The pharmacological profile of ELIC, a prokaryotic GABA-gated receptor

Abstract

The Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of vertebrate Cys-loop ligand-gated ion channels. It is activated by GABA, and this property, combined with its structural similarity to GABA(A) and other Cys-loop receptors, makes it potentially an excellent model to probe their structure and function. Here we characterise the pharmacological profile of ELIC, examining the effects of compounds that could activate or inhibit the receptor. We confirm that a range of amino acids and classic GABA(A) receptor agonists do not elicit responses in ELIC, and we show the receptor can be at least partially activated by 5-aminovaleric acid and γ-hydroxybutyric acid, which are weak agonists. A range of GABA(A) receptor non-competitive antagonists inhibit GABA-elicited ELIC responses including α-endosulfan (IC₅₀ = 17 μM), dieldrin (IC₅₀ = 66 μM), and picrotoxinin (IC₅₀ = 96 μM) which were the most potent. Docking suggested possible interactions at the 2' and 6' pore-lining residues, and mutagenesis of these residues supports this hypothesis for α-endosulfan. A selection of compounds that act at Cys-loop and other receptors also showed some efficacy at blocking ELIC responses, but most were of low potency (IC₅₀ > 100 μM). Overall our data show that a number of compounds can inhibit ELIC, but it has limited pharmacological similarity to GLIC and to Cys-loop receptors.

Fig. 1

An alignment of channel-lining residues for a range of eukaryotic Cys-loop receptors and prokaryotic homologues. As is common for these receptors, a prime notation is used to facilitate comparison between different subunits, with 0′ being the conserved charged residue at the start of M2. Grey indicates residue conservation. Accession numbers are: ELIC P0C7B7, GLIC Q7NDN8, 5-HT₃P46098, nACh α1 P02708, Gly P23415, GABA_A α1 P14867, GABA_A β2 P47870, GABA_A γ2 P18507, GluCl Q94900.

Fig. 2

GABA and 5-AV agonist concentration–response curves (A) and example responses (B). The black bar is the application of agonist. Data = mean ± SEM, n ≥ 4.

Fig. 3

ELIC antagonists. (A) Example traces showing inhibition by picrotoxinin (PXN). Concentration–inhibition curves for PXN (B), α-endosulfan (C), and rimantadine (D). Inhibition was measured at the GABA EC₅₀ (1.6 mM). Data = mean ± SEM, n ≥ 4. Values derived from the curves can be found in Table 2.

Fig. 4

(A) An overlay of all 10 docked poses for α-endosulfan. The channel volume occupied by 10 poses are calculated from the Van der Waals radii and are shown in wireframe. Inset Structure of α-endosulfan. Scale = 2.5 Å. (B) A single pose showing the channel from the side. There are hydrogen bond interactions with 6′ Thr residues from adjacent subunits. (C) The same pose is seen from above, looking down towards the cell intererior. (D) Concentration response curves show Q2′N and T6'S mutations caused a decrease in α-endosulfan potency.

Fig. 5

Comparison of pIC₅₀ values from ELIC with those previously reported at GLIC (Alqazzaz et al., 2011). Compounds are almost all less potent at ELIC and fewer compounds inhibit responses.