are highly similar in lipid vesicles and detergent micelles. These reorganizations are essentially completed within 2 ms and occur without channel opening at low proton concentration, indicating that they report a pre-active intermediate state in the transition pathway toward activation. This provides a template to investigate the gating of eukaryotic neurotransmitter receptors, for which intermediate states also participate in activation.DOI: http://dx.doi.org/10.7554/eLife.23955.001

Edited by Paul E. FraserBarbiturates induce anesthesia by modulating the activity of anionic and cationic pentameric ligand-gated ion channels (pLGICs). Despite more than a century of use in clinical practice, the prototypic binding site for this class of drugs within pLGICs is yet to be described. In this study, we present the first X-ray structures of barbiturates bound to GLIC, a cationic prokaryotic pLGIC with excellent structural homology to other relevant channels sensitive to general anesthetics and, as shown here, to barbiturates, at clinically relevant concentrations. Several derivatives of barbiturates containing anomalous scatterers were synthesized, and these derivatives helped us unambiguously identify a unique barbiturate binding site within the central ion channel pore in a closed conformation. In addition, docking calculations around the observed binding site for all three states of the receptor, including a model of the desensitized state, showed that barbiturates preferentially stabilize the closed state. The identification of this pore binding site sheds light on the mechanism of barbiturate inhibition of cationic pLGICs and allows the rationalization of several structural and functional features previously observed for barbiturates.The arrival of the first barbiturates into clinical practice at the beginning of the 20th century caused a revolution in the pharmacology-based treatments of psychiatric and neurological disorders due to their sedative and anxiolytic properties (1). As anticonvulsants, barbiturates were responsible for the first truly effective management regimens for epileptic seizures, whereas in the field of general anesthesia, they were the first injectable agents used for induction. Between 1920 and 1950, barbiturates were the most prominent class of drugs used as sedatives and hypnotics (2). As they are prone to cause respiratory depression, they have now been mostly replaced with the comparatively safer benzodiazepines (3). Despite this, barbiturates still retain several important sedative-hypnotic roles in medical treatment such as for asthmatic and gastrointestinal functional disorders, certain types of epilepsy, violent convulsions, and cerebral hemorrhages. Most importantly, they are still used for the induction of general anesthesia.Although barbiturates have enjoyed widespread use during the last century, insights into the molecular basis of their action have only arisen during the last couple of decades. Barbiturates are thought to modulate the action of various neural receptors, such as the AMPA/kainate receptors and the P/Q high voltageactivated calcium channels (4, 5), as well as members of the pentameric ligand-gated ion channel family, which are major mediators of synaptic transmission (6). It has been shown that barbiturates alter the action of anionic GABA A and glycine receptors (GABA A R 5 and GlyR, respectively), promoting their activation and subsequent polarization of neurons (7-9), as well as inhibiting cationic ion channels responsible for triggering ...