钙调蛋白突变体D130V与心肌CaV1.2通道IQ基序的相互作用  

Interaction between calmodulin mutant D130V and IQ motif of cardiac CaV1.2 channel

作　　者：唐路红 刘忆芳 潘雪婷 邢英美 郝丽英 苏敬阳 TANG Luhong;LIU Yifang;PAN Xueting;XING Yingmei;HAO Liying;SU Jingyang(Department of Clinical Pharmacology,School of Pharmacy,China Medical University,Shenyang 110012,China;Department of Pharmaceutical Toxicology,School of Pharmacy,China Medical University,Shenyang 110012,China)

机构地区：[1]中国医科大学药学院临床药理学教研室,沈阳110122 [2]中国医科大学药学院药物毒理学教研室,沈阳110122

出　　处：《中国医科大学学报》2025年第4期306-311,共6页Journal of China Medical University

基　　金：辽宁省自然科学基金(2022-BS-139);中国博士后科学基金(2021M693914)。

摘　　要：目的探讨钙调蛋白(CaM)突变体D130V与心肌CaV1.2通道IQ基序的结合作用。方法采用折叠识别建模、同源建模和蛋白对接方法预测突变体CaM-D130V与IQ基序的结合;利用42℃精确热击法将质粒转化至大肠杆菌BL-21感受态细胞中,诱导GST融合蛋白表达;超声破碎法提取蛋白,GS-4B beads纯化、PreScission蛋白酶切除GST标签;利用SDS-PAGE检测蛋白纯度;GST pull-down实验检测CaM-D130V与IQ基序的相互作用。结果蛋白对接结果表明,CaM-WT、CaM-D130V与心肌CaV1.2通道IQ基序均具有结合作用,但与CaM-WT相比,突变体CaM-D130V与IQ基序的结合位点减少,且结合构象发生变化,结合能减小(|S|由48.0866 kcal/mol下降至47.3095 kcal/mol)。GST pull-down实验结果显示,在2 mmol/L Ca^(2+)浓度下,与CaM-WT相比,CaM-D130V与IQ基序的结合量显著减少(P<0.01),亲和力显著降低。结论CaM-D130V与心肌CaV1.2通道IQ基序的结合能力降低,可能导致CaV1.2通道功能改变,为探讨CaM突变体相关的心血管疾病的发病机制和靶向治疗提供理论依据。Objective To investigate the binding interaction between the calmodulin(CaM)mutant D130V and the IQ motif of the cardiac CaV1.2 channel.Methods The binding of mutant CaM-D130V to the IQ motif was predicted by fold recognition modeling,homology modeling,and protein docking.The plasmid was transformed into Escherichia coli BL-21 sensory cells via heat shock at 42℃to induce the expression of glutathione S-transferase(GST)fusion protein.The protein was extracted by ultrasonic fragmentation and purified using GS-4B beads.PreScission protease was applied to remove the GST.SDS-PAGE was performed to detect the purity of protein.A GST pulldown assay was conducted to detect the interaction between CaM-D130V and IQ motif.Results Protein docking results showed that both CaM-WT and CaM-D130V could bind to the IQ motif of the cardiac CaV1.2 channel,but the binding sites of the mutant CaM-D130V to the IQ motif were reduced,and its binding conformation was changed compared with the CaM-WT,with decreased binding energy(|S|reduced from 48.0866 kcal/mol to 47.3095 kcal/mol).The GST pull-down assay indicated that the binding of CaM-D130V to IQ motif significantly decreased(P<0.01),and the affinity was significantly reduced at 2 mmol/L Ca^(2+)concentration compared with CaMWT.Conclusion The reduced binding ability of CaM-D130V to the IQ motif of the cardiac CaV1.2 channel may contribute to functional alterations in the channel.These findings provide a theoretical basis for understanding the pathogenesis of CaM mutant-associated cardiovascular diseases as well as targeted therapies.

关 键 词：钙调蛋白 突变体 心肌CaV1.2通道 结合能力 长QT综合征 

分 类 号：R96[医药卫生—药理学]