Mutations that stabilize the open state of the Erwinia chrisanthemi ligand-gated ion channel fail to change the conformation of the pore domain in crystals

Abstract

The determination of structural models of the various stable states of an ion channel is a key step toward the characterization of its conformational dynamics. In the case of nicotinic-type receptors, different structures have been solved but, thus far, these different models have been obtained from different members of the superfamily. In the case of the bacterial member ELIC, a cysteamine-gated channel from Erwinia chrisanthemi, a structural model of the protein in the absence of activating ligand (and thus, conceivably corresponding to the closed state of this channel) has been previously generated. In this article, electrophysiological characterization of ELIC mutants allowed us to identify pore mutations that slow down the time course of desensitization to the extent that the channel seems not to desensitize at all for the duration of the agonist applications (>20 min). Thus, it seems reasonable to conclude that the probability of ELIC occupying the closed state is much lower for the ligand-bound mutants than for the unliganded wild-type channel. To gain insight into the conformation adopted by ELIC under these conditions, we solved the crystal structures of two of these mutants in the presence of a concentration of cysteamine that elicits an intracluster open probability of >0.9. Curiously, the obtained structural models turned out to be nearly indistinguishable from the model of the wild-type channel in the absence of bound agonist. Overall, our findings bring to light the limited power of functional studies in intact membranes when it comes to inferring the functional state of a channel in a crystal, at least in the case of the nicotinicreceptor superfamily.electrophysiology | structure-function T he use of bacterial and archaeal ion channels as models of the structure and function of their eukaryotic counterparts has become one of the mainstays of ion-channel biophysics. It seems to us that this practice is particularly well justified whenever the use of noneukaryotic channels facilitates experiments that are otherwise too difficult or too cumbersome to perform. A clear case in point is the determination of structural models of these membrane proteins using X-ray crystallography.One of the main goals of direct structural approaches as applied to ion channels is to determine what these proteins look like in their different functional states. Assuming that all of these conformations can form well-diffracting crystals, the problem is reduced to finding the conditions under which the occupancy of each particular state is maximized. In general, for well-characterized ion channels, the experimental maneuvers that are needed to favor or disfavor at least some of these different conformations are known. For example, in the case of (wild-type) neurotransmitter-gated channels, the binding of the natural neurotransmitter strongly stabilizes the desensitized state (e.g., ref. 1), whereas the absence of neurotransmitter or the binding of a competitive antagonist keeps the channel mostly closed (2, 3). Similarly, mut...

Structural Basis for Allosteric Coupling at the Membrane-Protein Interface in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

“…However, the nAChR model from cryoelectron micrographs hugely benefits from the fact that these channels are in their native Torpedo membranes in contrast to the crystal structures of ELIC, GLIC, and GluCL, which are in a detergent-solubilized environment. There is also growing evidence that crystallization conditions may bias structures toward specific conformations (6,11,63). Gating in these channels is intrinsically modulated by membrane lipids (22,64,65), likely by an allosteric mechanism that alters the relay of communication through the interface between the ECD and the TMD.…”

“…Crystal structures of ELIC and GLIC have been proposed to represent the closed and the open states of the channel, respectively (3)(4)(5). However, significant controversies surround the functional states of the crystallographically trapped conformations (6). Interestingly, the structure of the agonist-bound glutamate-gated chloride channels from Caenorhabditis elegans closely resembles GLIC in its overall conformation.…”

Subunit stoichiometry and arrangement in a heteromeric glutamate-gated chloride channel

“…1A). These Glu-binding sites are homologous to the neurotransmitter/agonist-binding sites of other Cys-loop receptors (1,2,24), bacterial homologs of Cys-loop receptors (25)(26)(27)(28)(29)(30), and ACh-binding proteins (31)(32)(33)(34).…”

Asymmetric perturbations of signalling oligomers

“…3 and a recent review of Nys et al, 2013). Even though these homopentamers have proven to be good pLGIC models (Spurny et al, 2012), crystallization in the presence of saturating ligand concentrations has resulted in XRC structures with open or closed pore states not compatible with the functional effects of cocrystallized ligands (Gonzalez-Gutierrez et al, 2012). Furthermore, the XRC structures have revealed a perplexing heterogeneity of inter-and intra-subunit binding cavities both for anaesthetics in the TM domain and for benzodiazepines in the EC domain (Spurny et al, 2012;Spurny et al, 2013).…”