Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of ␣-cobratoxin (a long-chain ␣-neurotoxin) and heterodimers formed by ␣-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26 -30 in polypeptide loop II of ␣-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the ␣-cobratoxin capacity to compete with ␣-bungarotoxin for binding to Torpedo and ␣7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that ␣-cobratoxin dimer not only interacts with ␣7 nAChR but, in contrast to ␣-cobratoxin monomer, also blocks ␣3␤2 nAChR. In the latter activity it resembles -bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric ␣3␤2 nAChRs. Three-fingered toxins (TFTs)2 are the main components of the Elapidae snake venoms. TFTs consist of one polypeptide chain, their spatial structure being characterized by a hydrophobic core stabilized by four disulfide bridges, which confine three polypeptide loops (fingers). In cobra venom TFTs are represented mainly by ␣-neurotoxins and cytotoxins. So-called short-chain ␣-neurotoxins (60 -62 amino acid residues, 4 intramolecular disulfides) effectively block nicotinic acetylcholine receptors (nAChRs) of muscle-type, and long-chain ␣-neurotoxins (65-75 residues with an additional disulfide in central loop II) block neuronal homopentameric ␣7 nAChR as well (1, 2). These toxins are widely used as tools in the nAChR studies. Cytotoxins, structurally related to short-chain ␣-neurotoxins, manifest another activity; they non-selectively disrupt cell membranes and, thus, kill the cells (3).Another example of TFT interacting with nAChR are -bungarotoxins (-Bgts), minor components of the krait (Elapidae) venom (4, 5). All -Bgts consist of 66 amino acid residues and, similar to long-chain ␣-neurotoxins, contain five disulfide bonds. However, in contrast to ␣-neurotoxins, -Bgts practically do not block muscle-type nAChRs and only weakly act on ␣7 nAChR but with high efficiency interact with ␣3␤2 neuronal receptors (6). Despite the vast array of data on structure-activity relationship for ␣-neurotoxins and -Bgts, it is not yet clear what are the main structural features determining the specificity of a toxin to the particular receptor type. Recently, based on the x-ray structure of ␣-cobratoxin (␣-CT) with acetylcholinebinding protein, Bourne et al. (7) suggested that Lys-29 is the main residue determining the difference in specificity between ␣-neurotoxins and -bungarotoxins. However, A29K mut...

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Mitochondria are known to participate in the initiation of programmed cell death (PCD) in animals and in plants. The role of chloroplasts in PCD is still unknown. We describe a new system to study PCD in plants; namely, leaf epidermal peels. The peel represents a monolayer consisting of cells of two types: phototrophic (guard cells) and chemotrophic (epidermal cells). The peels from pea (Pisum sativum L.) leaves were treated by cyanide as an inducer of PCD. We found an apoptosis-enhancing effect of illumination on chloroplast-containing guard cells, but not on chloroplastless epidermal cells. Antioxidants and anaerobiosis prevented the CN(-)-induced apoptosis of cells of both types in the dark and in the light. On the other hand, methyl viologen and menadione known as ROS-generating reagents as well as the Hill reaction electron acceptors (BQ, DAD, TMPD, or DPIP) that are not oxidized spontaneously by O2 were shown to prevent the CN(-)-induced nucleus destruction in guard cells. Apoptosis of epidermal cells was potentiated by these reagents, and they had no influence on the CN- effect. The light-dependent activation of CN(-)-induced apoptosis of guard cells was suppressed by DCMU, stigmatellin or DNP-INT, by a protein kinase inhibitor staurosporine as well as by cysteine and serine protease inhibitors. The above data suggest that apoptosis of guard cells is initiated upon a combined action of two factors, i.e., ROS and reduced plastoquinone of the photosynthetic electron transfer chain. As to reduction of ubiquinone in the mitochondrial respiratory chain, it seems to be antiapoptotic for the guard cell.

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.

α-Cobratoxin is the main neurotoxin in the cobra Naja kaouthia venom; it binds efficiently and selectively with neuronal α7 and muscle type nicotinic acetylcholine receptor and can be used for specific labeling and visualization of these receptors in organs and tissues. For these applications we have prepared conjugates of α-cobratoxin with CdSe quantum dots which have many benefits as compared to organic fluorescent labels. To prepare the conjugate, CdSe quantum dots with ZnS shell were functionalized using a tripeptide glutathione and coupled to toxin using water soluble carbodiimide. The conjugate was purified by gel-filtration chromatography and tested for biological activity. It was found that conjugate preserved the capacity to interact with both neuronal α7 and muscle type nicotinic acetylcholine receptor. Its cytotoxicity to mammalian cells was not higher than that of functionalized quantum dots.

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