Effects of geographical heterogeneity in species interactions on the evolution of venom genes

Geographical heterogeneity in the composition of biotic interactions can create a mosaic of selection regimes that may drive the differentiation of phenotypes that operate at the interface of these interactions. Nonetheless, little is known about effects of these geographical mosaics on the evolution of genes encoding traits associated with species interactions. Predatory marine snails of the family Conidae use venom, a cocktail of conotoxins, to capture prey. We characterized patterns of geographical variation at five conotoxin genes of a vermivorous species, Conus ebraeus, at Hawaii, Guam and American Samoa, and evaluated how these patterns of variation are associated with geographical heterogeneity in prey utilization. All populations show distinct patterns of prey utilization. Three ‘highly polymorphic’ conotoxin genes showed significant geographical differences in allelic frequency, and appear to be affected by different modes of selection among populations. Two genes exhibited low levels of diversity and a general lack of differentiation among populations. Levels of diversity of ‘highly polymorphic’ genes exhibit a positive relationship with dietary breadth. The different patterns of evolution exhibited by conotoxin genes suggest that these genes play different roles in prey capture, and that some genes are more greatly affected by differences in predator–prey interactions than others. Moreover, differences in dietary breadth appear to have a greater influence on the differentiation of venoms than differences in the species of prey.     

Optimization of oxidative folding methods for cysteine‐rich peptides: a study of conotoxins containing three disulfide bridges

The oxidative folding of small, cysteine‐rich peptides to selectively achieve the native disulfide bond connectivities is critical for discovery and structure‐function studies of many bioactive peptides. As the propensity to acquire the native conformation greatly depends on the peptide sequence, numerous empirical oxidation methods are employed. The context‐dependent optimization of these methods has thus far precluded a generalized oxidative folding protocol, in particular for peptides containing more than two disulfides. Herein, we compare the efficacy of optimized solution‐phase and polymer‐supported oxidation methods using three disulfide‐bridged conotoxins, namely µ‐SIIIA, µ‐KIIIA and ω‐GVIA. The use of diselenide bridges as proxies for disulfide bridges is also evaluated. We propose the ClearOx‐assisted oxidation of selenopeptides as a fairly generalized oxidative folding protocol. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

芋螺毒素SO3对大鼠海马神经元缺氧后胞内游离钙离子浓度的影响

目的:研究芋螺毒素SO3对培养大鼠海马神经元缺氧后胞内游离钙离子浓度的影响.方法:运用激光共聚焦显微镜(CLSM)测定缺氧后大鼠海马神经元胞内游离钙离子浓度的变化.结果与结论:芋螺毒素SO3可以明显抑制因缺氧所致原代培养大鼠海马神经元胞内游离钙离子浓度的上升.     

Molecular basis for the differential sensitivity of rat and human α9α10 nAChRs to α-conotoxin RgIA

J. Neurochem. (2012) 122, 1137-1144. ABSTRACT: The α9α10 nicotinic acetylcholine receptor (nAChR) may be a potential target in pathophysiology of the auditory system, chronic pain, and breast and lung cancers. Alpha-conotoxins, from the predatory marine snail Conus, are potent nicotinic antagonists, some of which are selective for the α9α10 nAChR. Here, we report a two order of magnitude species difference in the potency of α-conotoxin RgIA for the rat versus human α9α10 nAChR. We investigated the molecular mechanism of this difference. Heterologous expression of the rat α9 with the human α10 subunit in Xenopus oocytes resulted in a receptor that was blocked by RgIA with potency similar to that of the rat α9α10 nAChR. Conversely, expression of the human α9 with that of the rat α10 subunit resulted in a receptor that was blocked by RgIA with potency approaching that of the human α9α10 receptor. Systematic substitution of residues found in the human α9 subunit into the homologous position in the rat α9 subunit revealed that a single point mutation, Thr56 to Ile56, primarily accounts for this species difference. Remarkably, although the α9 nAChR subunit has previously been reported to provide the principal (+) binding face for binding of RgIA, Thr56 is located in the (-) complementary binding face.     

Cannabinoid receptor agonists inhibit depolarization-induced calcium influx in cerebellar granule neurons.

Neuronal cannabinoid receptors (CB(1)) are coupled to inhibition of voltage-sensitive Ca(2+) channels (VSCCs) in several cell types. The purpose of these studies was to characterize the interaction between endogenous CB(1) receptors and VSCCs in cerebellar granule neurons (CGN). Ca(2+) transients were evoked by KCl-induced depolarization and imaged using fura-2. The CB(1) receptor agonists CP55940, Win 55212-2 and N-arachidonylethanolamine (anandamide) produced concentration-related decreases in peak amplitude of the Ca(2+) response and total Ca(2+) influx. Pre-treatment of CGN with pertussis toxin abolished agonist-mediated inhibition. The inhibitory effect of Win 55212-2 on Ca(2+) influx was additive with inhibition produced by omega-agatoxin IVA and nifedipine but not with omega-conotoxin GVIA, indicating that N-type VSCCs are the primary effector. Paradoxically, the CB(1) receptor antagonist, SR141716, also inhibited KCl-induced Ca(2+) influx into CGN in a concentration-related manner. SR141716 inhibition was pertussis toxin-insensitive and was not additive with the inhibition produced by Win 55212-2. Confocal imaging of CGN in primary culture demonstrate a high density of CB(1) receptor expression on CGN plasma membranes, including the neuritic processes. These data demonstrate that the CB(1) receptor is highly expressed by CGN and agonists serve as potent and efficacious inhibitory modulators of Ca(2+) influx through N-type VSCC.     

织锦芋螺ο家族芋螺毒素的序列分析

为了从织锦芋螺（Ｃｏｎｕｓｔｅｘｔｉｌｅ）中尽可能多地分离出ο家族的毒素序列和研究其应用价值,在克隆了织锦芋螺α芋螺毒素的基础上进行了织锦芋螺ο家族芋螺毒素基因的分离工作．从织锦芋螺毒管中提取ｍ ＲＮＡ,通过ＲＡＣＥ（ｒａｐｉｄ ａｍ ｐｌｉｆｉｃａｔｉｏｎ ｏｆｃＤＮＡ ｅｎｄｓ,ｃＤＮＡ 末端的快速扩增）－ＰＣＲ方法扩增获得ο家族芋螺毒素ｃＤＮＡ 片段,并进行克隆和序列分析．从织锦芋螺毒液中获得了６种新的芋螺毒素序列,且毒素序列的成熟肽部分均符合Ｃ－ Ｃ－ ＣＣ－ Ｃ－ Ｃ的保守半胱氨酸框架．这些是新的ο家族芋螺毒素序列,新序列的阐明为进一步研究其生物活性和应用打下了基础．     

Identification and characterization of a novel O‐superfamily conotoxin from Conus litteratus

A novel conotoxin named lt6c, an O‐superfamily conotoxin, was identified from the cDNA library of venom duct of Conus litteratus. The full‐length cDNA contains an open reading frame encoding a predicted 22‐residue signal peptide, a 22‐residue proregion and a mature peptide of 28 amino acids. The signal peptide sequence of lt6c is highly conserved in O‐superfamily conotoxins and the mature peptide consists of six cysteines arranged in the pattern of C? C? CC? C? C that is defined the O‐superfamily of conotoxins. The mature peptide fused with thioredoxin, 6‐His tag, and a Factor Xa cleavage site was successfully expressed in Escherichia coli. About 12 mg lt6c was purified from 1L culture. Under whole‐cell patch‐clamp mode, lt6c inhibited sodium currents on adult rat dorsal root ganglion neurons. Therefore, lt6c is a novel O‐superfamily conotoxin that is able to block sodium channels. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Glycerotoxin stimulates neurotransmitter release from N-type Ca2+ channel expressing neurons

Glycerotoxin (GLTx) is capable of stimulating neurotransmitter release at the frog neuromuscular junction by directly interacting with N-type Ca2+ (Cav2.2) channels. Here we have utilized GLTx as a tool to investigate the functionality of Cav2.2 channels in various mammalian neuronal preparations. We first adapted a fluorescent-based high-throughput assay to monitor glutamate release from rat cortical synaptosomes. GLTx potently stimulates glutamate secretion and Ca2+ influx in synaptosomes with an EC50 of 50 pm. Both these effects were prevented using selective Cav2.2 channel blockers suggesting the functional involvement of Cav2.2 channels in mediating glutamate release in this system. We further show that both Cav2.1 (P/Q-type) and Cav2.2 channels contribute equally to depolarization-induced glutamate release. We then investigated the functionality of Cav2.2 channels at the neonatal rat neuromuscular junction. GLTx enhances both spontaneous and evoked neurotransmitter release causing a significant increase in the frequency of postsynaptic action potentials. These effects were blocked by specific Cav2.2 channel blockers demonstrating that either GLTx or its derivatives could be used to selectively enhance the neurotransmitter release from Cav2.2-expressing mammalian neurons.     

芋螺毒素的药用价值研发进展

芋螺毒素是一种海洋软体动物芋螺分泌的一类用于自卫和捕食的小肽神经性毒素。芋螺毒素具有很高的药用开发价值和潜力。近年来,具有高度特异性生物活性的芋螺毒素一直广泛应用于研制特异性诊断试剂以及开发疗效特异的新药之中,并作为分子模型用于相关新药的设计。本文对近年来芋螺毒素药用开发研究的最新进展做一综述。     

A novel α-conotoxin MII-sensitive nicotinic acetylcholine receptor modulates [(3) H]-GABA release in the superficial layers of the mouse superior colliculus

Mouse superficial superior colliculus (SuSC) contains dense GABAergic innervation and diverse nicotinic acetylcholine receptor subtypes. Pharmacological and genetic approaches were used to investigate the subunit compositions of nicotinic acetylcholine receptors (nAChR) expressed on mouse SuSC GABAergic terminals. [(125) I]-Epibatidine competition-binding studies revealed that the α3β2* and α6β2* nicotinic subtype-selective peptide α-conotoxin MII-blocked binding to 40 ± 5% of SuSC nAChRs. Acetylcholine-evoked [(3) H]-GABA release from SuSC crude synaptosomal preparations is calcium dependent, blocked by the voltage-sensitive calcium channel blocker, cadmium, and the nAChR antagonist mecamylamine, but is unaffected by muscarinic, glutamatergic, P2X and 5-HT3 receptor antagonists. Approximately 50% of nAChR-mediated SuSC [(3) H]-GABA release is inhibited by α-conotoxin MII. However, the highly α6β2*-subtype-selective α-conotoxin PIA did not affect [(3) H]-GABA release. Nicotinic subunit-null mutant mouse experiments revealed that ACh-stimulated SuSC [(3) H]-GABA release is entirely β2 subunit-dependent. α4 subunit deletion decreased total function by >90%, and eliminated α-conotoxin MII-resistant release. ACh-stimulated SuSC [(3) H]-GABA release was unaffected by β3, α5 or α6 nicotinic subunit deletions. Together, these data suggest that a significant proportion of mouse SuSC nicotinic agonist-evoked GABA-release is mediated by a novel, α-conotoxin MII-sensitive α3α4β2 nAChR. The remaining α-conotoxin MII-resistant, nAChR agonist-evoked SuSC GABA release appears to be mediated via α4β2* subtype nAChRs.