Cholinergic dysfunction in Alzheimer’s disease (AD) can be mediated by the neuronal α7 nicotinic acetylcholine receptor (α7nAChR). Beta-amyloid peptide (Aβ) binds to the α7nAChR, disrupting the receptor’s function and causing neurotoxicity. In vivo not only Aβ but also its modified forms can drive AD pathogenesis. One of these forms, iso-Aβ (containing an isomerized Asp7 residue), shows an increased neurotoxicity in vitro and stimulates amyloidogenesis in vivo. We suggested that such effects of iso-Aβ are α7nAChR-dependent. Here, using calcium imaging and electrophysiology, we found that iso-Aβ is a more potent inhibitor of the α7nAChR-mediated calcium current than unmodified Aβ. However, Asp7 isomerization eliminated the ability of Aβ to decrease the α7nAChR levels. These data indicate differences in the interaction of the peptides with the α7nAChR, which we demonstrated using computer modeling. Neither Aβ nor iso-Aβ competed with ¹²⁵I-α-bungarotoxin for binding to the orthosteric site of the receptor, suggesting the allosteric binging mode of the peptides. Further we found that increased neurotoxicity of iso-Aβ was mediated by the α7nAChR. Thus, the isomerization of Asp7 enhances the inhibitory effect of Aβ on the functional activity of the α7nAChR, which may be an important factor in the disruption of the cholinergic system in AD.

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.

α-Conotoxins from Conus snails are capable of distinguishing muscle and neuronal nicotinic acetylcholine receptors (nAChRs). α-Conotoxin RgIA and αO-conotoxin GeXIVA, blocking neuronal α9α10 nAChR, are potential analgesics. Typically, α-conotoxins bind to the orthosteric sites for agonists/competitive antagonists, but αO-conotoxin GeXIVA was proposed to attach allosterically, judging by electrophysiological experiments on α9α10 nAChR. We decided to verify this conclusion by radioligand analysis in competition with α-bungarotoxin (αBgt) on the ligand-binding domain of the nAChR α9 subunit (α9 LBD), where, from the X-ray analysis, αBgt binds at the orthosteric site. A competition with αBgt was registered for GeXIVA and RgIA, IC₅₀ values being in the micromolar range. However, high nonspecific binding of conotoxins (detected with their radioiodinated derivatives) to His₆-resin attaching α9 LBD did not allow us to accurately measure IC₅₀s. However, IC₅₀s were measured for binding to Aplysia californica AChBP: the RgIA globular isomer, known to be active against α9α10 nAChR, was more efficient than the ribbon one, whereas all three GeXIVA isomers had similar potencies at low µM. Thus, radioligand analysis indicated that both conotoxins can attach to the orthosteric sites in these nAChR models, which should be taken into account in the design of analgesics on the basis of these conotoxins.

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 GABA_AR 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 GABA_AR 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-HT_3AR: 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.

Diverse ligands of the muscle nicotinic acetylcholine receptor (nAChR) are used as muscle relaxants during surgery. Although a plethora of such molecules exists in the market, there is still a need for new drugs with rapid on/off-set, increased selectivity, and so forth. We found that pyrroloiminoquinone alkaloid Makaluvamine G (MG) inhibits several subtypes of nicotinic receptors and ionotropic γ-aminobutiric acid receptors, showing a higher affinity and moderate selectivity toward muscle nAChR. The action of MG on the latter was studied by a combination of electrophysiology, radioligand assay, fluorescent microscopy, and computer modeling. MG reveals a combination of competitive and un-competitive inhibition and caused an increase in the apparent desensitization rate of the murine muscle nAChR. Modeling ion channel kinetics provided evidence for MG binding in both orthosteric and allosteric sites. We also demonstrated that theα1 (G153S) mutant of the receptor, associated with the myasthenic syndrome, is more prone to inhibition by MG. Thus, MG appears to be a perspective hit molecule for the design of allosteric drugs targeting muscle nAChR, especially for treating slow-channel congenital myasthenic syndromes.

With the use of surface plasmon resonance (SPR) it was shown that ws-Lynx1, a water-soluble analog of the three-finger membrane-bound protein Lynx1, that modulates the activity of brain nicotinic acetylcholine receptors (nAChRs), interacts with the acetylcholine-binding protein (AChBP) with high affinity, K D = 62 nM. This result agrees with the earlier demonstrated competition of ws-Lynx1 with radioiodinated α-bungarotoxin for binding to AChBP. For the first time it was shown that ws-Lynx1 binds to GLIC, prokaryotic Cys-loop receptor (K D = 1.3 μM). On the contrary, SPR revealed that α-cobratoxin, a three-finger protein from cobra venom, does not bind to GLIC. Obtained results indicate that SPR is a promising method for analysis of topography of ws-Lynx1 binding sites using its mutants and those of AChBP and GLIC.