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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。
The Kv2.1 potassium channel current expressed on the beta-islet cells is responsible for the repolarization of the action potential and thus the insulin secretion, which is an effective target for the treatment of type 2 diabetes. JZTX-XI (JZTX-XI) is a new type of peptide neurotoxin isolated and purified from the crude drug of Chilobrachys jingzhao, which inhibits the expression of potassium Kv2.1 on Xenopus laevis oocytes Channel current. In order to study the structure-function relationship of JZTX-XI, wild type JZTX-XI and mutant R3A-JZTX-XI were synthesized by Fomc solid-phase peptide synthesis. Of the oxidative refolding results were detected, resulting in the optimum conditions for the synthesis of the polypeptide oxidative refolding. The relative molecular mass of the refolded product was determined by mass spectrometry to be consistent with the theoretical molecular mass. Refolding JZTX-XI and natural toxins mixed with the same amount by high performance liquid chromatography analysis only get a single peak. Electrokinetic analysis of patch clamp showed that JZTX-XI had a very strong inhibitory effect on the potassium channel subtype hKv2.1 and the sodium channel subtype hNav1.5 transiently expressed in HEK293T cells. The half effective inhibitory concentration (IC50 ) Were 95.8 nmol / L and 437.1 nmol / L, respectively. The IC50 values of the mutant R3A-JZTX-XI for similarly expressed hKv2.1 and hNav1.5 channel currents were 1.22 and 1.96 μmol / L, respectively, when Arg at position 3 was replaced by Ala. The above experimental results show that the inhibitory activity of mutant R3A-JZTX-XI on hKv2.1 and hNav1.5 channels is reduced by 12.7 and 4.5 times compared with that of native JZTX-XI, respectively, indicating that Arg residue at position 3 is responsible for both JZTX-XI The key active residue that interacts with the hKv2.1 channel is also an amino acid residue that is related to the activity of the hNav1.5 channel. The current research results for the development of pancreatic potassium channel Kv2.1 molecular probes and Kv2.1 channel targeting drug design reference.