Please wait a minute...
浙江大学学报（医学版）
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
 全文: PDF(2077 KB)   HTML( 5 )
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
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　
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

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    或    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
1 SHAO D , ZHAO M , XU J et al. The individual N- and C-lobes of calmodulin tether to the Cav1.2 channel and rescue the channel activity from run-down in ventricular myocytes of guinea-pig heart[J]. FEBS Lett, 2014, 588 (21): 3855- 3861
doi: 10.1016/j.febslet.2014.09.029
2 KWONG K , CARR M J . Voltage-gated sodium channels[J]. Curr Opin Pharmacol, 2015, 22:131- 139
doi: 10.1016/j.coph.2015.04.007
3 BEN-JOHNY M , DICK I E , SANG L et al. Towards a unified theory of calmodulin regulation (calmodulation) of voltage-gated calcium and sodium channels[J]. Curr Mol Pharmacol, 2015, 8 (2): 188- 205
doi: 10.2174/1874467208666150507110359
4 EDINGTON S C , HALLING D B , BENNETT S M et al. Non-additive effects of binding site mutations in calmodulin[J]. Biochemistry, 2019, 58 (24): 2730- 2739
doi: 10.1021/acs.biochem.9b00096
5 KAWASAKI H , KRETSINGER R H . Conformational landscape mapping the difference between N-lobes and C-lobes of calmodulin[J]. J Inorg Biochem, 2017, 177:55- 62
doi: 10.1016/j.jinorgbio.2017.08.025
6 HE G , GUO F , ZH UT et al. Lobe-related concentration- and Ca2+-dependent interactions of calmodulin with C- and N-terminal tails of the CaV1.2 channel[J]. J Physiol Sci, 2013, 63 (5): 345- 353
doi: 10.1007/s12576-013-0270-y
7 郑敏, 王千慧, 封瑞 et al. Nav1.2 IQ及其癫痫突变体的表达纯化和活性鉴定[J]. 解剖科学进展, 2016, 22 (2): 176- 178
ZHENG Min , WANG Qianhui , FENG Rui et al. Expression, purification and bioactivity identification of Nav1.2 IQ and its epileptic mutant[J]. Progress of Anatomical Sciences, 2016, 22 (2): 176- 178
doi: 10.16695/j.cnki.1006-2947.2016.02.017
8 年青洋, 李佳宁, 佟欣 et al. 钙调蛋白对电压门控性钠通道NaV 1.1的调节作用[J]. 解剖科学进展, 2019, 25 (2): 213- 215, 219
NIAN Qingyang , LI Jianing , TONG Xin et al. The modulating effect of CaM on voltage-gated sodium channel Nav1.1[J]. Progress of Anatomical Sciences, 2019, 25 (2): 213- 215, 219
doi: 10.16695/j.cnki.1006-2947.2019.02.028
9 MAKINSON C D , DUTT K , LIN F et al. An Scn1a epilepsy mutation in Scn8a alters seizure susceptibility and behavior[J]. Exp Neurol, 2015, 275P3 (1): 46- 58
doi: 10.1016/j.expneurol.2015.09.008
10 MANTEGAZZA M , BROCCOLI V . SCN1A/Nav1.1 channelopathies:Mechanisms in expression systems, animal models, and human IPSC models[J]. Epilepsia, 2019, 60 (Suppl 3): S25- S38
doi: 10.1111/epi.14700.Review
11 BERECKI G , HOWELL K B , DEERASOORIYA Y H et al. Dynamic action potential clamp predicts functional separation in mild familial and severe de novo forms of SCN2A epilepsy[J]. Proc Natl Acad Sci U S A, 2018, 115 (24): E5516- E5525
doi: 10.1073/pnas.1800077115
12 XIE A , GALLANT B , GUO H et al. Functional cardiac Na+ channels are expressed in human melanoma cells[J]. Oncol Lett, 2018, 16 (2): 1689- 1695
doi: 10.3892/ol.2018.8865
13 GUAN G , ZHAO M , XU X et al. Abnormal changes in voltage-gated sodium channels subtypes NaV1.1, NaV1.2, NaV1.3, NaV1.6 and CaM/CaMKII pathway in low-grade astrocytoma[J]. Neurosci Lett, 2018, 674:148- 155
doi: 10.1016/j.neulet.2018.03.047
14 LI J , YU ZH , XU J et al. The effect of Ca2+, lobe-specificity, and CaMKII on CaM binding to NaV1.1[J]. Int J Mol Sci, 2018, 286 (14): 12308- 12316
doi: 10.3390/ijms19092495
15 ISBELL H M , KILPATRICK AM , LIN Z et al. Backbone resonance assignments of complexes of apo human calmodulin bound to IQ motif peptides of voltage-dependent sodium channels NaV1.1, NaV1.4 and NaV1.7[J]. Biomol Nmr Assign, 2018, 12 (2): 1- 7
doi: 10.1007/s12104-018-9824-5
16 HOVEY L , FOWLER C A , MAHLING R et al. Calcium triggers reversal of calmodulin on nested anti-parallel sites in the IQ motif of the neuronal voltage-dependent sodium channel NaV 1.2[J]. Biophys Chem, 2017, 224:1- 19
doi: 10.1016/j.bpc.2017.02.006
[1] 严敏,汪旭,郭锡汉. 早老蛋白1功能缺失性突变导致家族性阿尔茨海默病的研究进展[J]. 浙江大学学报（医学版）, 2020, 49(4): 487-499.
[2] 欧伟杰,林苏,吴奕隆,朱月永. UGT1A1基因复合杂合突变致吉尔伯特综合征一例[J]. 浙江大学学报（医学版）, 2020, 49(3): 406-409.
[3] 刘蓓,杨艳梅,严恺,陈敏,王丽雅,黄颖之,钱叶青,董旻岳. Ⅰ型神经纤维瘤一家系的基因检测及产前诊断[J]. 浙江大学学报（医学版）, 2019, 48(4): 367-372.
[4] 楼铁,黄颖之,董旻岳. CLN6基因复合杂合突变导致神经元蜡样脂褐质沉积症一家系遗传学研究[J]. 浙江大学学报（医学版）, 2019, 48(4): 373-377.
[5] 许雨晴,钱叶青,姚维妙,董旻岳. Van der Woude综合征一家系遗传学分析[J]. 浙江大学学报（医学版）, 2019, 48(4): 378-383.
[6] 叶青,张莹莹,王晶晶,毛建华. Ⅳ型胶原α5链基因突变致奥尔波特综合征两家系遗传学分析[J]. 浙江大学学报（医学版）, 2019, 48(4): 384-389.
[7] 洪萍萍,郭冰洁,林莉,林细华,周嘉强. 葡萄糖激酶W257R突变致青少年发病的成人型糖尿病一家系分析[J]. 浙江大学学报（医学版）, 2019, 48(2): 200-203.
[8] 唐思阳,叶佳,李月舟. I1363T突变致人骨骼肌电压门控钠通道快失活受损的机制[J]. 浙江大学学报（医学版）, 2019, 48(1): 12-18.
[9] 徐丽臻,杨帆. 瞬时受体电位M8型离子通道选择性滤器的三维结构计算建模[J]. 浙江大学学报（医学版）, 2019, 48(1): 19-24.
[10] 杜东芬,朱丽霞,王云贵,叶琇锦. 肾母细胞瘤1基因表达及其对急性髓系白血病患者预后的预测价值[J]. 浙江大学学报（医学版）, 2019, 48(1): 50-57.
[11] 肖梨,佟晓永. 肺动脉高压形成中的血管重构分子生物学机制研究进展[J]. 浙江大学学报（医学版）, 2019, 48(1): 102-110.
[12] 尹黎,李歌,沈健,刘震杰. 遗传性易栓症筛查及相关基因检测分析[J]. 浙江大学学报（医学版）, 2018, 47(6): 606-611.
[13] 史庭,叶琇锦. CCAAT增强子结合蛋白α与急性髓细胞白血病的发生[J]. 浙江大学学报（医学版）, 2018, 47(5): 552-557.
[14] 沈杰,陈闻东,姬开达,高平进,朱鼎良. Arg188Gln(G/A)突变对犬尿氨酸酶活性的影响[J]. 浙江大学学报（医学版）, 2017, 46(6): 643-648.
[15] 王梦嫣 等. 耶氏肺孢子菌对磺胺类药物耐药相关基因突变的研究进展[J]. 浙江大学学报（医学版）, 2017, 46(5): 563-569.