Phenylalanine in the pore of the Erwinia ligand-gated ion channel modulates picrotoxinin potency but not receptor function.

The Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of eukaryotic Cys-loop ligand-gated ion channels. This protein has the potential to be a useful model for Cys-loop receptors but is unusual in that it has an aromatic residue (Phe) facing into the pore, leading to some predictions that this protein is incapable of ion flux. Subsequent studies have shown this is not the case, so here we probe the role of this residue by examining the function of the ELIC in cases in which the Phe has been substituted with a range of alternative amino acids, expressed in Xenopus oocytes and functionally examined. Most of the mutations have little effect on the GABA EC50, but the potency of the weak pore-blocking antagonist picrotoxinin at F16'A-, F16'D-, F16'S-, and F16'T-containing receptors was increased to levels comparable with those of Cys-loop receptors, suggesting that this antagonist can enter the pore only when residue 16' is small. T6'S has no effect on picrotoxinin potency when expressed alone but abolishes the increased potency when combined with F16'S, indicating that the inhibitor binds at position 6', as in Cys-loop receptors, if it can enter the pore. Overall, the data support the proposal that the ELIC pore is a good model for Cys-loop receptor pores if the role of F16' is taken into consideration.This project was supported by the Wellcome Trust Grant 81925 to S.C.R.L. S.C.R.L. is a Wellcome Trust Senior Research Fellow in Basic Biomedical Studies. M.A. is funded by a Yousef Jameel Scholarship. D.A.W. was funded by an MRC studentship.This is the final published version. It first appeared at http://pubs.acs.org/doi/abs/10.1021/bi5008035

Cys-Loop Receptor Channel Blockers Also Block GLIC

AbstractThe 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 GABAA 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

The Prokaryote Ligand-Gated Ion Channel ELIC Captured in a Pore Blocker-Bound Conformation by the Alzheimer's Disease Drug Memantine

Pentameric ligand-gated ion channels (pLGIC) catalyze the selective transfer of ions across the cell membrane in response to a specific neurotransmitter. A variety of chemically diverse molecules, including the Alzheimer's drug memantine, block ion conduction at vertebrate pLGICs by plugging the channel pore. We show that memantine has similar potency in ELIC, a prokaryotic pLGIC, when it contains an F16'S pore mutation. X-ray crystal structures, using both memantine and its derivative, Br-memantine, reveal that the ligand is localized at the extracellular entryway of the channel pore, and the pore is in a more closed conformation than wild-type ELIC in both the presence and absence of memantine. However, using voltage clamp fluorometry we observe fluorescence changes in opposite directions during channel activation and pore block, revealing an additional conformational transition not apparent from the crystal structures. These results have important implications for drugs such as memantine, which block channel pores.publisher: Elsevier articletitle: The Prokaryote Ligand-Gated Ion Channel ELIC Captured in a Pore Blocker-Bound Conformation by the Alzheimer’s Disease Drug Memantine journaltitle: Structure articlelink: http://dx.doi.org/10.1016/j.str.2014.07.013 content_type: article copyright: Copyright © 2014 Elsevier Ltd. All rights reserved.status: publishe

The Prokaryote Ligand-Gated Ion Channel Elic Captured in a Pore Blocker-Bound Conformation by the Alzheimer's Disease Drug Memantine

Pentameric ligand-gated ion channels (pLGIC) catalyze the selective transfer of ions across the cell membrane in response to a specific neurotransmitter. A variety of chemically diverse molecules, including the Alzheimers drug memantine, block ion conduction at vertebrate pLGICs by plugging the channel pore. We show that memantine has similar potency in ELIC, a prokaryotic pLGIC, when it contains an F16S pore mutation. X-ray crystal structures, using both memantine and its derivative, Br-memantine, reveal that the ligand is localized at the extracellular entryway of the channel pore, and the pore is in a more closed conformation than wild-type ELIC in both the presence and absence of memantine. However, using voltage clamp fluorometry we observe fluorescence changes in opposite directions during channel activation and pore block, revealing an additional conformational transition not apparent from the crystal structures. These results have important implications for drugs such as memantine, which block channel pores