Inhibition of the prokaryotic pentameric ligand-gated ion channel ELIC by divalent cations.

The modulation of pentameric ligand-gated ion channels (pLGICs) by divalent cations is believed to play an important role in their regulation in a physiological context. Ions such as calcium or zinc influence the activity of pLGIC neurotransmitter receptors by binding to their extracellular domain and either potentiate or inhibit channel activation. Here we have investigated by electrophysiology and X-ray crystallography the effect of divalent ions on ELIC, a close prokaryotic pLGIC homologue of known structure. We found that divalent cations inhibit the activation of ELIC by the agonist cysteamine, reducing both its potency and, at higher concentrations, its maximum response. Crystal structures of the channel in complex with barium reveal the presence of several distinct binding sites. By mutagenesis we confirmed that the site responsible for divalent inhibition is located at the outer rim of the extracellular domain, at the interface between adjacent subunits but at some distance from the agonist binding region. Here, divalent cations interact with the protein via carboxylate side-chains, and the site is similar in structure to calcium binding sites described in other proteins. There is evidence that other pLGICs may be regulated by divalent ions binding to a similar region, even though the interacting residues are not conserved within the family. Our study provides structural and functional insight into the allosteric regulation of ELIC and is of potential relevance for the entire family.

The kinetic properties of the α3 rat glycine receptor make it suitable for mediating fast synaptic inhibition.

Glycine receptors mediate fast synaptic inhibition in spinal cord and brainstem. Two α subunits are present in adult neurones, α1, which forms most of the synaptic glycine receptors, and α3. The physiological role of α3 is not known, despite the fact that α3 expression is concentrated in areas involved in nociceptive processing, such as the superficial dorsal horn. In the present study, we characterized the kinetic properties of rat homomeric α3 glycine receptors heterologously expressed in HEK293 cells. We analysed steady state single channel activity at a range of different glycine concentrations by fitting kinetic schemes and found that α3 channels resemble α1 receptors in their high maximum open probability (99.1% cf. 98% for α1), but differ in that maximum open probability is reached when all five binding sites are occupied by glycine (cf. three out of five sites for α1). α3 activation was best described by kinetic schemes that allow the channel to open also when partially liganded and that contain more than the minimum number of shut states, either as desensitized distal states (Jones and Westbrook scheme) or as pre-open gating intermediates (flip scheme). We recorded also synaptic-like α3 currents elicited by the rapid application of 1 ms pulses of high concentration glycine to outside-out patches. These currents had fast deactivation, with a time constant of decay of 9 ms. Thus, if native synaptic currents can be mediated by α3 glycine receptors, they are likely to be very close in their kinetics to α1-mediated synaptic events.

Mechanism of activation of the prokaryotic channel ELIC by propylamine: a single-channel study.

Prokaryotic channels, such as Erwinia chrysanthemi ligand-gated ion channel (ELIC) and Gloeobacter violaceus ligand-gated ion channel, give key structural information for the pentameric ligand-gated ion channel family, which includes nicotinic acetylcholine receptors. ELIC, a cationic channel from E. chrysanthemi, is particularly suitable for single-channel recording because of its high conductance. Here, we report on the kinetic properties of ELIC channels expressed in human embryonic kidney 293 cells. Single-channel currents elicited by the full agonist propylamine (0.5-50 mM) in outside-out patches at -60 mV were analyzed by direct maximum likelihood fitting of kinetic schemes to the idealized data. Several mechanisms were tested, and their adequacy was judged by comparing the predictions of the best fit obtained with the observable features of the experimental data. These included open-/shut-time distributions and the time course of macroscopic propylamine-activated currents elicited by fast theta-tube applications (50-600 ms, 1-50 mM, -100 mV). Related eukaryotic channels, such as glycine and nicotinic receptors, when fully liganded open with high efficacy to a single open state, reached via a preopening intermediate. The simplest adequate description of their activation, the "Flip" model, assumes a concerted transition to a single intermediate state at high agonist concentration. In contrast, ELIC open-time distributions at saturating propylamine showed multiple components. Thus, more than one open state must be accessible to the fully liganded channel. The "Primed" model allows opening from multiple fully liganded intermediates. The best fits of this type of model showed that ELIC maximum open probability (99%) is reached when at least two and probably three molecules of agonist have bound to the channel. The overall efficacy with which the fully liganded channel opens was ∼ 102 (∼ 20 for α1ß glycine channels). The microscopic affinity for the agonist increased as the channel activated, from 7 mM for the resting state to 0.15 mM for the partially activated intermediate state.