Elena V. Kryukova

Mitochondria Express α7 Nicotinic Acetylcholine Receptors to Regulate Ca2+ Accumulation and Cytochrome c Release: Study on Isolated Mitochondria

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.

Azemiopsin from Azemiops feae Viper Venom, a Novel Polypeptide Ligand of Nicotinic Acetylcholine Receptor

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. Azemiopsin, a novel polypeptide, was isolated from the Azemiops feae viper venom by combination of gel filtration and reverse-phase HPLC. Its amino acid sequence (DNWWPKPPHQGPRPPRPRPKP) was determined by means of Edman degradation and mass spectrometry. It consists of 21 residues and, unlike similar venom isolates, does not contain cysteine residues. According to circular dichroism measurements, this peptide adopts a β-structure. Peptide synthesis was used to verify the determined sequence and to prepare peptide in sufficient amounts to study its biological activity. Azemiopsin efficiently competed with α-bungarotoxin for binding to Torpedo nicotinic acetylcholine receptor (nAChR) (IC50 0.18 ± 0.03 μm) and with lower efficiency to human α7 nAChR (IC50 22 ± 2 μm). It dose-dependently blocked acetylcholine-induced currents in Xenopus oocytes heterologously expressing human muscle-type nAChR and was more potent against the adult form (α1β1ϵδ) than the fetal form (α1β1γδ), EC50 being 0.44 ± 0.1 μm and 1.56 ± 0.37 μm, respectively. The peptide had no effect on GABAA (α1β3γ2 or α2β3γ2) receptors at a concentration up to 100 μm or on 5-HT3 receptors at a concentration up to 10 μm. Ala scanning showed that amino acid residues at positions 3–6, 8–11, and 13–14 are essential for binding to Torpedo nAChR. In biological activity azemiopsin resembles waglerin, a disulfide-containing peptide from the Tropidechis wagleri venom, shares with it a homologous C-terminal hexapeptide, but is the first natural toxin that blocks nAChRs and does not possess disulfide bridges.

Presence of α7 nicotinic acetylcholine receptors on dorsal root ganglion neurons proved using knockout mice and selective α‐neurotoxins in histochemistry

In complex tissues where multiple subtypes of nicotinic acetylcholine receptors (nAChRs) are expressed, immunohistochemistry has been the most popular tool for investigation of nAChR subunit distribution. However, recent studies with nAChR subunit knockout mice demonstrated that a large panel of antibodies is unsuitable. Thus, we aimed to develop a histochemical method for selective labeling of α7 nAChR with neurotoxins, utilizing α7 nAChR‐transfected cells, dorsal root ganglia (DRG) and spinal cord from wild‐type and knockout mouse. The specificity of Alexa Fluor 488‐conjugated α‐bungarotoxin (Alexa‐αBgt) was demonstrated in binding to α7‐transfected cells inhibited by long‐chain α‐cobratoxin (CTX), but not short‐chain α‐neurotoxin II (NTII). In contrast, binding to Torpedo muscle‐type nAChRs and to motor end plates in mouse tongue sections was prevented by both CTX and NTII. In tissue sections of DRG, expressing all neuronal nAChR subunits, only CTX precluded Alexa‐αBgt labeling of neurons, with no staining for α7 nAChR knockout tissue. It proved that α7 nAChRs are the major αBgt‐binding sites in mouse DRG. Corresponding results were obtained for terminals in the spinal cord. Thus, we present a protocol utilizing Alexa‐αBgt and non‐labeled CTX/NTII that allows specific histochemical detection of α7 nAChR with a spatial resolution at the level of single axon terminals.

Vaccination with Peptide 173-193 of Acetylcholine Receptor α7-Subunit Prevents Memory Loss in Olfactory Bulbectomized Mice

We studied the ability of four non-conjugated alpha7-subunit fragments of the nicotinic acetylcholine receptor to induce an immune response and to protect memory in olfactory bulbectomized mice which demonstrate abnormalities similar to Alzheimers disease (AD). Vaccination only with the alpha7-subunit fragment 173-193 was shown to rescue spatial memory, to restore the level of alpha7 acetylcholine receptors in the cortex, and to prevent an increase in the amyloid-beta (Abeta) level in brain tissue in these animals. Antibodies against the peptide 173-193 were revealed in blood serum and cerebrospinal liquid in the bulbectomized mice. Passive immunization with mouse blood sera containing antibodies to the peptide 173-193 also restored memory in bulbectomized animals. The observed positive effect of both active and passive immunization with the fragment of alpha7-subunit on memory of bulbectomized mice provides a new insight into an anti-AD drug design.

Neurotoxins from Snake Venoms and α-Conotoxin ImI Inhibit Functionally Active Ionotropic γ-Aminobutyric Acid (GABA) Receptors

Background: Different snake venom three-finger toxins interact with various receptors, channels, and membranes. Results: Here, we demonstrate that GABAA receptors are inhibited by α-cobratoxin, other long chain α-neurotoxins, nonconventional toxin from Naja kaouthia, and α-conotoxin ImI. Conclusion: Some toxin blockers of nicotinic acetylcholine receptors also inhibit GABAA receptors. Significance: Three-finger toxins offer new scaffolds for the design of GABAA receptor effectors. Ionotropic receptors of γ-aminobutyric acid (GABAAR) regulate neuronal inhibition and are targeted by benzodiazepines and general anesthetics. We show that a fluorescent derivative of α-cobratoxin (α-Ctx), belonging to the family of three-finger toxins from snake venoms, specifically stained the α1β3γ2 receptor; and at 10 μm α-Ctx completely blocked GABA-induced currents in this receptor expressed in Xenopus oocytes (IC50 = 236 nm) and less potently inhibited α1β2γ2 ≈ α2β2γ2 > α5β2γ2 > α2β3γ2 and α1β3δ GABAARs. The α1β3γ2 receptor was also inhibited by some other three-finger toxins, long α-neurotoxin Ls III and nonconventional toxin WTX. α-Conotoxin ImI displayed inhibitory activity as well. Electrophysiology experiments showed mixed competitive and noncompetitive α-Ctx action. Fluorescent α-Ctx, however, could be displaced by muscimol indicating that most of the α-Ctx-binding sites overlap with the orthosteric sites at the β/α subunit interface. Modeling and molecular dynamic studies indicated that α-Ctx or α-bungarotoxin seem to interact with GABAAR in a way similar to their interaction with the acetylcholine-binding protein or the ligand-binding domain of nicotinic receptors. This was supported by mutagenesis studies and experiments with α-conotoxin ImI and a chimeric Naja oxiana α-neurotoxin indicating that the major role in α-Ctx binding to GABAAR is played by the tip of its central loop II accommodating under loop C of the receptors.

Marine natural products acting on the acetylcholine-binding protein and nicotinic receptors: from computer modeling to binding studies and electrophysiology.

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.

Expression and chaperone-assisted refolding of a new cold-active lipase from Psychrobacter cryohalolentis K5T

We describe cloning and expression of genes coding for lipase Lip2Pc and lipase-specific foldase LifPc from a psychrotrophic microorganism Psychrobacter cryohalolentis K5(T) isolated from a Siberian cryopeg (the lense of overcooled brine within permafrost). Upon expression in Escherichiacoli Lip2Pc accumulated in inclusion bodies while chaperone was synthesized in a soluble form. An efficient protocol for solubilization and subsequent refolding of the recombinant lipase in the presence of the truncated chaperone was developed. Using this procedure Lip2Pc with specific activity of 6900U/mg was obtained. Contrary to published data on other lipase-chaperone complexes, refolded Lip2Pc was mostly recovered from the complex with chaperone by metal-affinity chromatography. Recombinant Lip2Pc displayed maximum lipolytic activity at 25°C and pH 8.0 with p-nitrophenyl palmitate (C16) as a substrate. Activity assays conducted at different temperatures revealed that the recombinant Lip2Pc is a cold-adapted lipase with ability to utilize substrates with long (C10-C16) hydrocarbon chains in the temperature range from +5 to +65°C. It demonstrated relatively high stability at temperatures above 60°C and in the presence of various metal ions or organic solvents (ethanol, methanol, etc.). Non-ionic detergents, such as Triton X-100 and Tween 20 decreased Lip2Pc activity and SDS completely inhibited it.

Nerve growth factor from cobra venom inhibits the growth of Ehrlich tumor in mice.

The effects of nerve growth factor (NGF) from cobra venom (cvNGF) on growth of Ehrlich ascites carcinoma (EAC) cells inoculated subcutaneously in mice have been studied. The carcinoma growth slows down, but does not stop, during a course of cvNGF injections and restores after the course has been discontinued. The maximal anti-tumor effect has been observed at a dose of 8 nmoles cvNGF/kg body weight. cvNGF does not impact on lifespan of mice with grafted EAC cells. K252a, a tyrosine kinase inhibitor, attenuates the anti-tumor effect of cvNGF indicating the involvement of TrkA receptors in the process. cvNGF has induced also increase in body weight of the experimental animals. In overall, cvNGF shows the anti-tumor and weight-increasing effects which are opposite to those described for mammalian NGF (mNGF). However in experiments on breast cancer cell line MCF-7 cvNGF showed the same proliferative effects as mNGF and had no cytotoxic action on tumor cells in vitro. These data suggest that cvNGF slows down EAC growth via an indirect mechanism in which TrkA receptors are involved.

Nicotinic receptor involvement in regulation of functions of mouse neutrophils from inflammatory site

Participation of nicotinic acetylcholine receptors (nAChRs) in functioning of polymorphonuclear neutrophils (PMNs) isolated from inflammatory site of mice and expression of different nAChR subunits were studied. Nicotine and acetylcholine (ACh) modified respiratory burst induced by a chemotactic peptide N-formyl-MLF in neutrophils of male (but not female) mice. Antagonists of nAChRs α-cobratoxin (αCTX), α-conotoxins MII and [A10L]PnIA at concentrations of 0.01-5μM, 0.2μM and 1μM, respectively, eliminated nAChR agonist effects. ACh also affected adhesion of PMNs, this effect was also prevented by αCTX (100nM) and MII (1nM). Neutrophils of female mice after chronic nicotine consumption acquired sensitivity to nAChR agonists. Changes of free intracellular Ca(2+) concentration in neutrophils under the action of nAChR ligands were analyzed. In cells with no Ca(2+) oscillations and relatively low resting level of intracellular Ca(2+), nicotine triggered Ca(2+)-spikes, the lag of the response shortened with increasing nicotine concentration. A nicotinic antagonist caramiphen strongly decreased the effect of nicotine. RT-PCR analysis revealed mRNAs of α2, α3, α4, α5, α6, α7, α9, β2, β3, and β4 nAChR subunits. Specific binding of [(125)I]-α-bungarotoxin was demonstrated. Thus in view of the effects and binding characteristics the results obtained suggest a regulatory role of α7, α3β2 or α6* nAChR types in specific functions of PMNs.

Expression of acetylcholine receptors in the brain of mice at the presymptomatic stage of Parkinson's disease.

69 The nigrostriatal dopaminergic (DA ergic) system plays a key role in the regulation of motor behavior of animals and humans [1]. In turn, DA ergic neurons of this system are highly responsive to the effect of nico tine, which stimulates the expression of tyrosine hydroxylase (the rate limiting enzyme of DA synthe sis) and promotes DA release [2, 3]. This effect is mediated by nicotinic acetylcholine receptors (nAChRs), which belong to the superfamily of Cys loop ligand gated ion channels [4]. Disturbances in the functioning of the nigrostriatal DA ergic system and its cholinergic regulation lead to disorders in the motor function. An illustrative example of such a pathology is Parkinson’s disease. The key element of its pathogenesis is degeneration of nigrostriatal DA ergic neurons accompanied by alterations in the expression and functional activity of different nAChR subtypes [5]. A noteworthy fact is that the incidence of Parkinson’s disease among tobacco smokers is mark edly lower than among nonsmokers [6, 7]. Studies on experimental models of the acute clinical stage of this disease have shown that nicotine, acting via α4 and α7 receptors, produces a neuroprotective effect on nigrostriatal DA ergic neurons [5, 8].