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Nuclear receptor-binding SET domain-containing 2 (NSD2) is the primary enzyme responsible for the dimethylation of lysine 36 of histone 3 (H3K36), a mark associated with active gene transcription and intergenic DNA methylation. In addition to a methyltransferase domain, NSD2 harbors two proline-tryptophan-tryptophan-proline (PWWP) domains and five plant homeodomains (PHDs) believed to serve as chromatin reading modules. Here, we report a chemical probe targeting the N-terminal PWWP (PWWP1) domain of NSD2. UNC6934 occupies the canonical H3K36me2-binding pocket of PWWP1, antagonizes PWWP1 interaction with nucleosomal H3K36me2 and selectively engages endogenous NSD2 in cells. UNC6934 induces accumulation of endogenous NSD2 in the nucleolus, phenocopying the localization defects of NSD2 protein isoforms lacking PWWP1 that result from translocations prevalent in multiple myeloma (MM). Mutations of other NSD2 chromatin reader domains also increase NSD2 nucleolar localization and enhance the effect of UNC6934. This chemical probe and the accompanying negative control UNC7145 will be useful tools in defining NSD2 biology.

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. ispartof: Structure vol:22 issue:10 pages:1399-1407 ispartof: location:United States status: published

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 GABAA 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 GABAA 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 GABAA 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., Highlights ► ELIC is structurally and functionally similar to Cys-loop receptors. ► ELIC can be activated by GABA but not other GABAA receptor agonists. ► ELIC responses are blocked by some compounds that block the channel of GABA-activated and other Cys-loop receptors. ► The potency of the compounds that block ELIC is lower than in Cys-loop receptors and in GLIC. ► ELIC pharmacology is distinct from that of related receptors.

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 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.

International audience; Chronic pain is a highly prevalent and poorly managed human health problem. We used microarray-based expression genomics in 25 inbred mouse strains to identify dorsal root ganglion (DRG)-expressed genetic contributors to mechanical allodynia, a prominent symptom of chronic pain. We identified expression levels of Chrna6, which encodes the α6 subunit of the nicotinic acetylcholine receptor (nAChR), as highly associated with allodynia. We confirmed the importance of α6* (α6-containing) nAChRs by analyzing both gain- and loss-of-function mutants. We find that mechanical allodynia associated with neuropathic and inflammatory injuries is significantly altered in α6* mutants, and that α6* but not α4* nicotinic receptors are absolutely required for peripheral and/or spinal nicotine analgesia. Furthermore, we show that Chrna6's role in analgesia is at least partially due to direct interaction and cross-inhibition of α6* nAChRs with P2X2/3 receptors in DRG nociceptors. Finally, we establish the relevance of our results to humans by the observation of genetic association in patients suffering from chronic postsurgical and temporomandibular pain.

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.

Pentameric ligand-gated ion channels (pLGICs) catalyze the selective transfer of ions across the cell membrane in response to a specific neurotransmitter. A variety of chemically diverse molecules block ion conduction by plugging the channel pore. Understanding the structural basis of pore block is important, as some of these molecules, including the antipsychotic chlorpromazine, the local anaesthetic lidocaine and the Alzheimer's disease drug memantine exert their therapeutic effects by specifically blocking these and other receptors. In this study, we report X-ray crystal structures of a prokaryote model ion channel ELIC containing a pore mutation F16'S, which is inhibited by pore blockers with affinities similar to human pLGICs. Structures in complex with memantine and its brominated derivative, Br-memantine, reveal that these pore blockers bind at the extracellular entryway of the channel pore, and cause a structural change, stabilizing the pore-lining helices in a conformation that is more closed than in the unbound structure. In addition, we observe that memantine binds at the agonist binding site, where it can act as a competitive antagonist. We further investigated the conformational dynamics of the pore domain by using voltage clamp fluorometry, which demonstrated that the pore helix orientation changes in opposite directions during pore block and channel activation, consistent with the crystal structures. These data are the first to reveal a conformational change in the pore initiated by a channel blocker, and have important implications for the action of drugs such as memantine which bind to channel pores.

The prokaryotic ligand-gated ion channels from Gloeobacter violaceus (GLIC) and Erwinia chrysanthemi (ELIC) have provided useful information about the homologous Cys-loop family of neurotransmitter receptors. GLIC is of particular interest for the study of gating transitions, in that its structure has been elucidated in multiple conformational states under a number of conditions. However its mechanism of action is controversial - the protonation of His11’ in M2 has been shown to be essential for the activation of GLIC(1,2), yet a GLIC-glycine chimera retains proton sensitivity without the GLIC M2 domain(3). Here we create an ELIC-GLIC and a GLIC-ELIC chimera, express them in Xenopus oocytes, and test them for function using TEVC. The ELIC-GLIC chimera was not activated by 100mM GABA or pH 4. The GLIC-ELIC chimera was not activated by GABA, but was activated by protons with a pH50 of 6.6, similar to the GLIC-glycine chimera but not WT GLIC (pH50=5.5). We then tested the GLIC-ELIC chimera with crotonic acid and picrotoxin. Crotonic acid inhibits GLIC with an IC50 of 110μM; our data indicate it binds to the extracellular domain. Picrotoxin (IC50=2.6μM) blocks the GLIC pore(4); it likely cannot access the ELIC pore(5), but may bind to the extracellular domain (IC50=96μM;6). These compounds were less potent than expected in the chimera (IC50>300μM). Overall the data suggest that domain specific effects may not be accurately reproduced in complex chimeras with intercommunicating domains, such as an orthosteric binding site and a pore in ligand-gated ion channels.1. Rienzo et al. (2104) Chem.Biol. (in press); 2.Wang et al. (2012) J.Biol.Chem. 287: 6482; 3.Duret et al. (2011). PNAS 108: 12143; 4.Alqazzaz et al., (2011) Biophys.J. 101: 2912; 5.Ulens et al.(2014) Structure(in press). 6.Thompson et al. (2012) Neuropharm. 63:761.

The Gloeobacter ligand-gated ion channel, GLIC, has up to 28% sequence identity with eukaryotic Cys-loop receptors, and many key residues are conserved, especially in the 2nd trans-membrane pore lining region, M2. The M2 region is responsible for ion selectivity, ion flux, and binding a wide range of non-competitive inhibitors (Alqazzaz et al., 2011). The aim of this work was to investigate the GLIC M2 region, especially His235 (or 11'), which has been proposed as essential in proton sensing linked to channel opening (Wang et al., 2012), and residues that are conserved across the Cys-loop receptor family. To probe the roles of specific amino acids in GLIC M2 trans-membrane domain, we substituted M2 residues lining the ion pore and M2 residues facing M3 trans-membrane domains. We generated over 40 mutations at various positions including those at Glu 222(-2'), Thr 226 (2'), Ser 230 (6'), Leu 232 (8'), Ile 233 (9'), Ala 234 (10'), Ile 236 (12'), Ala 237 (13') and Phe 238 (14') using site-directed mutagenesis, and tested their function using two-electrode voltage clamp as previously described (Alqazzaz et al., 2011). Our results showed that Ser6', Ile9' and His11' residues are very sensitive to subsitution with all substituents resulting in non-functional receptors. We conclude that His 11' has a role in channel opening/closing, and the residues Thr226, Ser230, and Ile233 also play an important role in receptor function.Alqazzaz M, Thompson AJ, Price KL, Breitinger HG, Lummis SC (2011). Cys-loop receptor channel blockers also block GLIC. Biophysical journal 101(12): 2912-2918.Wang HL, Cheng X, Sine SM (2012). Intramembrane proton binding site linked to activation of bacterial pentameric ion channel. The Journal of biological chemistry 287(9): 6482-6489.

Several neuronal cell types co-express nicotinic acetylcholine receptors (nAChRs) and P2X receptors (P2XRs); and previous experiments from several laboratories show that these two ligand-gated channel subtypes interact with each other, both functionally and physically. We extended these studies to interactions between alpha6 subunit-containing (alpha6∗) nAChRs and P2X2 or P2X3 receptors (P2X2Rs, P2X3Rs). In Xenopus oocytes, alpha6 subunits mutated in the M2 region co-expressed with beta4 subunits displayed ACh-induced currents. P2X2 or P2X3 receptors expressed in the same oocytes displayed ATP-induced currents. When ACh and ATP were applied simultaneously, the total current was less than the sum of the individual currents_the conventional definition of cross-inhibition_for alpha6beta4-P2X2 oocytes. Several combinations of fluorescent protein (FP)-fused alpha6, beta4, P2X2, and P2X3 subunits were expressed in cultured mouse cortical cells and Neuro2a cells. In Forster resonance energy transfer (FRET) measurements using fluorescent lifetime imaging microscopy (FLIM) and normalized FRET (NFRET), we detected physical contact between alpha6beta2 nAChRs and P2X2Rs, between alpha6beta4 nAChRs and P2X2Rs, and between alpha6beta4 nAChRs and P2X3Rs. Thus, two lines of evidence show interactions between alpha6∗ nAChRs and P2XRs.Support: Beckman fellowship to CIR, NIH (NRSA to CIR, DA-19375, NS034407), Wellcome Trust, Yousef Jameel Award.