Chi Yupeng, Deng Meichun, Wu Yuanyuan, Luo Ji, Rong Minqiang, Zhang Yiya, Zhang Dongyi, Zeng Xiongzhi, Liang Songping
Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, Hunan Normal University, Changsha 410081, China.
Sheng Wu Gong Cheng Xue Bao. 2011 Jun;27(6):900-8.
Kv2.1 channel currents in pancreatic beta-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 micromol/L and 1.96 micromol/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 hKv2.1 channel activity of JZTX-XI, but it is also an amino acid residue correlated with the binding activity of JZTX-XI to hNav1.5 channel. Our findings should be helpful to develop JZTX-XI into a molecular probe and drug candidate targeting to Kv2.1 potassium channel in the pancreas.
胰腺β细胞中的Kv2.1通道电流被认为有助于动作电位复极化，从而调节胰岛素分泌。由于其在这一重要生理过程中的核心作用，Kv2.1通道是治疗2型糖尿病的一个有前景的靶点。敬钊毒素-XI（JZTX-XI）是从蜘蛛敬钊缨毛蛛毒液中分离出的一种新型肽神经毒素。双电极电压钳实验表明，该毒素抑制非洲爪蟾卵母细胞中表达的Kv2.1钾电流。为了研究JZTX-XI 的结构-功能关系，在PS3自动肽合成仪上采用Fmoc保护的氨基酸通过固相化学方法合成了天然毒素以及将JZTX-XI的Arg3替换为Ala的突变体。采用反相高效液相色谱（RP-HPLC）和基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF/TOF MS）监测合成线性肽的氧化重折叠过程，以找到这些毒素的最佳复性条件。实验还证明重折叠肽的相对分子质量与其理论分子质量一致。共注射天然和重折叠JZTX-XI的RP-HPLC色谱图为单峰。在全细胞膜片钳模式下，JZTX-XI能完全抑制HEK293T细胞中表达的hKv2.1和hNav1.5通道电流，IC50值分别为95.8 nmol/L和437.1 nmol/L。突变体R3A-JZTX-XI也能抑制HEK293T细胞中表达的hKv2.1和hNav1.5通道电流，IC50值分别为1.22 μmol/L和1.96 μmol/L。然而，R3A-JZTX-XI对hKv2.1和hNav1.5通道的抑制水平分别降低了约12.7倍和4.5倍，表明Arg3是相对于JZTX-XI的hKv2.1通道活性的关键氨基酸残基，但它也是与JZTX-XI与hNav1.5通道结合活性相关的氨基酸残基。我们的研究结果应有助于将JZTX-XI开发成一种针对胰腺中Kv2.1钾通道的分子探针和候选药物。
