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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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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:;
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.

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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
[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
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