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TRPC1与Orai1的相互作用参与人脐静脉内皮细胞钙敏感受体介导的钙内流及一氧化氮生成

王腊梅¹, 唐娜¹, 钟华¹, 庞丽娟^1,2, 张春军¹, 何芳^1,*

¹新疆石河子大学医学院1病理生理学教研室/新疆地方与民族高发病教育部重点实验室，石河子 832002；²新疆石河子大学医学院病理学教研室，石河子 832002

摘要

本研究旨在探讨Ca2+感受蛋白经典瞬时感受器电位蛋白1 (canonical transient receptor potential channel 1, TRPC1)与钙释放激活钙通道调节分子1 (calcium release-activated calcium modulator 1, Orai1)的相互作用在人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)钙敏感受体(Ca-sensing receptor, CaR)介导的Ca2+内流和一氧化氮(NO)生成中的作用。取2~3代HUVECs，单独或联合用CaR激动剂精胺[Spermine，激活钙库活化的钙通道(store-operated calcium channels, SOC)和受体活化的钙通道(receptor-operated calcium channels, ROC)]、ROC模拟剂12-O-十四烷酰佛波醇-13-乙酸酯(TPA)+CaR负性变构调节剂Calhex231 (阻断SOC，激活ROC)、PKC抑制剂Ro31-8220 (激活SOC，阻断ROC)以及经典型PKCs和PKCµ抑制剂Go6967 (激活SOC，阻断ROC)处理。用免疫荧光技术检测HUVECs中TRPC1和Orai1的蛋白表达、共定位；用免疫共沉淀法检测TRPC1和Orai1之间的相互作用模式；随后用TRPC1和Orai1干扰质粒联合转染HUVECs，采用荧光探针同时检测HUVECs中[Ca2+]i和NO生成的变化。结果显示，HUVECs中TRPC1和Orai1蛋白表达共定位于胞膜。与Spermine+Ca2+组相比，Calhex231+ TPA+Spermine+Ca2+组、Ro31-8220+Spermine+Ca2+组和Go6976+Spermine+Ca2+组HUVECs中定位于胞浆中的TRPC1、Orai1蛋白表达均显著降低，二者的结合也减少。免疫共沉淀结果显示，Calhex231+TPA+Spermine+Ca2+组、Ro31-8220+Spermine+ Ca2+组和Go6976+Spermine+Ca2+组TRPC1/Orai1和Orai1/TRPC1相对比值百分数均明显低于对照组以及Spermine+Ca2+组(均P < 0.05)，表明Orai1和TRPC1相互作用均减弱。联合转染质粒敲低TRPC1和Orai1显著降低Spermine+Ca2+组、Calhex231+ TPA+Spermine+Ca2+组、Ro31-8220+Spermine+Ca2+组和Go6976+Spermine+Ca2+组的HUVECs中[Ca2+]i和NO生成。以上结果提示，TRPC1与Orai1以二元复合物的形式激活SOC和ROC，介导Ca2+内流及NO生成。

关键词： 经典瞬时感受器电位蛋白1; 钙释放激活钙通道调节分子1; 钙敏感受体; 钙离子; 一氧化氮

分类号：R363

Involvement of interaction between TRPC1 and Orai1 in calcium sensing receptor-mediated calcium influx and nitric oxide generation in human umbilical vein endothelial cells

WANG La-Mei¹, TANG Na¹, ZHONG Hua¹, PANG Li-Juan^1,2, ZHANG Chun-Jun¹, HE Fang^1,*

¹Department of Pathophysiology/Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Ministry of Education, Medical College of Shihezi University, Shihezi 832002, China；²Department of Pathology, Medical College of Shihezi University, Shihezi 832002, China

Abstract

The present study was to investigate the role of the interaction between canonical transient receptor potential channel 1 (TRPC1) and calcium release-activated calcium modulator 1 (Orai1) in extracellular Ca2+-sensing receptor (CaR)-induced extracellular Ca2+ influx and nitric oxide (NO) production. Human umbilical vein endothelial cells (HUVECs) were incubated with CaR agonist Spermine [activating store-operated calcium channels (SOC) and receptor-operated calcium channels (ROC)] alone or in combination with the following reagents: CaR negative allosteric modulator Calhex231 plus ROC analogue TPA (activating ROC and blocking SOC), Ro31-8220 (PKC inhibitor that activates SOC and blocks ROC) or Go6967 (PKCs and PKCµ inhibitor that activates SOC and blocks ROC). The protein expressions and co-localization of TRPC1 and Orai1 were determined using immunofluorescent staining. The interaction between TRPC1 and Orai1 was examined by co-immunoprecipitation. We silenced the expressions of their genes in the HUVECs by transfection of constructed TRPC1 and Orai1 shRNA plasmids. Intracellular Ca2+ concentration ([Ca2+]i) was detected using Ca2+ indicator Fura-2/AM, and NO production was determined by DAF-FM staining. The results showed that TRPC1 and Orai1 protein expressions were co-located on the cell membrane of the HUVECs. Compared with Spermine+Ca2+ group, Calhex231+ TPA+Spermine+Ca2+, Ro31-8220+Spermine+Ca2+ and Go6976+Spermine+Ca2+ groups exhibited down-regulated protein expressions of TRPC1 and Orai1 in cytoplasm and decreased co-localization on the cell membrane. Co-immunoprecipitation results showed that the interaction between TRPC1 and Orai1 was reduced by Calhex231 plus TPA, Ro31-8220 or Go6976 addition in the Spermine-stimulated HUVECs. Double knockdown of Trpc1 and Orai1 genes significantly decreased [Ca2+]i level and NO production in all of the Spermine+Ca2+, Calhex231+TPA+Spermine+Ca2+, Ro31-8220+Spermine+Ca2+ and Go6976+Spermine+Ca2+ groups. These results suggest that TRPC1/Orai1 may form a complex that mediates Ca2+ influx and No production via SOC and ROC activation.

Key words: canonical transient receptor potential channel 1; calcium release-activated calcium modulator 1; Ca2+-sensing receptor; Ca2+; nitric oxide

收稿日期：2017-09-22　　录用日期：2017-12-12

通讯作者：何芳　　E-mail: fangf2002shz@126.com

DOI: 10.13294/j.aps.2018.0022

引用本文：

王腊梅, 唐娜, 钟华, 庞丽娟, 张春军, 何芳. TRPC1与Orai1的相互作用参与人脐静脉内皮细胞钙敏感受体介导的钙内流及一氧化氮生成[J]. 生理学报 2018; 70 (3): 269-280.

WANG La-Mei, TANG Na, ZHONG Hua, PANG Li-Juan, ZHANG Chun-Jun, HE Fang. Involvement of interaction between TRPC1 and Orai1 in calcium sensing receptor-mediated calcium influx and nitric oxide generation in human umbilical vein endothelial cells. Acta Physiol Sin 2018; 70 (3): 269-280 (in Chinese with English abstract).