␣-Latrotoxin (LTX) stimulates massive exocytosis of synaptic vesicles and may help to elucidate the mechanism of regulation of neurosecretion. We have recently isolated latrophilin, the synaptic Ca 2؉ -independent LTX receptor. Now we demonstrate that latrophilin is a novel member of the secretin family of G protein-coupled receptors that are involved in secretion. Northern blot analysis shows that latrophilin message is present only in neuronal tissue. Upon expression in COS cells, the cloned protein is indistinguishable from brain latrophilin and binds LTX with high affinity. Latrophilin physically interacts with a G␣ o subunit of heterotrimeric G proteins, because the two proteins co-purify in a twostep affinity chromatography. Interestingly, extracellular domain of latrophilin is homologous to olfactomedin, a soluble neuronal protein thought to participate in odorant binding. Our findings suggest that latrophilin may bind unidentified endogenous ligands and transduce signals into nerve terminals, thus implicating G proteins in the control of synaptic vesicle exocytosis.

Spider venom, a factor that has played a decisive role in the evolution of one of the most successful groups of living organisms, is reviewed. Unique molecular diversity of venom components including substances of variable structure (from simple low molecular weight compounds to large multidomain proteins) with different functions is considered. Special attention is given to the structure, properties, and biosynthesis of toxins of polypeptide nature.

Seven novel short linear antimicrobial and cytolytic peptides named latarcins were purified from the venom of the spider Lachesana tarabaevi. These peptides were found to produce lytic effects on cells of diverse origin (Gram-positive and Gramnegative bacteria, erythrocytes, and yeast) at micromolar concentrations. In addition, five novel peptides that share considerable structural similarity with the purified latarcins were predicted from the L. tarabaevi venom gland expressed sequence tag data base. Latarcins were shown to adopt amphipathic ␣-helical structure in membrane-mimicking environment by CD spectroscopy. Planar lipid bilayer studies indicated that the general mode of action was scaled membrane destabilization at the physiological membrane potential consistent with the "carpet-like" model. Latarcins represent seven new structural groups of lytic peptides and share little homology with other known peptide sequences. For every latarcin, a precursor protein sequence was identified. On the basis of structural features, latarcin precursors were split into three groups: simple precursors with a conventional prepropeptide structure; binary precursors with a typical modular organization; and complex precursors, which were suggested to be cleaved into mature chains of two different types.

Venomous animals from distinct phyla such as spiders, scorpions, snakes, cone snails, or sea anemones produce small toxic proteins interacting with a variety of cell targets. Their bites often cause pain. One of the ways of pain generation is the activation of TRPV1 channels. Screening of 30 different venoms from spiders and sea anemones for modulation of TRPV1 activity revealed inhibitors in tropical sea anemone Heteractis crispa venom. Several separation steps resulted in isolation of an inhibiting compound. This is a 56-residue-long polypeptide named APHC1 that has a Bos taurus trypsin inhibitor (BPTI)/Kunitztype fold, mostly represented by serine protease inhibitors and ion channel blockers. APHC1 acted as a partial antagonist of capsaicin-induced currents (32 ؎ 9% inhibition) with half-maximal effective concentration (EC 50 ) 54 ؎ 4 nM. In vivo, a 0.1 mg/kg dose of APHC1 significantly prolonged tail-flick latency and reduced capsaicin-induced acute pain. Therefore, our results can make an important contribution to the research into molecular mechanisms of TRPV1 modulation and help to solve the problem of overactivity of this receptor during a number of pathological processes in the organism.During the evolutionary process, different poisonous animals combined a set of bioactive compounds in their venoms used mainly to paralyze prey and/or as a defense against predators (1, 2). Bites of these creatures may induce inflammation, pain, tissue necrosis, allergic reactions, and neurotoxic effects such as convulsions, paralysis, respiratory failure, and cardiovascular stroke (3). Numerous toxic peptides are found within these venoms, and some of them can discriminate between closely related cellular targets that make them attractive for drug development and scientific use (4). Molecules accounting for lethal and inflammation effects of venoms have been extensively characterized, but less is known about the properties of other compounds. We concentrated on searching the compounds able to reduce TRPV1 2 conductivity. These receptors are expressed in mammalians in small and medium size dorsal root ganglion neurons and are localized in peripheral and central neuronal system (5-7). At present, it is accepted that TRPV1 receptors are molecular integrators of pain stimulus and initiate neuronal response during inflammation. Experiments with knock-out mice lacking the gene of vanilloid receptor clearly demonstrate its role in pain perception (8, 9). Since vanilloid receptor had been disclosed and cloned in 1997, it became an object of numerous investigations as a potential target for novel drugs against pain of different origin (10). As recently reported, vanillotoxins from a tarantula Psalmopoeus cambridgei directly activate TRPV1 in micromolar concentrations, causing pain effect in the same way as capsaicin does (11). Venoms of several jellyfish also seem to interact with TRPV1, knocking down its desensitization (12). A number of small molecules were synthesized that selectively inhibit TRPV1 in nanomolar concentration ...

␣-Latrotoxin, a black widow spider neurotoxin, can bind to high affinity receptors on the presynaptic plasma membrane and stimulate massive neurotransmitter release in the absence of Ca 2؉. Neurexins, previously isolated as ␣-latrotoxin receptors, require Ca 2؉ for their interaction with the toxin and, thus, may not participate in the Ca 2؉ -independent ␣-latrotoxin activity. We now report the isolation of a novel protein that binds ␣-latrotoxin with high affinity in the presence of various divalent cations (Ca 2؉ , Mg 2؉ , Ba 2؉ , and Sr 2؉ ) as well as in EDTA. This protein, termed here latrophilin, has been purified from detergent-solubilized bovine brain membranes by affinity chromatography on immobilized ␣-latrotoxin and concentrated on a wheat germ agglutinin affinity column. The single polypeptide chain of latrophilin is N-glycosylated and has an apparent molecular weight of 120,000. Sucrose gradient centrifugations demonstrated that latrophilin and ␣-latrotoxin form a stable equimolar complex. In the presence of the toxin, anti-␣-latrotoxin antibodies precipitated iodinated latrophilin, whose binding to immobilized toxin was characterized by a dissociation constant of 0.5-0.7 nM. This presumably membrane-bound protein is localized to and differentially distributed among neuronal tissues, with about four times more latrophilin expressed in the cerebral cortex than in the cerebellum; subcellular fractionation showed that the protein is highly enriched in synaptosomal plasma membranes. Our data suggest that latrophilin may represent the Ca 2؉ -independent receptor and/or molecular target for ␣-latrotoxin.␣-Latrotoxin (LTX), 1 a neurotoxin from the black widow spider venom, potently stimulates neurotransmitter release from all vertebrate synapses tested (1). The toxin causes a massive discharge of synaptic vesicles (2) by acting upon nerve terminals hypothetically in two stages. Initially, it binds to a high affinity presynaptic receptor and then forms nonselective cation-permeable channels in the plasma membrane (reviewed in Ref.3). The subsequent entry of Ca 2ϩ through these channels triggers fast neurotransmitter release (4). However, accumulating evidence suggests that the mode of LTX action is more complex.First, studies in neuronal and PC12 cells demonstrate that the toxin-receptor interaction does not require Ca 2ϩ , although the removal of Ca 2ϩ appreciably decreases the binding (5, 6). This result is best explained by the existence of two classes of LTX receptors, Ca 2ϩ -dependent and independent, both presumably active when Ca 2ϩ is present. These receptor types possess similar high affinities to LTX (7) but display different toxin binding properties (e.g. the Ca 2ϩ -independent binding is more sensitive to high salt) (8). Furthermore, in at least one PC12 cell line only the Ca 2ϩ -independent binding was detectable (7), indicating that the heterogeneous LTX receptors are probably differentially regulated and, thus, may also be structurally different. Ca 2ϩ is also not essential during the second p...