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.     

Characteristics of Omega-conotoxin GVI A and MVIIC Binding to Ca<Subscript>v</Subscript> 2.1 and Ca<Subscript>v</Subscript> 2.2 Channels Captured by Anti-Ca<Superscript>2+</Superscript> Channel Peptide Antibodies

A New Binding Method (NBM) was used to investigate the characteristics of the specific binding of ¹²⁵I-omega-conotoxin (ω-CTX) GVIA and ¹²⁵I-ω-CTX MVIIC to Ca_v2.1 and Ca_v2.2 channels captured from chick brain membranes by antibodies against B1Nt (a peptide sequence in Ca_v2.1 and Ca_v2.2 channels). The results for the NBM were as follows. (1) The ED₅₀ values for specific binding of ¹²⁵I-ω-CTX GVIA and ¹²⁵I-ω-CTX MVIIC to Ca_v2.1 and Ca_v2.2 channels were about 68 and 60 pM, respectively, and very similar to those (87 and 35 pM, respectively) to crude membranes from chick brain. (2) The specific ¹²⁵I-ω-CTX GVIA (100 pM) binding was inhibited by ω-CTX GVIA (0.5 nM), dynorphine A (Dyn), gentamicin (Gen), neomycin (Neo) and tobramicin (Tob) (100 μM each), but not by ω-agaconotoxin (Aga) IVA, calciseptine, ω-CTX SVIB, ω-CTX MVIIC (0.5 nM each), PN200-110 (PN), diltiazem (Dil) or verapamil (Ver) (100 μM each). Calmodulin (CaM) inhibited the specific binding in a dose-dependent manner (IC₅₀ value of about 100 μg protein/ml). (3) The specific ¹²⁵I-ω-CTX MVIIC (60 pM) binding was inhibited by ω-CTX MVIIC, ω-CTX GVIA, ω-CTX SVIB (0.5 nM each), Dyn, Neo and Tob (100 μM, each), but not by ω-Aga IVA, calciseptine (0.5 nM each), PN, Dil, Ver (100 μM each) or 100 μg protein/ml CaM. These results suggested that the characteristics of the specific binding of ¹²⁵I-ω-CTX GVIA and ¹²⁵I-ω-CTX MVIIC to Ca_v2.1 and Ca_v2.2 channels in the NBM were very similar to those to crude membranes from chick brain, although the IC₅₀ values for CaM and free Ca²⁺ of CaM were about 33- and 5000-fold higher, respectively, than those for the specific binding of ¹²⁵I-ω-CTX GVIA and ¹²⁵I-ω-CTX MVIIC to crude membranes.     

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.     

Antigens Associated with N- and L-Type Calcium Channels in Lambert-Eaton Myasthenic Syndrome

Abstract: In Lambert-Eaton myasthenic syndrome neurotransmitter release is reduced by an autoimmune response directed against the calcium channel complex of the nerve terminal. Autoantibodies were detected by immunoprecipitation assays using solubilized receptors labeled with ligands selective for N-type (¹²⁵I-ω conotoxin GVIA) and L-type ([³H]PN200-110) calcium channels. Sera with a high antibody titer (>3 n M ) against rat brain N-type channels contained autoantibodies that immunoprecipitated neuronal and muscle L-type channels. These IgG fractions stained a 55-kDa protein in immunoblots of purified skeletal muscle dihydropyridine receptor, suggesting that they contain autoantibodies against the β subunit of the calcium channel. A distinct antibody population in the same fractions reacted with a nerve terminal 65-kDa protein that is unrelated to the β subunit and displays properties similar to those of synaptotagmin.     

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.     

Effects of Local Administration of L-, N-, and P/Q-Type Calcium Channel Blockers on Spontaneous Dopamine Release in the Striatum and the Substantia Nigra: A Microdialysis Study in Rat

Abstract: The pivotal role for voltage-sensitive calcium channels in initiating synaptic transmitter release is undisputed, but it is only partly known to what extent the different subtypes contribute in vivo. Their importance for the dendritic release of dopamine has not been investigated in vivo previously. To evaluate comprehensively the relative importance of different voltage-sensitive calcium channel subtypes for striatal dopamine release, and to further investigate the mechanism of dendritic dopamine release in the reticulate part of substantia nigra, dopamine was measured by in vivo microdialysis in the striatum or substantia nigra of awake rats. The calcium channel blockers nimodipine, ω-conotoxin-GVIA, ω-agatoxin-IVA, and neomycin were administered locally through the dialysis probes and compared with calcium-free perfusion. Results indicate that dopamine release in the striatum is mainly dependent on N- and P/Q-type channels, but the dendritic dopamine release in the substantia nigra is mediated mainly by some other calcium-dependent mechanism, for example, calcium mobilization through T-, O-, or R-type calcium channels. A portion of the dendritic release is calcium independent but can be inhibited partially by neomycin, which might suggest a role for inositol 4,5-bisphosphate breakdown products.     

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.