Purification and characterization of peptides Ap2, Ap3 and Ap5 (ω-toxins) from the venom of the Brazilian tarantula Acanthoscurria paulensis.

Peptides isolated from spider venoms are of pharmacological interest due to their neurotoxic activity, acting on voltage-dependent ion channels present in different types of human body tissues. Three peptide toxins titled as Ap2, Ap3 and Ap5 were purified by RP-HPLC from Acanthoscurria paulensis venom. They were partially sequenced by MALDI In-source Decay method and their sequences were completed and confirmed by transcriptome analysis of the venom gland. The Ap2, Ap3 and Ap5 peptides have, respectively, 42, 41 and 46 amino acid residues, and experimental molecular masses of 4886.3, 4883.7 and 5454.7 Da, with the Ap2 peptide presenting an amidated C-terminus. Amongst the assayed channels - NaV1.1, NaV1.5, NaV1.7, CaV1.2, CaV2.1 and CaV2.2 - Ap2, Ap3 and Ap5 inhibited 20-30 % of CaV2.1 current at 1 µM concentration. Ap3 also inhibited sodium current in NaV1.1, Nav1.5 and Nav1.7 channels by 6.6 ± 1.91 % (p = 0.0276), 4.2 ± 1.09 % (p = 0.0185) and 16.05 ± 2.75 % (p = 0.0282), respectively. Considering that Ap2, Ap3 and Ap5 belong to the 'U'-unknown family of spider toxins, which has few descriptions of biological activity, the present work contributes to the knowledge of these peptides and demonstrates this potential as channel modulators.

Multiple transcriptome mining coupled with tissue specific molecular cloning and mass spectrometry provide insights into agatoxin-like peptide conservation in decapod crustaceans.

Over the past decade, in silico genome and transcriptome mining has led to the identification of many new crustacean peptide families, including the agatoxin-like peptides (ALPs), a group named for their structural similarity to agatoxin, a spider venom component. Here, analysis of publicly accessible transcriptomes was used to expand our understanding of crustacean ALPs. Specifically, transcriptome mining was used to investigate the phylogenetic/structural conservation, tissue localization, and putative functions of ALPs in decapod species. Transcripts encoding putative ALP precursors were identified from one or more members of the Penaeoidea (penaeid shrimp), Sergestoidea (sergestid shrimps), Caridea (caridean shrimp), Astacidea (clawed lobsters and freshwater crayfish), Achelata (spiny/slipper lobsters), and Brachyura (true crabs), suggesting a broad, and perhaps ubiquitous, conservation of ALPs in decapods. Comparison of the predicted mature structures of decapod ALPs revealed high levels of amino acid conservation, including eight identically conserved cysteine residues that presumably allow for the formation of four identically positioned disulfide bridges. All decapod ALPs are predicted to have amidated carboxyl-terminals. Two isoforms of ALP appear to be present in most decapod species, one 44 amino acids long and the other 42 amino acids in length, both likely generated by alternative splicing of a single gene. In carideans, a gene or terminal exon duplication appears to have occurred, with alternative splicing producing four ALPs, two 44 and two 42 amino acid isoforms. The identification of ALP precursor-encoding transcripts in nervous system-specific transcriptomes (e.g., Homarus americanus brain, eyestalk ganglia, and cardiac ganglion assemblies, finding confirmed using RT-PCR) suggests that members of this peptide family may serve as locally-released and/or hormonally-delivered neuromodulators in decapods. Their detection in testis- and hepatopancreas-specific transcriptomes suggests that members of the ALP family may also play roles in male reproduction and innate immunity/detoxification.

A transgenic approach to control hemipteran insects by expressing insecticidal genes under phloem-specific promoters.

The first generation transgenic crops used strong constitutive promoters for transgene expression. However, tissue-specific expression is desirable for more precise targeting of transgenes. Moreover, piercing/sucking insects, which are generally resistant to insecticidal Bacillus thuringiensis (Bt) proteins, have emerged as a major pests since the introduction of transgenic crops expressing these toxins. Phloem-specific promoters isolated from Banana bunchy top virus (BBTV) were used for the expression of two insecticidal proteins, Hadronyche versuta (Blue Mountains funnel-web spider) neurotoxin (Hvt) and onion leaf lectin, in tobacco (Nicotiana tabacum). Here we demonstrate that transgenic plants expressing Hvt alone or in combination with onion leaf lectin are resistant to Phenacoccus solenopsis (cotton mealybug), Myzus persicae (green peach aphids) and Bemisia tabaci (silver leaf whitefly). The expression of both proteins under different phloem-specific promoters resulted in close to 100% mortality and provided more rapid protection than Hvt alone. Our results suggest the employment of the Hvt and onion leaf lectin transgenic constructs at the commercial level will reduce the use of chemical pesticides for control of hemipteran insect pests.

Agatoxin-like peptides in the neuroendocrine system of the honey bee and other insects.

We investigated the peptide inventory of the corpora cardiaca (CC) of the honey bee, Apis mellifera, by direct tissue profiling using MALDI-TOF MS combined with proteomic approaches focusing on cysteine-containing peptides. An agatoxin-like peptide (ALP) was identified as a component of the glandular part of the CC and was associated with the presence of the adipokinetic hormone in mass spectra. Although abundant in the CC, ALP does not belong to the toxins observed in the venom gland of A. mellifera. Homologs of ALP are highly conserved in major groups of arthropods and in line with this we detected ALP in the CC of non-venomous insects such as cockroaches and silverfish. In the American cockroach, Periplaneta americana, ALP was also identified in the CNS and stomatogastric nervous system. This is the first report that establishes the presence of ALPs in the neuroendocrine tissues of insects and further studies are necessary to reveal common functions of these peptides, e.g. as antimicrobial agents, ion channel modulators or classical neuropeptides. BIOLOGICAL SIGNIFICANCE: Among the messenger molecules of the nervous system, neuropeptides represent the structurally most diverse class and basically participate in the regulation of all physiological processes. The set of neuropeptides, their functions and spatial distribution are particularly well-studied in insects. Until now, however, several potential neuropeptide receptors remained orphan, which indicates the existence of so far unknown ligands. In our study, we used proteomic methods such as cysteine modification, enzymatic digestion and peptide derivatization, combined with direct tissue profiling by MALDI-TOF mass spectrometry, for the discovery of novel putative messenger molecules in the neuroendocrine system. The described presence of agatoxin-like peptides in the nervous system of the honey bee and other insects was overseen so far and is thus a remarkable addition to the very well studied neuropeptidome of insects. It is not yet clear, if these toxin-like peptides act as antimicrobial agents, ion channel modulators or classical neuropeptides.

Engineering agatoxin, a cystine-knot peptide from spider venom, as a molecular probe for in vivo tumor imaging.

BACKGROUND: Cystine-knot miniproteins, also known as knottins, have shown great potential as molecular scaffolds for the development of targeted therapeutics and diagnostic agents. For this purpose, previous protein engineering efforts have focused on knottins based on the Ecballium elaterium trypsin inhibitor (EETI) from squash seeds, the Agouti-related protein (AgRP) neuropeptide from mammals, or the Kalata B1 uterotonic peptide from plants. Here, we demonstrate that Agatoxin (AgTx), an ion channel inhibitor found in spider venom, can be used as a molecular scaffold to engineer knottins that bind with high-affinity to a tumor-associated integrin receptor. METHODOLOGY/PRINCIPAL FINDINGS: We used a rational loop-grafting approach to engineer AgTx variants that bound to αvß3 integrin with affinities in the low nM range. We showed that a disulfide-constrained loop from AgRP, a structurally-related knottin, can be substituted into AgTx to confer its high affinity binding properties. In parallel, we identified amino acid mutations required for efficient in vitro folding of engineered integrin-binding AgTx variants. Molecular imaging was used to evaluate in vivo tumor targeting and biodistribution of an engineered AgTx knottin compared to integrin-binding knottins based on AgRP and EETI. Knottin peptides were chemically synthesized and conjugated to a near-infrared fluorescent dye. Integrin-binding AgTx, AgRP, and EETI knottins all generated high tumor imaging contrast in U87MG glioblastoma xenograft models. Interestingly, EETI-based knottins generated significantly lower non-specific kidney imaging signals compared to AgTx and AgRP-based knottins. CONCLUSIONS/SIGNIFICANCE: In this study, we demonstrate that AgTx, a knottin from spider venom, can be engineered to bind with high affinity to a tumor-associated receptor target. This work validates AgTx as a viable molecular scaffold for protein engineering, and further demonstrates the promise of using tumor-targeting knottins as probes for in vivo molecular imaging.

Peptide neurotoxins that affect voltage-gated calcium channels: a close-up on ω-agatoxins.

Peptide neurotoxins found in animal venoms have gained great interest in the field of neurotransmission. As they are high affinity ligands for calcium, potassium and sodium channels, they have become useful tools for studying channel structure and activity. Peptide neurotoxins represent the clinical potential of ion-channel modulators across several therapeutic fields, especially in developing new strategies for treatment of ion channel-related diseases. The aim of this review is to overview the latest updates in the domain of peptide neurotoxins that affect voltage-gated calcium channels, with a special focus on ω-agatoxins.

Familial hemiplegic migraine Ca(v)2.1 channel mutation R192Q enhances ATP-gated P2X3 receptor activity of mouse sensory ganglion neurons mediating trigeminal pain.

BACKGROUND: The R192Q mutation of the CACNA1A gene, encoding for the α1 subunit of voltage-gated P/Q Ca2+ channels (Ca(v)2.1), is associated with familial hemiplegic migraine-1. We investigated whether this gain-of-function mutation changed the structure and function of trigeminal neuron P2X3 receptors that are thought to be important contributors to migraine pain. RESULTS: Using in vitro trigeminal sensory neurons of a mouse genetic model knockin for the CACNA1A R192Q mutation, we performed patch clamp recording and intracellular Ca2+ imaging that showed how these knockin ganglion neurons generated P2X3 receptor-mediated responses significantly larger than wt neurons. These enhanced effects were reversed by the Ca(v)2.1 blocker ω-agatoxin. We, thus, explored intracellular signalling dependent on kinases and phosphatases to understand the molecular regulation of P2X3 receptors of knockin neurons. In such cells we observed strong activation of CaMKII reversed by ω-agatoxin treatment. The CaMKII inhibitor KN-93 blocked CaMKII phosphorylation and the hyperesponsive P2X3 phenotype. Although no significant difference in membrane expression of knockin receptors was found, serine phosphorylation of knockin P2X3 receptors was constitutively decreased and restored by KN-93. No change in threonine or tyrosine phosphorylation was detected. Finally, pharmacological inhibitors of the phosphatase calcineurin normalized the enhanced P2X3 receptor responses of knockin neurons and increased their serine phosphorylation. CONCLUSIONS: The present results suggest that the CACNA1A mutation conferred a novel molecular phenotype to P2X3 receptors of trigeminal ganglion neurons via CaMKII-dependent activation of calcineurin that selectively impaired the serine phosphorylation state of such receptors, thus potentiating their effects in transducing trigeminal nociception.

Spider venom toxin protects plants from insect attack.

Many of the toxin proteins, that have been heterogeneously expressed in agricultural crops to provide resistance to insect pests, are too specific or are only mildly effective against the major insect pests. Spider venoms are a complex cocktail of toxins that have evolved specifically to kill insects. Here we show that the omega-ACTX-Hv1a toxin (Hvt), a component of the venom of the Australian funnel web spider (Hadronyche versuta) that is a calcium channel antagonist, retains its biological activity when expressed in a heterologous system. Expressed as a fusion protein in E. coli, the purified toxin fusion immobilized and killed Helicoverpa armigera and Spodoptera littoralis caterpillars when applied topically. Transgenic expression of Hvt in tobacco effectively protected the plants from H. armigera and S. littoralis larvae, with 100% mortality within 48 h. We conclude that the Hvt is an attractive and effective molecule for the transgenic protection of plants from herbivorous insects which should be evaluated further for possible application in agriculture.

Calcium current subtypes in GnRH neurons.

Calcium plays roles in excitability, rhythm generation, and neurosecretion. Identifying channel subtypes that regulate calcium influx is thus important to understanding rhythmic GnRH secretion, which is a prerequisite for reproduction. Whole-cell voltage-clamp recordings were made from short-term dissociated GnRH adult ovariectomized (OVX) mice (n = 21) to identify channel subtypes that carry calcium current using selective channel blockers and voltage characteristics. Low-voltage activated (LVA) currents were not observed in 42 GnRH neurons tested, although most non-GnRH neurons (4/6) displayed LVA current. The L-type component of the high-voltage activated (HVA) calcium current was 25% +/- 2%. The remaining HVA calcium current passed through N-type (27% +/- 3%), P-type (15% +/- 1%), Q-type (18% +/- 3%), and R-type (15% +/- 1%) channels. Because these data differ substantially from reports on cultured GnRH neurons, which may represent reproductively immature models, we also examined GnRH neurons from gonadal-intact young (Postnatal Days 4-10, n = 8 mice) mice. LVA currents were still rare (2/28) in young mice. Although the same HVA components were observed, the proportions were shifted toward significantly more L-type and less N-type current, suggesting a possible developmental shift in calcium currents in GnRH neurons. These data suggest that calcium channel subtypes in GnRH neurons prepared in the short term from brain slices differ substantially from those in long-term cultured GnRH models. These findings provide a vital foundation to examine the role of calcium channels in the secretory and rhythmic machinery of GnRH neurons.

Tumor necrosis factor alpha increases cytosolic calcium responses to AMPA and KCl in primary cultures of rat hippocampal neurons.

Acute behavioral effects of tumor necrosis factor alpha (TNFalpha) have been previously reported, however the cellular basis for these actions are unknown. To address this issue we examined the effects of TNFalpha on AMPA- and depolarization-induced changes in cytosolic Ca(2+) in cultured hippocampal neurons. Single cell Ca(2+) levels were determined with the fluorescent calcium indicator fura-2. TNFalpha caused an up-regulation of AMPA (10 microM)- and depolarization (55 mM KCl)-induced Ca(2+) responses. This effect occurred within a window of concentrations (1 and 10 ng/ml but not 0.1 or 100 ng/ml) and times (3 and 6 h but not 1 and 24 h). The effect was dependent upon protein synthesis (blocked by cycloheximide) and was prevented by the soluble TNF receptor and by a soluble TNF receptor fragment. Treatment with the soluble TNF receptor fragment also caused a decrease in the basal response. The TNFalpha treatment protocols did not appear to produce any toxicity to the neurons. Results are consistent with the hypothesis that TNFalpha regulates proteins known to be involved in neuronal communication (AMPA receptors) and cell regulation (voltage-dependent calcium channels) in a relatively rapid period of time (a few hours). These actions may be related to the behavioral effects produced by TNFalpha that occur within this time frame.

The presynaptic modulation of corticostriatal afferents by mu-opioids is mediated by K+ conductances.

Population spikes associated with the paired pulse ratio protocol were used to measure the presynaptic inhibition of corticostriatal transmission caused by mu-opioid receptor activation. A 1 microM of [D-Ala(2), N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO), a selective mu-opioid receptor agonist, enhanced paired pulse facilitation by 44+/-8%. This effect was completely blocked by 2 nM of the selective mu-receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-NH (CTOP). Antagonists of N- and P/Q-type Ca(2+) channels inhibited, whereas antagonists of potassium channels enhanced, synaptic transmission. A 1 microM of omega-conotoxin GVIA, a blocker of N-type Ca(2+) channels, had no effect on the action of DAMGO, but 400 nM omega-agatoxin TK, a blocker of P/Q-type Ca(2+)-channels, partially blocked the action of this opioid. However, 5 mM Cs(2+) and 400 microM Ba(2+), unselective antagonists of potassium conductances, completely prevented the action of DAMGO on corticostriatal transmission. These data suggest that presynaptic inhibition of corticostriatal afferents by mu-opioids is mediated by the modulation of K(+) conductances in corticostriatal afferents.

Phorbol ester stimulates a protein kinase C-mediated agatoxin-TK-sensitive calcium permeability pathway in human red blood cells.

Calcium entry into mature erythrocytes (red blood cells; RBCs) is associated with multiple changes in cell properties. At low intracellular Ca(2+), efflux of potassium and water predominates, leading to changes in erythrocyte rheology. At higher Ca(2+) content, activation of kinases and phosphatases, rupture of membrane-to-skeleton bridges, stimulation of a phospholipid scramblase and phospholipase C, and induction of transglutaminase-mediated protein cross-linking are also observed. Because the physiologic relevance of these latter responses depends partially on whether Ca(2+) entry involves a regulated channel or nonspecific leak, we explored mechanisms that initiate controlled Ca(2+) influx. Protein kinase C (PKC) was considered a prime candidate for the pathway regulator, and phorbol-12 myristate-13 acetate (PMA), a stimulator of PKC, was examined for its influence on erythrocyte Ca(2+). PMA was found to stimulate a rapid, dose-dependent influx of calcium, as demonstrated by the increased fluorescence of an entrapped Ca(2+)-sensitive dye, Fluo-3/AM. The PMA-induced entry was inhibited by staurosporine and the PKC-selective inhibitor chelerythrine chloride, but was activated by the phosphatase inhibitors okadaic acid and calyculin A. The PMA-promoted calcium influx was also inhibited by omega-agatoxin-TK, a calcium channel blocker specific for Ca(v)2.1 channels. To confirm that a Ca(v)2.1-like calcium channel exists in the mature erythrocyte membrane, RBC membrane preparations were immunoblotted with antiserum against the alpha(1A) subunit of the channel. A polypeptide of the expected molecular weight (190 kDa) was visualized. These studies indicate that an omega-agatoxin-TK-sensitive, Ca(v)2.1-like calcium permeability pathway is present in the RBC membrane and that it may function under the control of kinases and phosphatases.

Isolation, synthesis and pharmacological characterization of delta-palutoxins IT, novel insecticidal toxins from the spider Paracoelotes luctuosus (Amaurobiidae).

Four novel insecticidal toxins were isolated from the venom of the spider Paracoelotes luctuosus (Araneae: Amaurobiidae) and named delta-palutoxins IT1 to IT4. The four toxins are homologous 36-37 amino acid peptides reticulated by four disulfide bridges and three have amidated C-terminal residues. The delta-palutoxins are highly homologous with the previously described mu-agatoxins and curtatoxins (77-97%). The four peptides demonstrated significant toxicity against larvae of the crop pest Spodoptera litura (Lepidoptera: Noctuidae) in a microinjection bioassay, with LD50 values in the 9-50 microg per g of insect range. This level of toxicity is equivalent to that of several of the most active scorpion toxins used in the development of recombinant baculoviruses, and the delta-palutoxins appear to be insect specific. Electrophysiological experiments demonstrated that delta-palutoxin IT1, the most active toxin acts by affecting insect sodium channel inactivation, resulting in the appearance of a late-maintained sodium current, in a similar fashion to insecticidal scorpion alpha and alpha-like toxins and is thus likely to bind to channel receptor site 3. However, delta-palutoxin IT1 was distinguished by its lack of effect on peak sodium conductance, on the early phase of sodium current inactivation and the absence of a shift in the activation voltage of the sodium channels. delta-Palutoxins are thus proposed as new insecticidal toxins related to the alpha and alpha-like scorpion toxins. They will be useful both in the development of recombinant baculoviruses in agrochemical applications and also as molecular probes for the investigation of molecular mechanisms of insect selectivity and structure and function of sodium channels.

N-Type Ca(2+) channels trigger release of excitatory and inhibitory neurotransmitter from nerve endings in canine bronchi.

We set out to characterize the types of Ca(2+) channels that mediate release of the predominant excitatory (acetylcholine) and inhibitory (norepinephrine) neurotransmitters in canine bronchi, using electrically evoked contractions and relaxations, respectively, as indicators of this release. We found that the selective N-type Ca(2+) channel blocker (omega-conotoxin GVIA) eliminated electrically evoked contractions in a dose-dependent fashion (half-maximal inhibition in the presence of 1-5 nM) but had no significant effect on those evoked by exogenously added acetylcholine. Selective blockers of P-type Ca(2+) channels (omega-agatoxin TK; 10(-8) to 10(-7) M) or of L-type Ca(2+) channels (nifedipine; 10(-8) to 10(-6) M) had no significant effect on the responses to neurally released or exogenously added acetylcholine. Likewise, electrically evoked relaxations were blocked by omega-conotoxin GVIA (10(-7) M) but not by omega-agatoxin TK (10(-7) M) or nifedipine (10(-7) M); none of these Ca(2+) channel blockers had a significant inhibitory effect on isoproterenol-triggered relaxations. We conclude that excitatory and inhibitory neurotransmission in canine bronchi is mediated predominantly by N-type Ca(2+) channels, with little or no contribution from L-, P-, Q-, or T-type channels.

Regulation of neuropeptide Y release by neuropeptide Y receptor ligands and calcium channel antagonists in hypothalamic slices.

Neuropeptide Y (NPY) is an important regulator of energy balance in mammals through its orexigenic, antithermogenic, and insulin secretagogue actions. We investigated the regulation of endogenous NPY release from rat hypothalamic slices by NPY receptor ligands and calcium channel antagonists. High-potassium stimulation (60 mM) of the slices produced a calcium-dependent threefold increase in NPY release above basal release. The Y2 receptor agonists NPY(13-36) and N-acetyl[Leu28,Leu31]NPY(24-36), the Y4 agonist rat pancreatic polypeptide (rPP), and the Y4/Y5 agonist human pancreatic polypeptide (hPP) significantly reduced both basal and stimulated NPY release. NPY(13-36)-induced reduction of NPY release could be partially prevented in the presence of the weak Y2 antagonist T4-[NPY(33-36)]4, whereas the hPP- and rPP-induced inhibition of release was not affected by the Y5 antagonist CGP71683A or the Y1 antagonist BIBP3226. The selective Y1, Y2, and Y5 antagonists had no effect on either basal or potassium-stimulated release when administered alone. The calcium channel inhibitors omega-conotoxin GVIA (N-type), omega-agatoxin TK (P/Q-type), and omega-conotoxin MVIIC (Q-type) all significantly inhibited potassium-stimulated NPY release, without any effect on basal release, whereas nifedipine had no effect on either basal or stimulated release. Addition of both omega-conotoxin GVIA and omega-agatoxin TK together completely inhibited the potassium-stimulated release. In conclusion, we have demonstrated that NPY release from hypothalamic slices is calcium-dependent, involving N-, P-, and Q-type calcium channels. NPY release is also inhibited by Y2 agonists and rPP/hPP, suggesting that Y2 and Y4 receptors may act as autoreceptors on NPY-containing nerve terminals.

Calcium in sympathetic boutons of rat superior cervical ganglion during facilitation, augmentation and potentiation.

The sympathetic preganglionic nerve terminals of the rat superior cervical ganglion were loaded with the calcium indicator oregon green 488 BAPTA-1 to measure the change in calcium concentration in the terminal boutons, (delta[Ca2+]b) following short (1 or 5 impulses) and long (200 impulses) trains at 30 Hz. The delta[Ca2+]b after a single action potential or a short train declined in two phases: a fast phase with a time constant of 530+/-30 ms and a moderate phase with a time constant of 4.0+/-0.2 s. The delta[Ca2+]b following a long train eventually declined with a time constant of 127+/-34 s (slow phase). The addition of either omega-agatoxin TK (100 nM), omega-conotoxin GVIA (100 nM) or nifedipine (20 microM) to block P-type, N-type or L-type calcium channels respectively showed that the rise in delta[Ca2+ ]b in boutons was predominantly mediated by an influx of calcium through P-type (53+/-7%) and N-type (46+/-4%) calcium channels. Experiments with caffeine, ryanodine and thapsigargin indicate that intracellular caffeine-sensitive calcium stores have a small but statistically significant effect on the fast and moderate phases. The mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP; 2 microM) significantly decreased the amplitude of the slow phase of delta[Ca2+]b relaxation, and sped its time course, suggesting that mitochondria normally dump calcium during this phase. Adenosine reduced the amplitude of delta[Ca2+]b in response to single action potentials by 30+/-6%, suggesting that adenosine-mediated autoinhibition in these boutons reduces Ca2+ influx. Spontaneous increases in delta[Ca2+]b demonstrated Ca2+ coupling between adjacent boutons. The delta[Ca2+]b kinetics are compared with F2 facilitation, augmentation and post-tetanic potentiation.

The cystine knot structure of ion channel toxins and related polypeptides.

An increasing number of ion channel toxins and related polypeptides have been found to adopt a common structural motif designated the inhibitor cystine knot motif (Pallaghy P. K., Nielsen, K. J., Craik, D. J., Norton, R. S. (1994) A common structural motif incorporating a cystine knot and triple-stranded beta-sheet in toxic and inhibitory polypeptides. Protein Science 3, 1833-1839). These globular, disulfide-stabilized molecules come from phylogenetically diverse sources, including spiders, cone shells, plants and fungi, and have various functions, although many target voltage-gated ion-channels. The common motif consists of a cystine knot and a triple-stranded, anti-parallel beta-sheet. Examples of ion-channel toxins known to adopt this structure are the omega-conotoxins and omega-agatoxins, and, more recently, robustoxin, versutoxin and protein 5 from spiders, as well as kappa-conotoxin PVIIA and conotoxin GS from cone shells. The variations on the motif structure exemplified by these structures are described here. We also consider the sequences of several polypeptides that might adopt this fold, including SNX-325 from a spider, delta-conotoxin PVIA and the muO-conotoxins from cone shells, and various plant and fungal polypeptides. The interesting case of the two- and three-disulfide bridged binding domains of the cellobiohydrolases from the fungus Trichoderma reesei is also discussed. The compact and robust nature of this motif makes it an excellent scaffold for the design and engineering of novel polypeptides with enhanced activity against existing targets, or with activity against novel targets.

Determination of disulfide structure in agouti-related protein (AGRP) by stepwise reduction and alkylation.

The agouti-related protein gene (Agrp) plays an important role in body weight regulation. The mature human protein is a single polypeptide chain of 112 amino acid residues, consisting of an N-terminal acidic region and a unique C-terminal cysteine-rich domain. The disulfide structure of recombinant human AGRP was determined by chemical methods using partial reduction with tris(2-carboxyethyl)phosphine under acidic conditions, followed by direct alkylation with N-ethylmaleimide or fluorescein-5-maleimide. Partial reduction and alkylation provided several forms of AGRP that were modified in a stepwise fashion. The resulting proteins were characterized by peptide mapping, sequence analysis, and mass spectrometry, showing that AGRP contained a highly reducible disulfide bond, C85-C109, followed by less reactive ones, C90-C97, C74-C88, C67-C82, and C81-C99, respectively. The chemically defined disulfide connectivity of the recombinant human AGRP was homologous to that of omega-agatoxin IVB except for an additional disulfide bond, C85-C109.

Calcium channel types mediating synaptic transmission during aging.

The effects of omega-conotoxin (CTX) GVIA, omega-agatoxin (Aga) IVA, and the dihydropyridine nicardipine were studied on synaptic transmission in the hippocampus during aging. Field excitatory postsynaptic potentials (fEPSPs) were recorded in the CA1 region in slices from young and aged Fischer 344 rats. Peptide toxins reduced synaptic transmissions similarly in both age groups while nicardipine showed no effect. These results suggests that the well documented age-related changes in synaptic transmissions in the hippocampus cannot be explained by changes in the types of Ca2+ channel mediating synaptic transmission.

The structure of versutoxin (delta-atracotoxin-Hv1) provides insights into the binding of site 3 neurotoxins to the voltage-gated sodium channel.

BACKGROUND: Versutoxin (delta-ACTX-Hv1) is the major component of the venom of the Australian Blue Mountains funnel web spider, Hadronyche versuta. delta-ACTX-Hv1 produces potentially fatal neurotoxic symptoms in primates by slowing the inactivation of voltage-gated sodium channels; delta-ACTX-Hv1 is therefore a useful tool for studying sodium channel function. We have determined the three-dimensional structure of delta-ACTX-Hv1 as the first step towards understanding the molecular basis of its interaction with these channels. RESULTS: The solution structure of delta-ACTX-Hv1, determined using NMR spectroscopy, comprises a core beta region containing a triple-stranded antiparallel beta sheet, a thumb-like extension protruding from the beta region and a C-terminal 310 helix that is appended to the beta domain by virtue of a disulphide bond. The beta region contains a cystine knot motif similar to that seen in other neurotoxic polypeptides. The structure shows homology with mu-agatoxin-I, a spider toxin that also modifies the inactivation kinetics of vertebrate voltage-gated sodium channels. More surprisingly, delta-ACTX-Hv1 shows both sequence and structural homology with gurmarin, a plant polypeptide. This similarity leads us to suggest that the sweet-taste suppression elicited by gurmarin may result from an interaction with one of the downstream ion channels involved in sweet-taste transduction. CONCLUSIONS: delta-ACTX-Hv1 shows no structural homology with either sea anemone or alpha-scorpion toxins, both of which also modify the inactivation kinetics of voltage-gated sodium channels by interacting with channel recognition site 3. However, we have shown that delta-ACTX-Hv1 contains charged residues that are topologically related to those implicated in the binding of sea anemone and alpha-scorpion toxins to mammalian voltage-gated sodium channels, suggesting similarities in their mode of interaction with these channels.