Curare alkaloids from Matis Dart Poison: Comparison with d-tubocurarine in interactions with nicotinic, 5-HT3 serotonin and GABAA receptors.

Several novel bisbenzylisoquinoline alkaloids (BBIQAs) have recently been isolated from a Matis tribe arrow poison and shown by two-electrode voltage-clamp to inhibit mouse muscle nicotinic acetylcholine receptors (nAChR). Here, using radioligand assay with Aplysia californica AChBP and radioiodinated α-bungarotoxin ([125I]-αBgt), we show that BBIQA1, BBIQA2, and d-tubocurarine (d-TC) have similar affinities to nAChR orthosteric site. However, a competition with [125I]-αBgt for binding to the Torpedo californica muscle-type nAChR revealed that BBIQAs1, 2, and 3 are less potent (IC50s = 26.3, 8.75, and 17.0 μM) than d-TC (IC50 = 0.39 μM), while with α7 nAChR in GH4C1 cells, BBIQA1 was less potent that d-TC (IC50s = 162 μM and 7.77 μM, respectively), but BBIQA2 was similar (IC50 = 5.52 μM). In inhibiting the Ca2+ responses induced by acetylcholine in Neuro2a cells expressing the mouse adult α1β1εδ nAChR or human α7 nAChR, BBIQAs1 and 2 had similar potencies to d-TC (IC50s in the range 0.75-3.08 μM). Our data suggest that BBIQA1 and BBIQA2 can inhibit adult muscle α1β1εδ nAChR by both competitive and noncompetitive mechanisms. Further experiments on neuronal α3β2, α4β2, and α9α10 nAChRs, expressed in Xenopus laevis oocytes, showed that similar potencies for BBIQAs1, 2, and d-TC. With α3β2γ2 GABAAR currents were almost completely inhibited by d-TC at a high (100 μM) concentration, but BBIQAs1 and 2 were less potent (only 40-50% inhibition), whereas in competition with Alexa Fluor 546-α-cobratoxin for binding to α1β3γ2 GABAAR in Neuro2a cells, d-TC and these analogs had comparable affinities. Especially interesting effects of BBIQAs1 and 2 in comparison with d-TC were observed for 5-HT3AR: BBIQA1 and BBIQA2 were 5- and 87-fold less potent than d-TC (IC50 = 22.63 nM). Thus, our results reveal that these BBIQAs differ from d-TC in their potencies towards certain Cys-loop receptors, and we suggest that understanding the reasons behind this might be useful for future drug design.The work of ENS, IAI, DSK, IVS, AIG, LVS, and VIT was supported by the Russian Science Foundation Grant 16-14-00215 (http://rscf. ru/en). The work of IEK was supported by the Russian Foundation for Basic Research Grant 18- 04-01366 (http://www.rfbr.ru/rffi/eng). The work of SCRL was supported by a Medical Research Council Grant MR L021676 (https://mrc.ukri.org/)

Point Mutations of Nicotinic Receptor α1 Subunit Reveal New Molecular Features of G153S Slow-Channel Myasthenia

Slow-channel congenital myasthenic syndromes (SCCMSs) are rare genetic diseases caused by mutations in muscle nicotinic acetylcholine receptor (nAChR) subunits. Most of the known SCCMS-associated mutations localize at the transmembrane region near the ion pore. Only two SCCMS point mutations are at the extracellular domains near the acetylcholine binding site, α1(G153S) being one of them. In this work, a combination of molecular dynamics, targeted mutagenesis, fluorescent Ca2+ imaging and patch-clamp electrophysiology has been applied to G153S mutant muscle nAChR to investigate the role of hydrogen bonds formed by Ser 153 with C-loop residues near the acetylcholine-binding site. Introduction of L199T mutation to the C-loop in the vicinity of Ser 153 changed hydrogen bonds distribution, decreased acetylcholine potency (EC50 2607 vs. 146 nM) of the double mutant and decay kinetics of acetylcholine-evoked cytoplasmic Ca2+ rise (τ 14.2 ± 0.3 vs. 34.0 ± 0.4 s). These results shed light on molecular mechanisms of nAChR activation-desensitization and on the involvement of such mechanisms in channelopathy genesis

Data from: Calcium imaging with genetically encoded sensor Case12: facile analysis of α7/α9 nAChR mutants

Elucidation of the structural basis of pharmacological differences for highly homologous α7 and α9 nicotinic acetylcholine receptors (nAChRs) may shed light on their involvement in different physiological functions and diseases. Combination of site-directed mutagenesis and electrophysiology is a powerful tool to pinpoint the key amino-acid residues in the receptor ligand-binding site, but for α7 and α9 nAChRs it is complicated by their poor expression and fast desensitization. Here, we probed the ligand-binding properties of α7/α9 nAChR mutants by a proposed simple and fast calcium imaging method. The method is based on transient co-expression of α7/α9 nAChR mutants in neuroblastoma cells together with Ric-3 or NACHO chaperones and Case12 fluorescent calcium ion sensor followed by analysis of their pharmacology using a fluorescence microscope or a fluorometric imaging plate reader (FLIPR) with a GFP filter set. The results obtained were confirmed by electrophysiology and by calcium imaging with the conventional calcium indicator Fluo-4. The affinities for acetylcholine and epibatidine were determined for human and rat α7 nAChRs, and for their mutants with homologous residues of α9 nAChR incorporated at positions 117–119, 184, 185, 187, and 189, which are anticipated to be involved in ligand binding. The strongest decrease in the affinity was observed for mutations at positions 187 and 119. The L119D mutation of α7 nAChR, showing a larger effect for epibatidine than for acetylcholine, may implicate this position in pharmacological differences between α7 and α9 nAChRs

pdb

PDB file WT.pdb contains the last frame from 10 ns molecular dynamics simulation alpha7-AChBP chimera (PDB 3SQ6). Only two adjacent subunits were subjected to simulations. PDB files with names E185V, E189G, F187S, L119D, Q117T, R186I, S184N and Y118W contain the last frame from molecular dynamics simulation of alpha7-AChBP chimera with the respective mutations (residues numbered according to human alpha7 nAChR). PDB files F104.pdb and V104_mutant.pdb contain structures in which 104th residue of alpha7 AChBP chimera was changed either to phenylalanine (as in actual human alpha7 nAChR) or to valine (as in alpha9 nAChR)

Calcium imaging with genetically encoded sensor Case12: Facile analysis of α7/α9 nAChR mutants.

Elucidation of the structural basis of pharmacological differences for highly homologous α7 and α9 nicotinic acetylcholine receptors (nAChRs) may shed light on their involvement in different physiological functions and diseases. Combination of site-directed mutagenesis and electrophysiology is a powerful tool to pinpoint the key amino-acid residues in the receptor ligand-binding site, but for α7 and α9 nAChRs it is complicated by their poor expression and fast desensitization. Here, we probed the ligand-binding properties of α7/α9 nAChR mutants by a proposed simple and fast calcium imaging method. The method is based on transient co-expression of α7/α9 nAChR mutants in neuroblastoma cells together with Ric-3 or NACHO chaperones and Case12 fluorescent calcium ion sensor followed by analysis of their pharmacology using a fluorescence microscope or a fluorometric imaging plate reader (FLIPR) with a GFP filter set. The results obtained were confirmed by electrophysiology and by calcium imaging with the conventional calcium indicator Fluo-4. The affinities for acetylcholine and epibatidine were determined for human and rat α7 nAChRs, and for their mutants with homologous residues of α9 nAChR incorporated at positions 117-119, 184, 185, 187, and 189, which are anticipated to be involved in ligand binding. The strongest decrease in the affinity was observed for mutations at positions 187 and 119. The L119D mutation of α7 nAChR, showing a larger effect for epibatidine than for acetylcholine, may implicate this position in pharmacological differences between α7 and α9 nAChRs

New quinoline derivatives as nicotinic receptor modulators

none10siAs a continuation of previous work on quinoline derivatives, which showed some preference (2-3 times) for the α7 with respect to α4β2 acetylcholine nicotinic receptors (nAChRs), we synthesized a series of novel azabicyclic or diazabicyclic compounds carrying a quinoline or isoquinoline ring, with the aim of searching for more selective α7 nAChR compounds. Radioligand binding studies on α7* and α4β2* nAChRs (rat brain homogenate) revealed one compound (7) with a 2-fold higher affinity for the α4β2*-subtype, and four compounds (11, 13, 14 and 16) with at least 3-fold higher affinity for α7* nAChR. The most promising was 11, showing Ki∼100 nM and over 10-fold selectivity for α7* nAChR. Compounds 7, 11, 13 and 16 at 50 μM suppressed ion currents induced in the rat α4β2 nAChR and the chimeric nAChR composed of the ligand-binding domain of the chick α7 and transmembrane domain of the α1 glycine receptor, expressed in Xenopus oocytes. Calcium imaging experiments on the human α7 nAChR expressed in the Neuro2a cells and potentiated by PNU-120596 confirmed the antagonistic activity for 7; on the contrary, 11, 13 and 16 were agonists with the EC50 values in the range of 1.0-1.6 μM. Thus, the introduced modifications allowed us to enhance the selectivity of quinolines towards α7 nAChR and to get novel compounds with agonistic activity.mixedManetti, Dina; Bellucci, Cristina; Dei, Silvia; Teodori, Elisabetta; Varani, Katia; Spirova, Ekaterina; Kudryavtsev, Denis; Shelukhina, Irina; Tsetlin, Victor; Romanelli, Maria NovellaManetti, Dina; Bellucci, Cristina; Dei, Silvia; Teodori, Elisabetta; Varani, Katia; Spirova, Ekaterina; Kudryavtsev, Denis; Shelukhina, Irina; Tsetlin, Victor; Romanelli, Maria Novell

Electrophysiology and Ca_imaging normalized data

The file contains the normalized electrophysiological (TEVC (two-electrode voltage clamp)) and calcium imaging (Ca-img) data for oocyte and Neuro2a cells responses, respectively. WT human α7 nAChR (nicotinic acetylcholine receptor) and mouse muscle nAChR were expressed in oocytes and Neuro2a cells. Besides, their mutants (at positions 117-119, 184, 185, 187 and 189 in α7 nAChR and at positions 153 and 190 in muscle nAChR) were heterologously expressed as well and their responses were estimated accordingly. Responses to different concentrations of acetylcholine (Ach) and epibatidine (Epi) were measured. For calcium imaging studies two calcium sensors (genetically encoded Case12 and low-molecular weight Fluo-4) were used. Calcium imaging analysis was carried out in the presence of a positive allosteric modulator PNU120596

Photos 101-115

Photos 101-115 – Cytochemical fluorescent and bright field images were generated for quantification of Case12 and Alexa Fluor 555-α-bungarotoxin fluorescence in Neuro2a cells expressing human α7 nAChR (nicotinic acetylcholine receptor); Photos 201-210 – Cytochemical fluorescent and bright field images were generated for quantification of Alexa Fluor 555-α-bungarotoxin fluorescence and background fluorescence (in green channel) in Neuro2a cells expressing human α7 nAChR; Photos 301-316 – Cytochemical fluorescent and bright field images were generated for quantification of Case12 fluorescence and background fluorescence (in red channel) in Neuro2a cells expressing human α7 nAChR; Photos 401-413 – Cytochemical fluorescent and bright field images were generated for quantification of background fluorescence (in green and red channels) in Neuro2a cells expressing human α7 nAChR; Photos 501-505 – Cytochemical fluorescent and bright field images were generated for quantification of Case12 and Alexa Fluor 555-α-bungarotoxin fluorescence in Neuro2a cells expressing mouse muscle (WT nAChR; Photos 604-612 – Cytochemical fluorescent and bright field images were generated for quantification of Alexa Fluor 555-α-bungarotoxin fluorescence and background fluorescence (in green channel) in Neuro2a cells expressing mouse muscle (WT) nAChR; Photos 701-710 – Cytochemical fluorescent and bright field images were generated for quantification of TMRE (Tetramethylrhodamine, ethyl ester; 20 nM) labeling of Neuro2a cells expressing human α7 nAChR; Photos 801-805 – Cytochemical fluorescent and bright field images were generated for quantification of PI (Propidium iodide; 50 ng/ml) labeling of Neuro2a cells expressing human α7 nACh

6-Bromohypaphorine from Marine Nudibranch Mollusk Hermissenda crassicornis is an Agonist of Human α7 Nicotinic Acetylcholine Receptor

6-Bromohypaphorine (6-BHP) has been isolated from the marine sponges Pachymatisma johnstoni, Aplysina sp., and the tunicate Aplidium conicum, but data on its biological activity were not available. For the nudibranch mollusk Hermissenda crassicornis no endogenous compounds were known, and here we describe the isolation of 6-BHP from this mollusk and its effects on different nicotinic acetylcholine receptors (nAChR). Two-electrode voltage-clamp experiments on the chimeric α7 nAChR (built of chicken α7 ligand-binding and glycine receptor transmembrane domains) or on rat α4β2 nAChR expressed in Xenopus oocytes revealed no action of 6-BHP. However, in radioligand analysis, 6-BHP competed with radioiodinated α-bungarotoxin for binding to human α7 nAChR expressed in GH4C1 cells (IC50 23 ± 1 μM), but showed no competition on muscle-type nAChR from Torpedo californica. In Ca2+-imaging experiments on the human α7 nAChR expressed in the Neuro2a cells, 6-BHP in the presence of PNU120596 behaved as an agonist (EC50 ~80 μM). To the best of our knowledge, 6-BHP is the first low-molecular weight compound from marine source which is an agonist of the nAChR subtype. This may have physiological importance because H. crassicornis, with its simple and tractable nervous system, is a convenient model system for studying the learning and memory processes