Cys-loop receptor channel blockers also block GLIC

Abstract

The Gloeobacter ligand-gated ion channel (GLIC) is a bacterial homolog of vertebrate Cys-loop ligand-gated ion channels. Its pore-lining region in particular has a high sequence homology to these related proteins. Here we use electrophysiology to examine a range of compounds that block the channels of Cys-loop receptors to probe their pharmacological similarity with GLIC. The data reveal that a number of these compounds also block GLIC, although the pharmacological profile is distinct from these other proteins. The most potent compound was lindane, a GABA(A) receptor antagonist, with an IC₅₀ of 0.2 μM. Docking studies indicated two potential binding sites for this ligand in the pore, at the 9' or between the 0' and 2' residues. Similar experiments with picrotoxinin (IC₅₀ = 2.6 μM) and rimantadine (IC₅₀ = 2.6 μM) reveal interactions with 2'Thr residues in the GLIC pore. These locations are strongly supported by mutagenesis data for picrotoxinin and lindane, which are less potent in a T2'S version of GLIC. Overall, our data show that the inhibitory profile of the GLIC pore has considerable overlap with those of Cys-loop receptors, but the GLIC pore has a unique pharmacology.

Figure 1

Alignment of the pore lining regions of GLIC and a selection of related proteins. The residues that line the pore are highlighted. Comparison of the sequences of GLIC and nACh α1 from 0′ to 18′ reveals 28% identity and 61% similarity. A/M2 = Influenza A M2 Channel.

Figure 2

Activation of GLIC. (A) pH dependence of GLIC activation (typical of seven similar experiments) with concentration response curve shown in panel B. (Data = mean ± SE, n = 7.)

Figure 3

GLIC antagonists. (A) Example traces showing inhibition by picrotoxinin (PXN) and concentration-inhibition curves for PXN (B), rimantadine (C), and lindane (D). Inhibition was measured at the pH₅₀. (Data = mean ± SE, n = 3–5.)

Figure 4

An overlay of the 10 most energetically favorable docked poses for lindane, picrotoxinin, tetracaine, and rimantadine. For lindane, the poses are almost equally distributed between two quite distinct binding locations, whereas the locations of the docked poses of the other compounds are broadly similar. The channel volume occupied is calculated from the van der Waals radii and shown in wireframe. (Inset) Structures of the docked ligands; scale bar = 2.5 Å.

Figure 5

Examples of orientations of lindane, picrotoxinin, and rimantadine docked into the GLIC channel near the 2′ residue, the location supported by our mutagenesis data. There are no predicted H-bonds with lindane, but several with 2′Thr residues for both picrotoxinin and rimantadine (dashed lines).