Stabilization of the GluCl ligand-gated ion channel in the presence and absence of ivermectin.

Improving our understanding of the mechanisms and effects of anesthetics is a critically important part of neuroscience. The currently dominant theory is that anesthetics and similar molecules act by binding to Cys-loop receptors in the postsynaptic terminal of nerve cells and potentiate or inhibit their function. Although structures for some of the most important mammalian channels have still not been determined, a number of important results have been derived from work on homologous cationic channels in bacteria. However, partly due to the lack of a nervous system in bacteria, there are a number of questions about how these results relate to higher organisms. The recent determination of a structure of the eukaryotic chloride channel, GluCl, is an important step toward accurate modeling of mammalian channels, because it is more similar in function to human Cys-loop receptors such as GABAAR or GlyR. One potential issue with using GluCl to model other receptors is the presence of the large ligand ivermectin (IVM) positioned between all five subunits. Here, we have performed a series of microsecond molecular simulations to study how the dynamics and structure of GluCl change in the presence versus absence of IVM. When the ligand is removed, subunits move at least 2 Å closer to each other compared to simulations with IVM bound. In addition, the pore radius shrinks to 1.2 Å, all of which appears to support a model where IVM binding between subunits stabilizes an open state, and that the relaxed nonIVM conformations might be suitable for modeling other channels. Interestingly, the presence of IVM also has an effect on the structure of the important loop C located at the neurotransmitter-binding pocket, which might help shed light on its partial agonist behavior.

Inhibition versus potentiation of ligand-gated ion channels can be altered by a single mutation that moves ligands between intra- and intersubunit sites.

Pentameric ligand-gated ion channels (pLGICs) are similar in structure but either inhibited or potentiated by alcohols and anesthetics. This dual modulation has previously not been understood, but the determination of X-ray structures of prokaryotic GLIC provides an ideal model system. Here, we show that a single-site mutation at the F14' site in the GLIC transmembrane domain turns desflurane and chloroform from inhibitors to potentiators, and that this is explained by competing allosteric sites. The F14'A mutation opens an intersubunit site lined by N239 (15'), I240 (16'), and Y263. Free energy calculations confirm this site is the preferred binding location for desflurane and chloroform in GLIC F14'A. In contrast, both anesthetics prefer an intrasubunit site in wild-type GLIC. Modulation is therefore the net effect of competitive binding between the intersubunit potentiating site and an intrasubunit inhibitory site. This provides direct evidence for a dual-site model of allosteric regulation of pLGICs.