小龙虾机械敏感压电离子通道的克隆与功能表征。

IF 2.5 Q3 CELL BIOLOGY Cellular Physiology and Biochemistry Pub Date : 2023-07-29 DOI:10.33594/000000640
Bora Ergin, Berk Saglam, Kaan Arslan, Nazlı Coskun Beyatli, Zihni Ekim Taskiran, Turgut Bastug, Nuhan Purali
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引用次数: 0

摘要

背景/目的:机械敏感离子通道是机械力向神经活动传导的主要因素。迄今为止,发表的关于机械敏感通道的分子和结构特性的研究相当少。压电通道是真核生物中唯一一种由膜张力选择性门控的离子通道家族。在哺乳动物和其他一些真核生物中也发现了压电通道。然而,关于甲壳类动物的信息并不多。方法:采用常规克隆方法克隆小龙虾神经节中推定的PIEZO通道mRNA。用克隆基因质粒转染HEK293T细胞进行功能研究。计算了翻译成蛋白质的mRNA的CDS序列和通道的三维结构。结果:从小龙虾中首次克隆到全长9378 bp的mRNA,该mRNA编码2674个残基。克隆的序列与其他物种中报道的压电通道mrna相似。对编码蛋白的序列进行了分析,并确定了一些功能域。参考小鼠压电1通道蛋白数据,成功计算出编码蛋白的三维结构。合成了带荧光蛋白指示剂的质粒,并在HEK293T细胞中进行了异源表达。机械刺激引起的钙反应与对照细胞无明显差异。然而,转染后的细胞对门控修饰物YODA-1更敏感。结论:基于其序列、结构和功能特性与其他已知压电通道的相似性,克隆的mRNA可能在小龙虾中编码一个压电样离子通道。
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De Novo Cloning and Functional Characterization of a Mechanosensitive Piezo-Like Ion Channel in the Crayfish.

Background/aims: Mechanosensitive ion channels are the principal elements in the transduction of mechanical force to neural activity. To date, considerably fewer studies have been published about the molecular and structural properties of mechanosensitive channels. Piezo channels are the only ion channel family in eukaryotes which is selectively gated by the membrane tension. Piezo channels have been described in mammals and some other eukaryotes. However, not much information is available for the crustaceans.

Methods: Conventional cloning methods were used to clone the putative PIEZO channel mRNA in crayfish ganglia samples. HEK293T cells were transfected by the plasmid of the cloned gene for functional studies. The CDS of the mRNA translated into the protein sequence and three-dimensional structure of the channel has been calculated.

Results: An mRNA, 9378 bp, was firstly cloned from crayfish which codes a 2674 residues protein. The cloned sequence is similar to the piezo channel mRNAs reported in the other species. The sequence of the coded protein has been analyzed, and some functional domains have been identified. A three-dimensional structure of the coded protein was successfully calculated in reference to mouse piezo 1 channel protein data. A plasmid with a fluorescent protein indicator was synthesized for heterologous expression in HEK293T cells. The evoked calcium response to mechanical stimulation was not different from those observed in the control cells. However, the transfected cells were more sensitive to the gating modifier YODA-1.

Conclusion: Based on the apparent similarity in sequence, structure and functional properties to other known piezo channels, it has been proposed that cloned mRNA may code a piezo-like ion channel in crayfish.

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来源期刊
CiteScore
5.80
自引率
0.00%
发文量
86
审稿时长
1 months
期刊介绍: Cellular Physiology and Biochemistry is a multidisciplinary scientific forum dedicated to advancing the frontiers of basic cellular research. It addresses scientists from both the physiological and biochemical disciplines as well as related fields such as genetics, molecular biology, pathophysiology, pathobiochemistry and cellular toxicology & pharmacology. Original papers and reviews on the mechanisms of intracellular transmission, cellular metabolism, cell growth, differentiation and death, ion channels and carriers, and the maintenance, regulation and disturbances of cell volume are presented. Appearing monthly under peer review, Cellular Physiology and Biochemistry takes an active role in the concerted international effort to unravel the mechanisms of cellular function.
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