芋螺毒素与钙离子通道相互作用的计算机模拟

电压门控N－型钙离子通道是与神经元中释放的神经信号传递有关的跨细胞膜的特殊蛋白质分子,它由好几个蛋白质亚基组成,其中的α1亚基包含了电压敏感器和钙离子的选择性孔道,该亚基的一级结构已经发表,一般认为α1亚基包含4个重复单位（I－Ⅳ）,每个重复单位包括6段跨膜区（S1－S6）,其中跨膜区S4上有很多正电荷,被认为是通道的电压敏感器,S5和S6之间的连接区（P区）被认为是形成通道的门孔的部分,N－型钙离子通道能够被一些w-芋螺毒素特异性在阻断,这些ω－芋螺毒素的三维结构已经由二维核磁共振方法测定,尽管还没有被证实,但一般认为w-芋螺毒素占据了通道的孔道,有实验证明,钙离子第三个重复单位的P区（ⅢP区）是通道的芋螺毒素结合的主要部位,在本中,我们用分子模拟程序建模了ⅢP区的结构,为了通道的阻断机理有一个清楚的了解,我们利用分子对接程序模拟了IIIP区和三种芋螺毒素GVA,MVIIA和S03作用的理论模型,在我们的模型中,GVIA与钙通道的作用方式可能与MVIIA不同,而M VII A和SO3与钙通道的作用方式可能相同,我们还讨论了这些芋螺毒素中的关键残基的作用。     

Regulation of protein kinase C Apl II by serotonin receptors in Aplysia

Serotonin (5-hydroxytryptamine, 5HT) is the neurotransmitter that mediates dishabituation in Aplysia. Serotonin mediates this behavioral change through the reversal of synaptic depression in sensory neurons (SNs). However, the 5HT receptors present in SNs and in particular, the receptor important for activation of protein kinase C (PKC) have not been fully identified. Using a recent genome assembly of Aplysia, we identified new receptors from the 5HT(2) , 5HT(4) , and 5HT(7) families. Using RT-PCR from isolated SNs, we found that three 5HT receptors, 5HT(1Apl(a)) , 5HT(2Apl) , and 5HT(7Apl) were expressed in SNs. These receptors were cloned and expressed in a heterologous system. In this system, 5HT(2Apl) could significantly translocate PKC Apl II in response to 5HT and this was blocked by pirenperone, a 5HT(2) receptor antagonist. Surprisingly, pirenperone did not block 5HT-mediated translocation of PKC Apl II in SNs, nor 5HT-mediated reversal of depression. Expression of 5HT(1Apl(a)) in SNs or genistein, an inhibitor of tyrosine kinases inhibited both PKC translocation and reversal of depression. These results suggest a non-canonical mechanism for the translocation of PKC Apl II in SNs.     

Characterization of a T-superfamily conotoxin TxVC from Conus textile that selectively targets neuronal nAChR subtypes

T-superfamily conotoxins have a typical cysteine pattern of “CC–CC”, and are known to mainly target calcium or sodium ion channels. Recently, we screened the targets of a series of T-superfamily conotoxins and found that a new T-superfamily conotoxin TxVC (KPCCSIHDNSCCGL-NH₂) from the venom of Conus textile. It selectively targeted the neuronal nicotinic acetylcholine receptor (nAChR) subtypes α4β2 and α3β2, with IC₅₀ values of 343.4 and 1047.2 nM, respectively, but did not exhibit obvious pharmacological effects on voltage-gated potassium, sodium or calcium channel in DRG cells, the BK channels expressed in HEK293 cells, or the Kv channels in LβT2 cells. The changes in the inhibitory activities of its Ala mutants, the NMR structure, and molecular simulation results based on other conotoxins targeting nAChR α4β2, all demonstrated that the residues Ile⁶ and Leu¹⁴ were the main hydrophobic pharmacophores. To our best knowledge, this is the first T-superfamily conotoxin that inhibits neuronal nAChRs and possesses high binding affinity to α4β2. This finding will expand the knowledge of the targets of T-superfamily conotoxins and the motif information could help the design of new nAChR inhibitors.     

Re‐engineering the μ‐conotoxin SIIIA scaffold

Voltage‐gated sodium (Na_v) channels are responsible for generation and propagation of action potentials throughout the nervous system. Their malfunction causes several disorders and chronic conditions including neuropathic pain. Potent subtype specific ligands are essential for deciphering the molecular mechanisms of Na_v channel function and development of effective therapeutics. µ‐Conotoxin SIIIA is a potent mammalian Na_v1.2 channel blocker that exhibits analgesic activity in rodents. We undertook to reengineer loop 1 through a strategy involving charge alterations and truncations which led to the development of µ‐SIIIA mimetics with novel selectivity profiles. A novel [N5K/D15A]SIIIA(3–20) mutant with enhanced net positive charge showed a dramatic increase in its Na_v1.2 potency (IC₅₀ of 0.5 nM vs. 9.6 nM for native SIIIA) though further truncations led to loss of potency. Unexpectedly, it appears that SIIIA loop 1 significantly influences its Na_v channel interactions despite loop 2 and 3 residues constituting the pharmacophore. This minimal functional conotoxin scaffold may allow further development of selective Na_V blockers. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 347–354, 2014.     

Two Potent α3/5 Conotoxins from Piscivorous Conus achatinus

Every cone snail produces a mixture of different conotoxins and secretes them to immobilize their prey and predators. α3/5 Conotoxins, isolated from fish-hunting cone snails, target muscle nicotinic acetylcholine receptors. The structure and function of α3/5 conotoxin from the piscivorous Conus achatinus have not been studied. We synthesized two pentadecamer peptides, Ac 1.1 a and Ac 1.1 b, with appropriate disulfide bonding, based on cDNA sequences of α3/5 conotoxins from C. achatinus. Ac 1.1 a and Ac 1.1 b differ by only one amino acid residue. They have similar potency on blocking recombinant mouse muscle acetylcholine receptor expressed in Xenopus laevis oocytes, with IC_(50) values of 36 nM and 26 nM, respectively. For Ac 1.1b, deletion of the first three N-terminal amino acids did not change its activity, indicating that the Nterminus is not involved in the interaction with its receptor. Furthermore, our experiments indicate that both toxins strongly prefer the α1-δ subunit interface instead of the α1-γ binding site on the mouse muscle nicotinic acetylcholine receptor. These peptides provide additional tools for the study of the structure and function of nicotinic receptor.     

Interactions among toxins that inhibit N-type and P-type calcium channels

A number of peptide toxins from venoms of spiders and cone snails are high affinity ligands for voltage-gated calcium channels and are useful tools for studying calcium channel function and structure. Using whole-cell recordings from rat sympathetic ganglion and cerebellar Purkinje neurons, we studied toxins that target neuronal N-type (Ca(V)2.2) and P-type (Ca(V)2.1) calcium channels. We asked whether different toxins targeting the same channels bind to the same or different sites on the channel. Five toxins (omega-conotoxin-GVIA, omega-conotoxin MVIIC, omega-agatoxin-IIIA, omega-grammotoxin-SIA, and omega-agatoxin-IVA) were applied in pairwise combinations to either N- or P-type channels. Differences in the characteristics of inhibition, including voltage dependence, reversal kinetics, and fractional inhibition of current, were used to detect additive or mutually occlusive effects of toxins. Results suggest at least two distinct toxin binding sites on the N-type channel and three on the P-type channel. On N-type channels, results are consistent with blockade of the channel pore by omega-CgTx-GVIA, omega-Aga-IIIA, and omega-CTx-MVIIC, whereas grammotoxin likely binds to a separate region coupled to channel gating. omega-Aga-IIIA produces partial channel block by decreasing single-channel conductance. On P-type channels, omega-CTx-MVIIC and omega-Aga-IIIA both likely bind near the mouth of the pore. omega-Aga-IVA and grammotoxin each bind to distinct regions associated with channel gating that do not overlap with the binding region of pore blockers. For both N- and P-type channels, omega-CTx-MVIIC binding produces complete channel block, but is prevented by previous partial channel block by omega-Aga-IIIA, suggesting that omega-CTx-MVIIC binds closer to the external mouth of the pore than does omega-Aga-IIIA.     

Conus peptides--a rich pharmaceutical treasure

Marine predatory cone snails (genus Conus) with over 500 species represent what is arguably the largest single genus of marine animals alive today. All Conus are venomous and utilize a complex mixture of Conus peptides to capture their preys and for other biological purposes. Each component of Conus peptides selectively     

Disulfide structures of highly bridged peptides: a new strategy for analysis.

A new approach is described for analyzing disulfide linkage patterns in peptides containing tightly clustered cystines. Such peptides are very difficult to analyze with traditional strategies, which require that the peptide chain be split between close or adjacent Cys residues. The water-soluble tris-(2-carboxyethyl)-phosphine (TCEP) reduced disulfides at pH 3, and partially reduced peptides were purified by high performance liquid chromatography with minimal thiol-disulfide exchange. Alkylation of free thiols, followed by sequencer analysis, provided explicit assignment of disulfides that had been reduced. Thiol-disulfide exchange occurred during alkylation of some peptides, but correct deductions were still possible. Alkylation competed best with exchange when peptide solution was added with rapid mixing to 2.2 M iodoacetamide. Variants were developed in which up to three alkylating agents were used to label different pairs of thiols, allowing a full assignment in one sequencer analysis. Model peptides used included insulin (three bridges, intra- and interchain disulfides; -Cys.Cys- pair), endothelin and apamin (two disulfides; -Cys.x.Cys- pair), conotoxin GI and isomers (two disulfides; -Cys.Cys- pair), and bacterial enterotoxin (three bridges within 13 residues; two -Cys.Cys- pairs). With insulin, all intermediates in the reduction pathway were identified; with conotoxin GI, analysis was carried out successfully for all three disulfide isomers. In addition to these known structures, the method has been applied successfully to the analysis of several previously unsolved structures of similar complexity. Rates of reduction of disulfide bonds varied widely, but most peptides did not show a strongly preferred route for reduction.     

O2-芋螺毒素Tx7.29抑制钙通道电流及镇痛活性研究（英文）

目的 海洋肉食性软体动物芋螺的毒液是生物活性多肽的一个宝贵来源。这些活性多肽大多是富含二硫键的神经毒素,通常称为芋螺毒素。在本研究中,发现了一个全新的O2超家族芋螺毒素Tx7.29,通过对其进行功能研究,期望发现新的镇痛药候选物。方法 从织锦芋螺毒管c DNA文库中克隆得到Tx7.29的c DNA序列。通过化学合成,制得了Tx7.29的成熟肽,并通过质谱鉴定了其分子质量。通过膜片钳实验和动物实验确定Tx7.29的生物学功能。结果 Tx7.29的c DNA序列编码了一个包含68个氨基酸残基的芋螺毒素前体,由19个残基的信号肽、28个残基的前片段和22个残基的成熟肽组成。圆二色谱分析表明,β转角和反平行片层是Tx7.29二级结构中的主要组分。通过膜片钳实验发现,Tx7.29可以显著抑制大鼠背根神经节细胞的钙通道电流,但对钠电流和钾电流没有明显作用。在小鼠热板疼痛试验中,从0.5到4小时,Tx7.29以剂量依赖性的方式,增加了试验小鼠的热板潜伏时间。Tx7.29对ND7/23细胞无明显细胞毒性。结论 Tx7.29有望成为一种镇痛药物先导化合物,同时它的发现也扩大了O2-芋螺毒素的作用范围。     

Decreased calcium accumulation in isolated nerve endings during hibernation in ground squirrels

Resting and depolarization-induced⁴⁵CaCl₂ accumulation was compared for synaptosomes isolated from hibernating and nonhibernating ground squirrels. Channel subtype antagonists were used to identify the active voltage-sensitive calcium channel subtypes in these preparations. There was significantly less⁴⁵Ca²⁺ accumulation in synaptosomes isolated from hibernating as compared to cold-adapted nonhibernating ground squirrels in both basal (p<0.005) and depolarizing (p<0.03) media over a 30 sec to 5 min incubation period. The elevation in⁴⁵Ca²⁺ accumulation triggered by K⁺ depolarization was blocked by 50 μM CdCl₂, 1 μM ω-conotoxin MVIIC or 1 μM ω-agatoxin IVA. Inhibition was not observed with 1 μM nifedipine or with 1 μM ω-conotoxin GVIA. These results suggest that hibernation is associated with reduced presynaptic⁴⁵Ca²⁺ conductance via voltage-sensitive channels with a pharmacological sensitivity that is different from the established L-, N-, and P-types in other systems but share features of the recently described Q-type calcium channel. This decrease may reflect a cellular adaptation that helps confer tolerance to the near total cerebral ischemia associated with hibernation.