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J Zhejiang Univ (Med Sci)  2020, Vol. 49 Issue (1): 71-75    DOI: 10.3785/j.issn.1008-9292.2020.02.06
    
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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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 wordsCalmodulin      Voltage-gated sodium channels      DNA, circular/analysis      Amino acid sequence      Mutation      Isoleucine      Calcium/metabolism     
Received: 20 September 2019      Published: 08 June 2020
CLC:  R35  
Corresponding Authors: GUO Feng     E-mail: yujun_wan@163.com;aforge@126.com
Cite this article:

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.

URL:

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


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

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 
Fig 1 Schematic illustrations of calmodulin and its mutants
Fig 2 Purification and combination of CaM and GST-IQ determined by pull-down assay
Fig 3 Binding affinity of CaM and GST-IQ at calcium free and at 100 nmol/L
Fig 4 Binding affinity of GST-IQ and CaM or its mutants at calcium free
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