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浙江大学学报(医学版)  2020, Vol. 49 Issue (1): 71-75    DOI: 10.3785/j.issn.1008-9292.2020.02.06
原著     
钙调蛋白及其突变体与电压门控钠离子通道1.2异亮氨酸-谷氨酰胺基序的结合作用
宛昱君1(),刘君妍2,王妤婷2,成小雨1,沙莎1,贾婉莹2,胡德邻1,李昕宇1,郭凤2,*()
1. 中国医科大学临床医学专业, 辽宁 沈阳 110122
2. 中国医科大学药学院药物毒理教研室, 辽宁 沈阳 110122
Effect of calmodulin and its mutants on binding to NaV1.2 IQ
WAN Yujun1(),LIU Junyan2,WANG Yuting2,CHENG Xiaoyu1,SHA Sha1,JIA Wanying2,HU Delin1,LI Xinyu1,GUO Feng2,*()
1. Department of Clinical Medicine, China Medical University, Shenyang 110122, China
2. Department of Pharmaceutical Toxicology, School of Pharmacy, China Medical University, Shenyang 110122, China
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摘要:

目的: 探讨在不同钙离子浓度下电压门控钠离子通道(NaV)1.2与钙调蛋白(CaM)的结合,并分析CaM的钙离子结合位点突变后与NaV1.2结合能力的变化。方法: 应用PCR技术构建NaV1.2蛋白片段异亮氨酸-谷胺酰胺(IQ)基序的cDNA,采用QIAGEN点突变技术构建CaM突变体(CaM12、CaM34、CaM1234),应用牵出(pull-down)试验技术检测有钙(100 nmol/L钙离子浓度)和无钙条件下NaV1.2 IQ与CaM及其突变体(CaM12、CaM34、CaM1234)的结合情况。结果: CaM与NaV1.2 IQ在无钙和有钙情况下均可互相结合,而单独重组谷胱甘肽-S-转移酶(GST)不具有与CaM结合的能力。无钙条件下CaM与NaV1.2 IQ的结合量大于有钙条件下两者的结合量(P < 0.05);无钙条件下,CaM野生型与NaV1.2 IQ结合量大于CaM突变体与NaV1.2 IQ的结合量,其中CaM1234的结合能力在三个突变体中最弱(P < 0.05)。结论: CaM及其突变体对NaV1.2 IQ的结合具有钙离子依赖性,这一CaM调控NaV1.2新机制为离子通道疾病研究提供了一定依据。

关键词: 钙调蛋白电压门控钠通道DNA, 环状/分析氨基酸序列突变异亮氨酸钙/代谢    
Abstract:

Objective: To investigate the effect of calmodulin (CaM) and its mutants on binding to voltage-gated Na channel isoleucine-glutamine domain (NaV1.2 IQ). Methods: The cDNA of NaV1.2 IQ was constructed by PCR technique, CaM mutants CaM12, CaM34 and CaM1234 were constructed with QuickchangeTM site-directed mutagenesis kit (QIAGEN). The binding of NaV1.2 IQ to CaM and CaM mutants under calcium and calcium free conditions were detected by pull-down assay. Results: NaV1.2 IQ and CaM were bound to each other at different calcium concentrations, while GST alone did not bind to CaM. The binding affinity of CaM and NaV1.2 IQ at [Ca2+]-free was greater than that at 100 nmol/L [Ca2+] (P < 0.05). In the absence of calcium, the binding amount of CaM wild-type to NaV1.2 IQ was greater than that of its mutant, and the binding affinity of CaM1234 to NaV1.2 IQ was the weakest among the three mutants (P < 0.05). Conclusions: The binding ability of CaM and CaM mutants to NaV1.2 IQ is Ca2+-dependent. This study has revealed a new mechanism of NaV1.2 regulated by CaM, which would be useful for the study of ion channel related diseases.

Key words: Calmodulin    Voltage-gated sodium channels    DNA, circular/analysis    Amino acid sequence    Mutation    Isoleucine    Calcium/metabolism
收稿日期: 2019-09-20 出版日期: 2020-06-08
CLC:  R35  
基金资助: 国家自然科学基金(81971212, 81471323);辽宁省大学生创新创业训练计划资助(201910159106)
通讯作者: 郭凤     E-mail: yujun_wan@163.com;aforge@126.com
作者简介: 宛昱君(1998-), 女, 大学生; E-mail:yujun_wan@163.com; https://orcid.org/0000-0002-4025-7180
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引用本文:

宛昱君,刘君妍,王妤婷,成小雨,沙莎,贾婉莹,胡德邻,李昕宇,郭凤. 钙调蛋白及其突变体与电压门控钠离子通道1.2异亮氨酸-谷氨酰胺基序的结合作用[J]. 浙江大学学报(医学版), 2020, 49(1): 71-75.

WAN Yujun,LIU Junyan,WANG Yuting,CHENG Xiaoyu,SHA Sha,JIA Wanying,HU Delin,LI Xinyu,GUO Feng. Effect of calmodulin and its mutants on binding to NaV1.2 IQ. J Zhejiang Univ (Med Sci), 2020, 49(1): 71-75.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2020.02.06        http://www.zjujournals.com/med/CN/Y2020/V49/I1/71

图 1  点突变技术构建钙调蛋白突变体示意图
图 2  CaM与GST-IQ的纯化及结合蛋白电泳图
图 3  无钙条件和100 nmol/L钙离子浓度下GST-IQ蛋白与CaM结合蛋白电泳图
图 4  无钙条件下GST-IQ蛋白与CaM及其突变体的结合作用
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