Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate synaptic transmission in the muscle and autonomic ganglia and regulate transmitter release in the brain. The nAChRs composed of α7 subunits are also expressed in non-excitable cells to regulate cell survival and proliferation. Up to now, functional α7 nAChRs were found exclusively on the cell plasma membrane. Here we show that they are expressed in mitochondria and regulate early pro-apoptotic events like cytochrome c release. The binding of α7-specific antibody with mouse liver mitochondria was revealed by electron microscopy. Outer membranes of mitochondria from the wild-type and β2−/− but not α7−/− mice bound α7 nAChR-specific antibody and toxins: FITC-labeled α-cobratoxin or Alexa 555-labeled α-bungarotoxin. α7 nAChR agonists (1 µM acetylcholine, 10 µM choline or 30 nM PNU-282987) impaired intramitochondrial Ca2+ accumulation and significantly decreased cytochrome c release stimulated with either 90 µM CaCl2 or 0.5 mM H2O2. α7-specific antagonist methyllicaconitine (50 nM) did not affect Ca2+ accumulation in mitochondria but attenuated the effects of agonists on cytochrome c release. Inhibitor of voltage-dependent anion channel (VDAC) 4,4′-diisothio-cyano-2,2′-stilbene disulfonic acid (0.5 µM) decreased cytochrome c release stimulated with apoptogens similarly to α7 nAChR agonists, and VDAC was co-captured with the α7 nAChR from mitochondria outer membrane preparation in both direct and reverse sandwich ELISA. It is concluded that α7 nAChRs are expressed in mitochondria outer membrane to regulate the VDAC-mediated Ca2+ transport and mitochondrial permeability transition.
A novel "weak toxin" (WTX) from Naja kaouthia snake venom competes with [125 I]␣-bungarotoxin for binding to the membrane-bound Torpedo californica acetylcholine receptor (AChR), with an IC 50 of ϳ2.2 M. In this respect, it is ϳ300 times less potent than neurotoxin II from Naja oxiana and ␣-cobratoxin from N. kaouthia, representing short-type and long-type ␣-neurotoxins, respectively. WTX and ␣-cobratoxin displaced [125 I]␣-bungarotoxin from the Escherichia coli-expressed fusion protein containing the rat ␣7 AChR N-terminal domain 1-208 preceded by glutathione S-transferase with IC 50 values of 4.3 and 9.1 M, respectively, whereas for neurotoxin II the IC 50 value was >100 M. Micromolar concentrations of WTX inhibited acetylcholine-activated currents in Xenopus oocyte-expressed rat muscle AChR and human and rat ␣7 AChRs, inhibiting the latter most efficiently (IC 50 of ϳ8.3 M). Thus, a virtually nontoxic "three-fingered" protein WTX, although differing from ␣-neurotoxins by an additional disulfide in the N-terminal loop, can be classified as a weak ␣-neurotoxin. It differs from the short chain ␣-neurotoxins, which potently block the muscle-type but not the ␣7 AChRs, and is closer to the long ␣-neurotoxins, which have comparable potency against the above-mentioned AChR types.
Background: Information was not available about prototoxin LYNX1 amino acid residues involved in binding to muscle and neuronal nicotinic receptors. Results: A series of water-soluble LYNX1 (ws-LYNX1) mutants was obtained and their interaction with nicotinic receptors was analyzed. Conclusion: There are both common and selective ws-LYNX1 residues recognizing distinct receptor types. Significance: For the first time, several functionally important residues in ws-LYNX1 are identified.
Background: Different snake venom three-finger toxins interact with various receptors, channels, and membranes. Results: Here, we demonstrate that GABA A receptors are inhibited by ␣-cobratoxin, other long chain ␣-neurotoxins, nonconventional toxin from Naja kaouthia, and ␣-conotoxin ImI. Conclusion: Some toxin blockers of nicotinic acetylcholine receptors also inhibit GABA A receptors. Significance: Three-finger toxins offer new scaffolds for the design of GABA A receptor effectors.
Background: Venoms from rare snake species may contain toxins of new structural or/and pharmacological types.Results: Amino acid sequence of the new polypeptide azemiopsin isolated from Azemiops feae viper venom was established, and its biological activity was determined.Conclusion: Azemiopsin is the first natural toxin that blocks nicotinic acetylcholine receptors and does not contain disulfide bridges.Significance: Azemiopsin is the first member of a new toxin group.
Incubation of gangliosides with natural killer (NK) cells from various sources was found to inhibit NK activity in vitro whereas incubation of the same gangliosides with human or mouse lymphoma cells prior to their exposure to NK effectors resulted in a sharp increase in the NK sensitivity of the tumor cells. These effects depended on the oligosaccharide structure of the gangliosides and on the origin of the NK effector cells. The lysis of YAC cells by mouse splenocytes or of MOLT-4 cells by NK cells isolated from the peripheral blood of Syrian hamsters or humans was inhibited most strongly by pre-incubation of the effector cells with gangliosides GM3 and GD3 which are known to be elevated in the serum of tumor-bearing hosts. It is suggested that target cell-associated gangliosides may function as target structures recognized by NK cells while serum gangliosides may contribute to the inhibition of NK cells during tumor development and thus help the tumor to escape NK surveillance.
For a small library of natural products from marine sponges and ascidians, in silico docking to the Lymnaea stagnalis acetylcholine-binding protein (AChBP), a model for the ligand-binding domains of nicotinic acetylcholine receptors (nAChRs), was carried out and the possibility of complex formation was revealed. It was further experimentally confirmed via competition with radioiodinated α-bungarotoxin ([125I]-αBgt) for binding to AChBP of the majority of analyzed compounds. Alkaloids pibocin, varacin and makaluvamines С and G had relatively high affinities (Ki 0.5–1.3 μM). With the muscle-type nAChR from Torpedo californica ray and human neuronal α7 nAChR, heterologously expressed in the GH4C1 cell line, no competition with [125I]-αBgt was detected in four compounds, while the rest showed an inhibition. Makaluvamines (Ki ~ 1.5 μM) were the most active compounds, but only makaluvamine G and crambescidine 359 revealed a weak selectivity towards muscle-type nAChR. Rhizochalin, aglycone of rhizochalin, pibocin, makaluvamine G, monanchocidin, crambescidine 359 and aaptamine showed inhibitory activities in electrophysiology experiments on the mouse muscle and human α7 nAChRs, expressed in Xenopus laevis oocytes. Thus, our results confirm the utility of the modeling studies on AChBPs in a search for natural compounds with cholinergic activity and demonstrate the presence of the latter in the analyzed marine biological sources.
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