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Nicotinic acetylcholine receptors (nAChRs) present as many different subtypes in the nervous and immune systems, muscles and on the cells of other organs. In the immune system, inflammation is regulated via the vagus nerve through the activation of the non-neuronal α7 nAChR subtype, affecting the production of cytokines. The analgesic properties of α7 nAChR-selective compounds are mostly based on the activation of the cholinergic anti-inflammatory pathway. The molecular mechanism of neuropathic pain relief mediated by the inhibition of α9-containing nAChRs is not fully understood yet, but the role of immune factors in this process is becoming evident. To obtain appropriate drugs, a search of selective agonists, antagonists and modulators of α7- and α9-containing nAChRs is underway. The naturally occurring three-finger snake α-neurotoxins and mammalian Ly6/uPAR proteins, as well as neurotoxic peptides α-conotoxins, are not only sophisticated tools in research on nAChRs but are also considered as potential medicines. In particular, the inhibition of the α9-containing nAChRs by α-conotoxins may be a pathway to alleviate neuropathic pain. nAChRs are involved in the inflammation processes during AIDS and other viral infections; thus they can also be means used in drug design. In this review, we discuss the role of α7- and α9-containing nAChRs in the immune processes and in pain.

Oligoarginine peptides, known mostly for their cell-penetrating properties, are also inhibitors of the nicotinic acetylcholine receptors (nAChRs). Since octa-arginine (R8) inhibits α9α10 nAChR and suppresses neuropathic pain, we checked if other polycationic compounds containing amino and/or guanidino groups could be effective and tested the activity of the disulfide-fixed “cyclo”R8, a series of biogenic polyamines (putrescine, spermidine, and spermine), C -methylated spermine analogs, agmatine and its analogs, as well as acylpolyamine argiotoxin-636 from spider venom. Their inhibitory potency on muscle-type, α7 and α9α10 nAChRs was determined using radioligand analysis, electrophysiology, and calcium imaging. “Cyclo”R8 showed similar activity to that of R8 against α9α10 nAChR (IC 50 ≈ 60 nM). Biogenic polyamines as well as agmatine and its analogs displayed low activity on muscle-type Torpedo californica , as well as α7 and α9α10 nAChRs, which increased with chain length, the most active being spermine and its C -methylated derivatives having IC 50 of about 30 μM against muscle-type T. californica nAChR. Argiotoxin-636, which contains a polyamine backbone and terminal guanidino group, also weakly inhibited T. californica nAChR (IC 50 ≈ 15 μM), but it revealed high potency against rat α9α10 nAChR (IC 50 ≈ 200 nM). We conclude that oligoarginines and similar polycationic compounds effectively inhibiting α9α10 nAChR may serve as a basis for the development of analgesics to reduce neuropathic pain.

Hypaphorines, tryptophan derivatives, have anti-inflammatory activity, but their mechanism of action was largely unknown. Marine alkaloid L-6-bromohypaphorine with EC50 of 80 μM acts as an agonist of α7 nicotinic acetylcholine receptor (nAChR) involved in anti-inflammatory regulation. We designed the 6-substituted hypaphorine analogs with increased potency using virtual screening of their binding to the α7 nAChR molecular model. Fourteen designed analogs were synthesized and tested in vitro by calcium fluorescence assay on the α7 nAChR expressed in neuro 2a cells, methoxy ester of D-6-iodohypaphorine (6ID) showing the highest potency (EC50 610 nM), being almost inactive toward α9α10 nAChR. The macrophages cytometry revealed an anti-inflammatory activity, decreasing the expression of TLR4 and increasing CD86, similarly to the action of PNU282987, a selective α7 nAChR agonist. 6ID administration in doses 0.1 and 0.5 mg/kg decreased carrageenan-induced allodynia and hyperalgesia in rodents, in accord with its anti-inflammatory action. Methoxy ester of D-6-nitrohypaphorine demonstrated anti-oedemic and analgesic effects in arthritis rat model at i.p. doses 0.05–0.26 mg/kg. Tested compounds showed excellent tolerability with no acute in vivo toxicity in dosages up to 100 mg/kg i.p. Thus, combining molecular modelling and natural product-inspired drug design improved the desired activity of the chosen nAChR ligand.

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.

SLURP-1 is a three-finger human protein targeting nicotinic acetylcholine receptors (nAChRs). The recombinant forms of SLURP-1 produced in E. coli differ in added fusion fragments and in activity. The closest in sequence to the naturally occurring SLURP-1 is the recombinant rSLURP-1, differing by only one additional N-terminal Met residue. sSLURP-1 can be prepared by peptide synthesis and its amino acid sequence is identical to that of the natural protein. In view of recent NMR analysis of the conformational mobility of rSLURP-1 and cryo-electron microscopy structures of complexes of α-bungarotoxin (a three-finger snake venom protein) with Torpedo californica and α7 nAChRs, we compared conformations of sSLURP-1 and rSLURP-1 by Raman spectroscopy and CD-controlled thermal denaturation, analyzed their competition with α-bungarotoxin for binding to the above-mentioned nAChRs, compared the respective receptor complexes with computer modeling and compared their inhibitory potency on the α9α10 nAChR. The CD revealed a higher thermostability of sSLURP-1; some differences between sSLURP-1 and rSLURP-1 were observed in the regions of disulfides and tyrosine residues by Raman spectroscopy, but in binding, computer modeling and electrophysiology, the proteins were similar. Thus, sSLURP-1 and rSLURP-1 with only one additional Met residue appear close in structure and functional characteristics, being appropriate for research on nAChRs.

SLURP-1 is a three-finger human protein targeting nicotinic acetylcholine receptors (nAChRs). The recombinant forms of SLURP-1 produced in E. coli differ in added fusion fragments and in activity. The closest in sequence to the naturally occurring SLURP-1 is the recombinant rSLURP-1, differing by only one additional N-terminal Met residue. sSLURP-1 can be prepared by peptide synthesis and its amino acid sequence is identical to that of the natural protein. In view of recent NMR analysis of the conformational mobility of rSLURP-1 and cryo-electron microscopy structures of complexes of α-bungarotoxin (a three-finger snake venom protein) with Torpedo californica and α7 nAChRs, we compared conformations of sSLURP-1 and rSLURP-1 by Raman spectroscopy and CD-controlled thermal denaturation, analyzed their competition with α-bungarotoxin for binding to the above-mentioned nAChRs, compared the respective receptor complexes with computer modeling and compared their inhibitory potency on the α9α10 nAChR. The CD revealed a higher thermostability of sSLURP-1; some differences between sSLURP-1 and rSLURP-1 were observed in the regions of disulfides and tyrosine residues by Raman spectroscopy, but in binding, computer modeling and electrophysiology, the proteins were similar. Thus, sSLURP-1 and rSLURP-1 with only one additional Met residue appear close in structure and functional characteristics, being appropriate for research on nAChRs.

Elucidation of the structural basis of pharmacological differences for highly homologous [alpha]7 and [alpha]9 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 [alpha]7 and [alpha]9 nAChRs it is complicated by their poor expression and fast desensitization. Here, we probed the ligand-binding properties of [alpha]7/[alpha]9 nAChR mutants by a proposed simple and fast calcium imaging method. The method is based on transient co-expression of [alpha]7/[alpha]9 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 [alpha]7 nAChRs, and for their mutants with homologous residues of [alpha]9 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 [alpha]7 nAChR, showing a larger effect for epibatidine than for acetylcholine, may implicate this position in pharmacological differences between [alpha]7 and [alpha]9 nAChRs.