Bora Ergin, Berk Saglam, Kaan Arslan, Nazlı Coskun Beyatli, Zihni Ekim Taskiran, Turgut Bastug, Nuhan Purali
{"title":"小龙虾机械敏感压电离子通道的克隆与功能表征。","authors":"Bora Ergin, Berk Saglam, Kaan Arslan, Nazlı Coskun Beyatli, Zihni Ekim Taskiran, Turgut Bastug, Nuhan Purali","doi":"10.33594/000000640","DOIUrl":null,"url":null,"abstract":"<p><strong>Background/aims: </strong>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.</p><p><strong>Methods: </strong>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.</p><p><strong>Results: </strong>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.</p><p><strong>Conclusion: </strong>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.</p>","PeriodicalId":9845,"journal":{"name":"Cellular Physiology and Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.5000,"publicationDate":"2023-07-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"De Novo Cloning and Functional Characterization of a Mechanosensitive Piezo-Like Ion Channel in the Crayfish.\",\"authors\":\"Bora Ergin, Berk Saglam, Kaan Arslan, Nazlı Coskun Beyatli, Zihni Ekim Taskiran, Turgut Bastug, Nuhan Purali\",\"doi\":\"10.33594/000000640\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background/aims: </strong>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.</p><p><strong>Methods: </strong>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.</p><p><strong>Results: </strong>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.</p><p><strong>Conclusion: </strong>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.</p>\",\"PeriodicalId\":9845,\"journal\":{\"name\":\"Cellular Physiology and Biochemistry\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.5000,\"publicationDate\":\"2023-07-29\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Cellular Physiology and Biochemistry\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.33594/000000640\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"CELL BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cellular Physiology and Biochemistry","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.33594/000000640","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"CELL BIOLOGY","Score":null,"Total":0}
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.
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.