中文摘要：

表达于β-胰岛细胞上的Kv2.1钾通道电流负责动作电位的复极化,从而调节胰岛素的分泌,是治疗2型糖尿病的有效作用靶点。敬钊毒素-XI（JZTX-XI）是从敬钊缨毛蛛Chilobrachys jingzhao粗毒中分离纯化到的一种新型的肽类神经毒素,能够抑制非洲爪蟾卵母细胞上表达的Kv2.1钾通道电流。为了研究JZTX-XI的结构与功能关系,用芴甲氧羰基（Fomc）固相多肽合成方法合成了野生型JZTX-XI和突变体R3A-JZTX-XI,结合反相HPLC和质谱对不同条件下的氧化复性结果进行检测,从而得到合成多肽的最佳氧化复性条件。复性产物经质谱测定其相对分子质量与理论分子质量相符。复性JZTX-XI与天然毒素等量混合后用高效液相色谱分析也只得到单一峰。膜片钳电生理活性分析结果显示,JZTX-XI对瞬时表达于HEK293T细胞的钾通道亚型hKv2.1和钠通道亚型hNav1.5电流具有非常强的抑制作用,其半数有效抑制浓度（IC50）分别为95.8 nmol/L和437.1 nmol/L。当第3位Arg被Ala取代后,突变体R3A-JZTX-XI对同样表达的hKv2.1和hNav1.5通道电流的IC50值分别为1.22μmol/L和1.96μmol/L。以上实验结果表明突变体R3A-JZTX-XI对hKv2.1和hNav1.5通道的抑制活性分别比天然JZTX-XI降低了12.7倍和4.5倍,说明第3位的Arg残基既是负责JZTX-XI与hKv2.1通道相互作用的关键活性残基,也是与hNav1.5通道活性相关的氨基酸残基。目前的研究结果对研发胰脏钾通道Kv2.1的分子探针和以Kv2.1通道为靶点的药物设计具有借鉴作用。

英文摘要：

Kv2.1 channel currents in pancreatic ？-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion.Because of its central role in this important physiological process,Kv2.1 channel is a promising target for the treatment of type 2 diabetes.Jingzhaotoxin-XI（JZTX-XI） is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao.Two-microelectrode voltage clamp experiments had showed that the toxin inhibited Kv2.1 potassium currents expressed in Xenopus Laevis oocytes.In order to investigate the structure-function relationship of JZTX-XI,the natural toxin and a mutant of JZTX-XI in which Arg3 was replaced by Ala,were synthesized by solid-phase chemistry method with Fmoc-protected amino acids on the PS3 automated peptide synthesizer.Reverse-phase high performance liquid chromatography（RP-HPLC） and matrix assisted laser desorption/ ionization time-of-flight mass spectrometry（MALDI-TOF/TOF MS） were used to monitor the oxidative refolding process of synthetic linear peptides to find the optimal renaturation conditions of these toxins.The experiments also proved that the relative molecular masses of refolded peptides were in accordance with their theoretical molecular masses.RP-HPLC chromatogram of co-injected native and refolded JZTX-XI was a single peak.Under the whole-cell patch-clamp mode,JZTX-XI could completely inhibit hKv2.1 and hNav1.5 channels currents expressed in HEK293T cells with IC50 values of 95.8 nmol/L and 437.1 nmol/L respectively.The mutant R3A-JZTX-XI could also inhibit hKv2.1 and hNav1.5 channel currents expressed in HEK293T cells with IC50 values of 1.22 ？mol/L and 1.96 ？mol/L respectively.However,the prohibitive levels of R3A-JZTX-XI on hKv2.1 and hNav1.5 channels were reduced by about 12.7 times and 4.5 times respectively,indicating that Arg3 was a key amino acid residue relative to the hKv2.1channel activity of JZTX-XI,but it is also an amino acid residue correlated with the binding activity of JZTX-XI to