Structural models of ligand-gated ion channels: sites of action for anesthetics and ethanol.

The molecular mechanism(s) of action of anesthetic, and especially, intoxicating doses of alcohol (ethanol [EtOH]) have been of interest even before the advent of the Research Society on Alcoholism. Recent physiological, genetic, and biochemical studies have pin-pointed molecular targets for anesthetics and EtOH in the brain as ligand-gated ion channel (LGIC) membrane proteins, especially the pentameric (5 subunit) Cys-loop superfamily of neurotransmitter receptors including nicotinic acetylcholine (nAChRs), GABAA (GABAA Rs), and glycine receptors (GlyRs). The ability to demonstrate molecular and structural elements of these proteins critical for the behavioral effects of these drugs on animals and humans provides convincing evidence for their role in the drugs' actions. Amino acid residues necessary for pharmacologically relevant allosteric modulation of LGIC function by anesthetics and EtOH have been identified in these channel proteins. Site-directed mutagenesis revealed potential allosteric modulatory sites in both the trans-membrane domain (TMD) and extracellular domain (ECD). Potential sites of action and binding have been deduced from homology modeling of other LGICs with structures known from crystallography and cryo-electron microscopy studies. Direct information about ligand binding in the TMD has been obtained by photoaffinity labeling, especially in GABAA Rs. Recent structural information from crystallized procaryotic (ELIC and GLIC) and eukaryotic (GluCl) LGICs allows refinement of the structural models including evaluation of possible sites of EtOH action.

Picrotoxin dramatically speeds the mammalian circadian clock independent of Cys-loop receptors.

Picrotoxin is extensively and specifically used to inhibit GABAA receptors and other members of the Cys-loop receptor superfamily. We find that picrotoxin acts independently of known Cys-loop receptors to shorten the period of the circadian clock markedly by specifically advancing the accumulation of PERIOD2 protein. We show that this mechanism is surprisingly tetrodotoxin-insensitive, and the effect is larger than any known chemical or genetic manipulation. Notably, our results indicate that the circadian target of picrotoxin is common to a variety of human and rodent cell types but not Drosophila, thereby ruling out all conserved Cys-loop receptors and known regulators of mammalian PERIOD protein stability. Given that the circadian clock modulates significant aspects of cell physiology including synaptic plasticity, these results have immediate and broad experimental implications. Furthermore, our data point to the existence of an important and novel target within the mammalian circadian timing system.

Anesthetic sites and allosteric mechanisms of action on Cys-loop ligand-gated ion channels.

PURPOSE: The Cys-loop ligand-gated ion channel superfamily is a major group of neurotransmitter-activated receptors in the central and peripheral nervous system. The superfamily includes inhibitory receptors stimulated by γ-aminobutyric acid (GABA) and glycine and excitatory receptors stimulated by acetylcholine and serotonin. The first part of this review presents current evidence on the location of the anesthetic binding sites on these channels and the mechanism by which binding to these sites alters their function. The second part of the review addresses the basis for this selectivity, and the third part describes the predictive power of a quantitative allosteric model showing the actions of etomidate on γ-aminobutyric acid type A receptors (GABA(A)Rs). PRINCIPAL FINDINGS: General anesthetics at clinical concentrations inhibit the excitatory receptors and enhance the inhibitory receptors. The location of general anesthetic binding sites on these receptors is being defined by photoactivable analogues of general anesthetics. The receptor studied most extensively is the muscle-type nicotinic acetylcholine receptor (nAChR), and progress is now being made with GABA(A)Rs. There are three categories of sites that are all in the transmembrane domain: 1) within a single subunit's four-helix bundle (intrasubunit site; halothane and etomidate on the δ subunit of AChRs); 2) between five subunits in the transmembrane conduction pore (channel lumen sites; etomidate and alcohols on nAChR); and 3) between two subunits (subunit interface sites; etomidate between the α1 and ß2/3 subunits of the GABA(A)R). CONCLUSIONS: These binding sites function allosterically. Certain conformations of a receptor bind the anesthetic with greater affinity than others. Time-resolved photolabelling of some sites occurs within milliseconds of channel opening on the nAChR but not before. In GABA(A)Rs, electrophysiological data fit an allosteric model in which etomidate binds to and stabilizes the open state, increasing both the fraction of open channels and their lifetime. As predicted by the model, the channel-stabilizing action of etomidate is so strong that higher concentrations open the channel in the absence of agonist. The formal functional paradigm presented for etomidate may apply to other potent general anesthetic drugs. Combining photolabelling with structure-function mutational studies in the context of allosteric mechanisms should lead us to a more detailed understanding of how and where these important drugs act.