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敬钊毒素-V 对Kv4.3 通道的抑制作用

蔡丽君, 徐德宏, 罗吉, 陈仁忠, 池宇朋, 曾雄智^*, 王贤纯, 梁宋平

湖南师范大学蛋白质化学与发育生物学教育部重点实验室，长沙 410081

摘要

K+ 通道亚型Kv4.3 在调节心肌细胞动作电位的幅度与时程方面具有重要作用，是治疗心律失常的有效作用靶点，但目前世界上该通道的特异性抑制剂非常缺乏。敬钊毒素-V (Jingzhaotoxin-V, JZTX-V)是从敬钊缨毛蜘蛛粗毒中纯化到的一种新型肽类神经毒素，能够部分抑制大鼠背根神经节细胞上的瞬时外向K+ 电流，其半数有效抑制浓度(IC50 值)为52.3 nmol/L。为了研究JZTX-V 对Kv4.3 通道的作用，本实验通过多肽固相化学合成的方法得到JZTX-V，并用双电极杆电压钳技术检测JZTX-V 对表达在非洲爪蟾卵母细胞上的Kv4.3 通道电流的作用。结果显示，JZTX-V 能够完全抑制Kv4.3 通道电流，并且这种抑制作用具有浓度依赖性和时间依赖性，其IC50 值为425.1 nmol/L，JZTX-V 还能够使通道的电流- 电压关系曲线和稳态失活曲线分别向去极化方向漂移大约29 mV 和10 mV，改变Kv4.3 通道的动力学特征，因此我们推测JZTX-V 是一种Kv4.3 通道门控调制毒素。以上研究结果对于开发心肌Kv4.3 通道的分子探针及以Kv4.3 通道为靶点的药物设计具有借鉴作用。

关键词： 敬钊毒素-V; Kv4 .3 通道; 电压钳技术; 非洲爪蟾

分类号：R329

[Inhibition of Jingzhaotoxin-V on Kv4.3 channel.] [Ariticle in Chinese]

CAI Li-Jun, XU De-Hong, LUO Ji, CHEN Ren-Zhong, CHI Yu-Peng, ZENG Xiong-Zhi^*, WANG Xian-Chun, LIANG Song-Ping

Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, Hunan Normal University, Changsha 410081, China

Abstract

Kv4.3 channel is present in many mammalian tissues, predominantly in the heart and central nervous system. Its currents aretransient, characterized by rapid activation and inactivation. In the hearts of most mammals, it is responsible for repolarization of theaction potential of ventricular myocytes and is important in the regulation of the heart rate. Because of its central role in this importantphysiological process, Kv4.3 channel is a promising target for anti-arrhythmic drug development. Jingzhaotoxin-V (JZTX-V) is a novelpeptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. Whole-cell patch clamp recording showed that itpartly blocked the transient outward potassium channels in dorsal root ganglion neurons of adult rats with an IC50 value of 52.3 nmol/L.To investigate the effect of JZTX-V on Kv4.3 channel, JZTX-V was synthesized using the solid-phase chemical synthesis andseparated by reverse phase high performance liquid chromatography (HPLC). The purity was tested by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MOLDI-TOF mass spectrometry). Two-electrode voltage-clamp technique wasused to characterize the action of JZTX-V on Kv4.3 channels expressed in Xenopus laevis oocytes. As a result, JZTX-V displayed fastkinetics of inhibition and recovery from inactivation. Furthermore, it could inhibit Kv4.3 channel current in a time- and concentrationdependentmanner with an IC50 value of 425.1 nmol/L. The application of JZTX-V affected the activation and inactivation characteristicsof Kv4.3 channel and caused a shift of the current-voltage relationship curve and the steady-state inactivation curve to depolar izing direction by approximately 29 mV and 10 mV, respectively. So we deduced that JZTX-V is a gating modifier toxin of Kv4.3channel. Present findings should be helpful to develop JZTX-V into a molecular probe and drug candidate targeting to Kv4.3 channelin the myocardium.

Key words: Jingzhaotoxin-V; Kv4.3 channel; voltage clamp techniques; Xenopus laevis

收稿日期：2010-03-01　　录用日期：2010-04-15

通讯作者：曾雄智　　E-mail: xiongzhizeng@yahoo.com.cn

引用本文：

蔡丽君, 徐德宏, 罗吉, 陈仁忠, 池宇朋, 曾雄智, 王贤纯, 梁宋平. 敬钊毒素-V 对Kv4.3 通道的抑制作用[J]. 生理学报 2010; 62 (3): 255-260.

CAI Li-Jun, XU De-Hong, LUO Ji, CHEN Ren-Zhong, CHI Yu-Peng, ZENG Xiong-Zhi, WANG Xian-Chun, LIANG Song-Ping. [Inhibition of Jingzhaotoxin-V on Kv4.3 channel.] [Ariticle in Chinese] . Acta Physiol Sin 2010; 62 (3): 255-260 (in Chinese with English abstract).