Barbara Serrano-Flores, Edith Garay, Francisco G Vázquez-Cuevas, Rogelio O Arellano
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引用次数: 3
摘要
背景:非洲爪蟾卵母细胞是研究Ca2+稳态和细胞周期调节这两个高度相关的过程的有用细胞模型。在这里,我们使用反义寡核苷酸来研究基质相互作用分子(STIM)蛋白在卵母细胞中的作用,STIM蛋白是储存操作钙进入(SOCE)现象的基本要素,因为它们既是细胞内储层中Ca2+浓度的传感器,也是允许Ca2+内流的膜通道的激活剂。结果:证实了内源性的STIM1和STIM2的表达,并且在注射特定反义序列的卵母细胞后48-72 h,它们的合成被抑制。通过PCR和Western blot分析证实了它们的mRNA和蛋白表达的选择性消除,然后我们评估了它们的缺失对两种内源性反应的影响:G蛋白偶联受体(GPCR)激活的Ca2+释放引发的SOC通道打开,以及黄体酮刺激的成熟过程。通过测量T响应来监测SOC通道的激活,这是一种依赖于Ca2+流入的Cl-电流,而成熟度通过生发囊泡破裂(GVBD)评分和电生理学来评估。结论:发现在这两种反应中,STIM2而不是STIM1是必需的,并且在缺乏STIM2的细胞中,T in current和GVBD强烈减少或消除;STIM1敲低对成熟过程没有影响,但它使T反应降低了15%至70%。由此可见,非洲爪蟾卵母细胞内源性SOCE反应主要依赖于STIM2,其表达是进入黄体酮诱导的减数分裂所必需的。
Differential role of STIM1 and STIM2 during transient inward (T in) current generation and the maturation process in the Xenopus oocyte.
Background: The Xenopus oocyte is a useful cell model to study Ca2+ homeostasis and cell cycle regulation, two highly interrelated processes. Here, we used antisense oligonucleotides to investigate the role in the oocyte of stromal interaction molecule (STIM) proteins that are fundamental elements of the store-operated calcium-entry (SOCE) phenomenon, as they are both sensors for Ca2+ concentration in the intracellular reservoirs as well as activators of the membrane channels that allow Ca2+ influx.
Results: Endogenous STIM1 and STIM2 expression was demonstrated, and their synthesis was knocked down 48-72 h after injecting oocytes with specific antisense sequences. Selective elimination of their mRNA and protein expression was confirmed by PCR and Western blot analysis, and we then evaluated the effect of their absence on two endogenous responses: the opening of SOC channels elicited by G protein-coupled receptor (GPCR)-activated Ca2+ release, and the process of maturation stimulated by progesterone. Activation of SOC channels was monitored electrically by measuring the T in response, a Ca2+-influx-dependent Cl- current, while maturation was assessed by germinal vesicle breakdown (GVBD) scoring and electrophysiology.
Conclusions: It was found that STIM2, but not STIM1, was essential in both responses, and T in currents and GVBD were strongly reduced or eliminated in cells devoid of STIM2; STIM1 knockdown had no effect on the maturation process, but it reduced the T in response by 15 to 70%. Thus, the endogenous SOCE response in Xenopus oocytes depended mainly on STIM2, and its expression was necessary for entry into meiosis induced by progesterone.
BMC PhysiologyBiochemistry, Genetics and Molecular Biology-Physiology
CiteScore
9.60
自引率
0.00%
发文量
0
期刊介绍:
BMC Physiology is an open access journal publishing original peer-reviewed research articles in cellular, tissue-level, organismal, functional, and developmental aspects of physiological processes. BMC Physiology (ISSN 1472-6793) is indexed/tracked/covered by PubMed, MEDLINE, BIOSIS, CAS, EMBASE, Scopus, Zoological Record and Google Scholar.