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The concentration-and Ca2+-dependent interactions of long QT syndrome-associated calmodulin C-lobe mutant E141G with CaV1.2 channel

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The concentration-and Ca2+-dependent interactions of long QT...

2019中国生理学会学术年会暨张锡钧基金第十五届全国青年优秀生理学学术论文交流会及第十三届全国青年生理学工作者学术会议

作者：张晨阳 dongxue shao Xi Zheng Liying Hao Department of Pharmaceutical Toxicology China Medical University

Calmodulin(CaM) molecule, consisting of the N-terminal lobe(N-lobe) and the Cterminal lobe(C-lobe), mediates the positive Ca2+-dependent facilitation(CDF) and negative Ca2+-dependent inactivation(CDI) of calcium channnels, thus maintaining intracellular calcium *** than a dozen human calmodulin(CaM) missense mutations that cause long-QT syndrome(LQTS) have recently been ***, all the LQTS mutations reside at or near Ca2+ coordinating residues within the C-lobe of CaM, including CaM E141 G mutation (CaME141 G) that has been reported to cause LQTS due to impaired CDI of *** verify that impaired CDI is due to that the CaME141 G mutant reduces the Ca2+-dependence of CaM binding to CaV1.2, We examined the binding of C-lobe and C-lobe mutant(C-lobeE141 G) to preIQ, IQ and NT peptide of CaV1.2 at different Ca2+ concentration([Ca2+]) from 100 nM to 2 mM by GST pull-down *** found that C-lobe could tether to pre-IQ, IQ and NT in a concentration-and Ca2+-dependence manner.C-lobeE141 G was still able to bind to CaV1.2 weakly in a concentrationdependent manner, but the Ca2+-dependence significantly *** binding of C-lobeE141 G to preIQ or IQ was stronger than the binding of C-lobe at 100 nM [Ca2+], however the binding of ClobeE141 G was weaker than that of C-lobe when [Ca2+] increased to 10 μM and 2 *** concentration-and Ca2+-dependence binding behavior of NT is similar to preIQ and *** binding of C-lobeE141 G and NT was stronger than C-lobe at both 100 nM and 10 μM [Ca2+], but weaker than C-lobe at 2 mM [Ca2+].These results suggested that the CaME141 G mutant reduces the Ca2+-dependence of CaM binding to CaV1.2, which may induce LQTS.

关键词： calmodulin C-lobe mutant E141G CaV1.2 channel long QT Syndrome

Calmodulin Mutant CaM-F142L May Induce the Changes of the Ca2+-depentment Regulation by Its Decreased Binding to CaV1.2 channel

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Calmodulin Mutant CaM-F142L May Induce the Changes of the Ca...

2019年中国生理学会心血管生理学术研讨会

作者： Jingyang Su Ming Lei Zhengnan Zhu Xuanxuan Sun Qinghua Gao Rui Feng dongxue shao Xuefei Sun Liying Hao Department of Pharmaceutical toxicology School of PharmacyChina Medical University Institute of Cardiovascular Research of Southwest Medical University Department of Physiology Graduate School of Medical and Dental SciencesKagoshima University

It is reported that calmodulin(CaM) mutations are associated with severe long QT syndrome(LQTS),and induced a weakening of Ca-dependent inactivation(CDI) of Cav1.2 *** refers to the prolonged period of QT interval in the electrocardiogram,the clinical manifestation of recurrent syncope or sudden ***-F142 L is a critical missense variant associated with LQTS,which located in EF-hand IV region of ***,the interaction of CaM-F142 L and Cav1.2 channel remaining *** our previous study,we have already found that CaM-WT can bind with N terminus(NT) and C terminus(CT) of Cav1.2 channel under deferent *** this study,we tried to check the combining of the mutant with the Cav1.2 channel in ***,the 142 th amino F was mutated into L of the wild type CaM by using the site-specific mutagenesis *** the GST-CaM-F142 L fusion protein was purified by GS-4 B *** Protease was applied to cut off the *** we utilized GST pull-down assay to explore the interaction between CaM-F142 L and Cavl.2 channel protein fregments compared with *** found that CaM-F142 L still have the ability to bind with different Cav1.2 channel fregments(CT1,PreIQ and NT) at different *** binding of CaM-F142 L to CTl/PreIQ/NT were decreased compared to CaM-WT in high Caconcentration(2 mM,10μM) and in a protein-concentration dependent *** maximal binding(B) and the K value of CaM-F142 L to CT1,PreIQ and NT were reduced in≈2 mM,10μM [Ca].These data imply that the identified LQTS-associated variants CaM-F142 L disrupts the binding with cardiac Cav1.2 channel,which may attenuate Cav1.2 inactivation through the loss of CDI.

关键词： Calmodulin mutant Cav1.2 channel Binding Long QT syndrome

Persistent phosphorylation of calmodulin-dependent protein kinase Ⅱ in cardiac hypertrophy remodeling

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Persistent phosphorylation of calmodulin-dependent protein k...

首届东方药理论坛暨2018年全国心脑血管药理学术会议、上海市药理学会第十九届学术年会

作者： Jingyuan Li dongxue shao Siqi Wang Ze Kang Shuai Lei Xuanxuan Sun Meimi Zhao Huiyuan Hu Liying Hao Department of Pharmaceutical Toxicology School of PharmacyChina Medical University

Rationale:Long term stimulation of β-adrenoceptor（β-AR） activates calmodulin-dependent protein kinase Ⅱ（CaMKⅡ） and enhances the Cainflux,which may further induce the imbalance of the intracellular Cahomeostasis on cardiac myocytes and may eventually result in cardiac hypertrophy,and shown the importance of Caand CaMKII in the progress of cardiac ***,the mechanism of CaMKII contribution to the process of hypertrophy is obviously ***,cardiac hypertrophy is hard to get *** the underlying mechanism is mysteriously unknown which should be found out ***:To examine the characteristics of CaMKII activation and its interactions with Cav1.2 channels during the development and reversal of cardiac *** and Results:Isoproterenol（ISO） was administrated to SD rats by subcutaneous injection for 2 weeks and 3 weeks to prepare the animal models with different level of cardiac hypertrophy,and then the ISO was withdrawn and the model animals were cultivated for another 3 weeks to investigate reversal possibility of cardiac *** results shown that withdrawing ISO did not improve hypertrophy reversal,and the hypertrophy even became worse after the withdrawal of *** importantly,expression and activity of CaMKII also persisted at extremely high level during ISO withdrawing ***,protein expression levels of Cav1.2(α1 C subunit of L-type Cachannel) and Cavβ(β subunit) proteins did not change in ISO 2 w and ISO 3 w groups,however,Cav1.2 obviously increased during the three weeks after ISO *** binding to Cav1.2 also increased during the administration of ISO and persisted at high level after ISO withdrawing,which is coincidentally consistent with the pathological process of the development of the cardiac ***:The persisted CaMKII activity may be the cause of the continuous progress of cardiac *** the coincidentally increased Cav1.2 expression an

关键词： calmodulin-dependent protein kinase Ⅱ Cav1.2 cardiac hypertrophy phosphorylation persistent

Searching for putative binding sites on the calmodulin while interacting with Cav1.2 channel

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Searching for putative binding sites on the calmodulin while...

2019年中国生理学会心血管生理学术研讨会

作者： Yan Liu Sichong Chen Shan Yan Guilin He Jie Zhang Yixuan Shen Jingyang Su Rui Feng dongxue shao Liying Hao Department of Pharmaceutical Toxicology School of PharmacyChina Medical University

Objective In order to investigate the interaction between Calmodulin(CaM) and C-terminal fragment of Cav1.2 channels(Pre-IQ,IQ).Methods We have examined the bindings of CaM and its mutants to Pre-IQ and IQ by GST pull-down *** then we used MOE(Molecular Operating Environment) to analyze the most likely residues to play an important role in the *** We have found that the wild type CaM(CaM) and CaM binding to Pre-IQ and IQ in a [CaM] and [Ca]dependent *** the MOE results showed that amino acids of CaM that play a key role in binding with Pre-IQ and IQ are concentrated in the central helix *** we used GST pull-down assays to verify the competitive effect of peptide-Linker(amino acids 70-85) on CaM/IQ and CaM/PreIQ *** We conclude that though C-lobe and N-lobe play vital role in sensing the change of calcium ion,the central helix of CaM is involved in the binding of CaM to Cav1.2,not only participates in the combination with IQ,but also with *** the role of central helix of CaM is very important which may be the key binding site of CaM to calcium channels.

关键词： Calcium channels Calmodulin(CaM) GST pull-down assays Molecular docking

Different properties and cooperative effects of calmodulin N-and Clobes in regulations of Cav1.2 channel

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Different properties and cooperative effects of calmodulin N...

第十一届全国钙信号和细胞功能研讨会

作者： dongxue shao Jianjun Xu Meimi Zhao Etsuko Minobe Jingyang Su Yi Du Jiayao Sun Mo Li Zijian Tang Masaki Kameyama Liying Hao Department of Pharmaceutical Toxicology School of Pharmacy China Medical University Department of Physiology Graduate School of Medical and Dental Sciences Kagoshima University

Voltage-gated Ca channels(Cavs) are regulated by Ca/calmodulin(Ca M) through two opposing feedback mechanisms, Ca-dependent facilitation(CDF) and inactivation(CDI). A Ca M molecule consists of two connected lobes, the N-terminal lobe(N-lobe) and the C-terminal lobe(C-lobe). It is currently quite controversial whether the two lobes of Ca M mediate the opposing feedback systems independently. By using Ca M N-and C-lobe fragments, we confirmed that the N-lobe was bound to the IQ motif, a Ca M-binding site on the C-terminus of Cav1.2 in free or low Ca conditions, revealing that N-lobe played a more important role when intracellular Ca concentration is free or low. While, neither individual N-lobe nor C-lobe but equimolar mixture of N-and C-lobe(N&C) could induce CDI under our experimental condition, indicating that CDI is a process that starts by the binding of the single N-lobe to the channel and ends with the co-existence of N-and C-lobes. The binding affinities of the individual lobes were much lower than that of the full-structured Ca M, implying that the linker may contribute to the effective cooperation between the N-and C-lobes. We propose a model wherein the CDI process is akin to double touching magic locks controlled by N-and C-lobe bindings, which provides an important perspective to understand the regulatory mechanisms of Cavs and the control of intracellular Ca homeostasis.

关键词：

The effect of Ca MK II and CIP on the binding of calmodulin to cardiac Cav1.2 channel

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The effect of Ca MK II and CIP on the binding of calmodulin ...

第十一届全国钙信号和细胞功能研讨会

作者： Jingyang Su Liting Lyu Jianwei Mao Guigui Wei dongxue shao Meimi Zhao Feng Guo Huiyuan HU Xuefei Sun Guangyu Jiao Rui Feng Hao Liying Department of Pharmaceutical Toxicology School of Pharmacy China Medical University Respiratory Department and Intensive Care Unit Shengjing Hospital of China Medical University

In the present study, the roles of Ca/Ca M dependent protein kinase II(Ca MKII) and Calf Intestinal Alkaline Phosphatase(CIP) on the binding of calmodulin(Ca M) to cardiac Cav1.2 channel were examined by pull-down assay method. We examined the binding of Ca M to cardiac Cav1.2 fragments after treated by Ca MKII and CIP. The experiments showed that the Cav1.2 C-terminal fragment CT1 still has the ability to bind with Ca M after treatments at different Ca concentration([Ca]) from ≈ free to 2 m M. The binding effects of Ca M to CT1 were increased by CaM KII phosphorylation with CaM-concentration dependent manner, and the binding effects of Ca M to CT1 were decreased by CIP dephosphorylation with Ca Mconcentration dependent manner. To further find the key regions in CT1 for Ca MKII and CIP regulation, we examined the binding effect of Ca M to IQ after Ca MKII and CIP treatment. The results showed that the binding effect were increased after Ca MKII treatment, and were decreased after CIP treatment in 2 mM, 10μM Ca, in additon, the effect were Ca Mconcentration dependent manner. The data imply that at higher Ca, Ca M can easily bind to CT1 after Ca MKII phosphorylation, and the binding effect was easily inhibited after CIP dephosphorylation. Furthermore, the IQ motif might be the important region for Ca MKII and CIP to mediate CaM binding.

关键词： calmodulin Ca2+/Ca M dependent protein kinase II L-type calcium channel phosphorylation

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不锈钢在海水中腐蚀特性研究进展

辽宁石油化工大学学报 2018年第1期38卷 26-30页

作者：邵冬雪吴明谢飞王丹辽宁石油化工大学石油天然气工程学院辽宁抚顺113001

不锈钢因具有优良的抗氧化、耐腐蚀等特性被广泛地应用于工业生产中。但是,不锈钢材料在海洋环境中容易受到海水的影响而降低其耐腐蚀特性。分析了不锈钢在海水中的腐蚀机理,并从溶解氧浓度、海水pH、海水流速、微生物等角度讨论了影响... 详细信息

不锈钢因具有优良的抗氧化、耐腐蚀等特性被广泛地应用于工业生产中。但是,不锈钢材料在海洋环境中容易受到海水的影响而降低其耐腐蚀特性。分析了不锈钢在海水中的腐蚀机理,并从溶解氧浓度、海水pH、海水流速、微生物等角度讨论了影响不锈钢在海水中腐蚀的因素,探讨了当前不锈钢防腐技术的研究情况。

关键词：不锈钢海水腐蚀特性溶解氧

钙调蛋白镁结合位点突变体载体的构建、表达纯化及鉴定

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中国医科大学学报 2016年第5期45卷 394-397页

作者：赵美眯李卓邵冬雪梁洪玥晏珊封瑞孙雪菲郭凤郝丽英中国医科大学药学院药物毒理学教研室沈阳110122

目的构建钙调蛋白3种镁离子结合位点突变体的质粒载体,并进行表达纯化及鉴定,为深入研究钙调蛋白的生物学功能奠定基础。方法利用钙调蛋白cDNA进行点突变,制备3种镁离子结合位点突变的cDNA。将突变后的cDNA分别插入pGEX-6P-3载体,制备... 详细信息

目的构建钙调蛋白3种镁离子结合位点突变体的质粒载体,并进行表达纯化及鉴定,为深入研究钙调蛋白的生物学功能奠定基础。方法利用钙调蛋白cDNA进行点突变,制备3种镁离子结合位点突变的cDNA。将突变后的cDNA分别插入pGEX-6P-3载体,制备钙调蛋白突变体载体质粒。质粒转化大肠杆菌BL21感受态细胞,培养大肠杆菌,并诱导GST融合蛋白表达。利用Glutathione-Sepharose 4B珠子和PreScission蛋白酶分离纯化蛋白。结果酶切鉴定和DNA测序证实成功构建钙调蛋白突变体质粒;表达纯化得到的钙调蛋白突变体经电泳图鉴定纯度较高,浓度约1.0mg/mL。结论本研究成功构建了钙调蛋白镁结合位点突变体融合蛋白原核表达质粒,分离纯化可获得较高浓度较高纯度的钙调蛋白突变体。

关键词：钙调蛋白突变体融合蛋白 pGEX-6P-3

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钙调蛋白结构域来源小分子多肽的构建

中国医科大学学报 2021年第8期50卷 714-718页

作者：张晨阳邵冬雪郑曦苏敬阳胡聪佟欣田兴古丽仙常心怡郝丽英中国医科大学药学院药物毒理学教研室沈阳110122

目的构建钙调蛋白结构域来源小分子多肽(EF-hand 4,a.a 113-148),建立一种简便、直观、快速检测EF-hand 4的电泳方法。方法通过固相合成法构建EF-hand 4。采用分离胶为16.5%T的Tricine-SDS-PAGE凝胶电泳法检测多肽,CS Analyzer 3.0软件... 详细信息

目的构建钙调蛋白结构域来源小分子多肽(EF-hand 4,a.a 113-148),建立一种简便、直观、快速检测EF-hand 4的电泳方法。方法通过固相合成法构建EF-hand 4。采用分离胶为16.5%T的Tricine-SDS-PAGE凝胶电泳法检测多肽,CS Analyzer 3.0软件对EF-hand 4多肽条带进行纯度分析,GST pull-down方法检测EF-hand 4的生物学活性。结果Tricine-SDS-PAGE凝胶电泳结果显示,在约4×10^(3)处可见EF-hand 4清晰蛋白条带;CS Analyzer 3.0软件分析其纯度为100%;GST pull-down结果表明,EF-hand 4可以和CaV1.2钙通道结合,具有生物学活性。结论Tricine-SDS-PAGE凝胶电泳法可简便、直观、快速检测EF-hand 4,固相合成法得到的EF-hand 4具有与钙通道结合的生物学活性,本研究为CaM结构域来源EF-hand 4生物学功能的深入研究提供了材料基础。

关键词：钙调蛋白小分子多肽构建