State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, People's Republic of China.
PLoS One. 2012;7(2):e31830. doi: 10.1371/journal.pone.0031830. Epub 2012 Feb 15.
DrTx(1-42) (a carboxyl-terminally truncated version of drosotoxin) is a potent and selective blocker of tetrodotoxin-resistant (TTX-R) Na(+) channels in rat dorsal root ganglion neurons with analgesic activity. This purpose is to identify key amino acids which are responsible for both blocking and analgesic effects of DrTx(1-42).
On the basis of previous study, we designed five mutants of DrTx(1-42) (delN, D8A, D8K, G9A, and G9R) in the amino-terminal turn (N-turn) region, a proposed functional region located in the amino-terminus of the molecule. All these mutants were expressed in E.coli and purified by RP-HPLC. Electrophysiological properties of these analogues were examined by whole-cell patch-clamp recordings and their antinociceptive effects were investigated by the formalin test and acetic acid induced writhing test.
All the mutants except for G9A possess a similar secondary structure to that of DrTx(1-42), as identified by circular dichroism analysis. Three mutants (delN, D8A and G9A) were found almost inactive to TTX-R Na(+) channels, whereas D8K retains similar activity and G9R showed decreased potency when compared with the wild-type molecule. Consistent with the electrophysiological observations, D8K and G9R exhibited antinociceptive effects in the second phase (inflammatory pain) of the formalin test and the acetic acid induced writhing test, while delN, D8A and G9A lack such effects.
Our results show that the N-turn is closely related to function of DrTx(1-42). The mutant (D8A) as a control peptide further reveals that a charged residue at site 8 of the N-terminus is important for channel blockade and analgesic activity. This study indicates that blocking of voltage-gated TTX-R Na(+) channel in DRG neurons contributes to analgesic effect in rat inflammatory pain. Structural and functional data described here offers support for the development of novel analgesic drugs through targeting TTX-R Na(+) channels.
DrTx(1-42)（河豚毒素抗性（TTX-R）Na+通道的羧基端截断版本）是一种在大鼠背根神经节神经元中对河豚毒素抗性（TTX-R）Na+通道具有高选择性和阻断作用的有效物质，具有镇痛活性。本研究旨在鉴定既能阻断河豚毒素抗性（TTX-R）Na+通道又具有镇痛活性的关键氨基酸。
基于先前的研究，我们设计了 DrTx(1-42)（N 端环，分子氨基端的一个假定功能区）中 5 个突变体（delN、D8A、D8K、G9A 和 G9R）。所有突变体均在大肠杆菌中表达并通过反相高效液相色谱法（RP-HPLC）纯化。通过全细胞膜片钳记录技术检测这些类似物的电生理特性，并通过福尔马林试验和醋酸诱导扭体试验研究其镇痛作用。
除 G9A 外，所有突变体的二级结构均与 DrTx(1-42)相似，这一点通过圆二色谱分析得以确认。3 个突变体（delN、D8A 和 G9A）对 TTX-R Na+通道几乎没有活性，而 D8K 保留了与野生型分子相似的活性，G9R 则表现出活性降低。与电生理观察结果一致，D8K 和 G9R 在福尔马林试验的第二阶段（炎症性疼痛）和醋酸诱导的扭体试验中具有镇痛作用，而 delN、D8A 和 G9A 则没有这种作用。
我们的结果表明，N 端环与 DrTx(1-42)的功能密切相关。突变体（D8A）作为对照肽进一步表明，N 端第 8 位的带电荷残基对于通道阻断和镇痛活性很重要。本研究表明，DRG 神经元电压门控 TTX-R Na+通道的阻断作用有助于大鼠炎症性疼痛的镇痛作用。这里描述的结构和功能数据为通过靶向 TTX-R Na+通道开发新型镇痛药物提供了支持。
