Michèle Bachmann, Audrey Ortega-Ramírez, Lilia Leisle, Stefan Gründer
{"title":"在哺乳动物细胞中高效表达网状肽门控离子通道。","authors":"Michèle Bachmann, Audrey Ortega-Ramírez, Lilia Leisle, Stefan Gründer","doi":"10.1080/19336950.2021.1882762","DOIUrl":null,"url":null,"abstract":"<p><p>Hydra Na<sup>+</sup> channels (HyNaCs) are peptide-gated ion channels of the DEG/ENaC gene family that are directly activated by neuropeptides of the <i>Hydra</i> nervous system. They have previously been successfully characterized in <i>Xenopus</i> oocytes. To establish their expression in mammalian cells, we transiently expressed heteromeric HyNaC2/3/5 in human HEK 293 and monkey COS-7 cells. We found that the expression of HyNaC2/3/5 using native cDNAs was inefficient and that codon optimization strongly increased protein expression and current amplitude in patch-clamp experiments. We used the improved expression of codon-optimized channel subunits to perform Ca<sup>2+</sup> imaging and to demonstrate their glycosylation pattern. In summary, we established efficient expression of a cnidarian ion channel in mammalian cell lines.</p>","PeriodicalId":72555,"journal":{"name":"Channels (Austin, Tex.)","volume":" ","pages":"273-283"},"PeriodicalIF":0.0000,"publicationDate":"2021-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7889164/pdf/","citationCount":"0","resultStr":"{\"title\":\"Efficient expression of a cnidarian peptide-gated ion channel in mammalian cells.\",\"authors\":\"Michèle Bachmann, Audrey Ortega-Ramírez, Lilia Leisle, Stefan Gründer\",\"doi\":\"10.1080/19336950.2021.1882762\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Hydra Na<sup>+</sup> channels (HyNaCs) are peptide-gated ion channels of the DEG/ENaC gene family that are directly activated by neuropeptides of the <i>Hydra</i> nervous system. They have previously been successfully characterized in <i>Xenopus</i> oocytes. To establish their expression in mammalian cells, we transiently expressed heteromeric HyNaC2/3/5 in human HEK 293 and monkey COS-7 cells. We found that the expression of HyNaC2/3/5 using native cDNAs was inefficient and that codon optimization strongly increased protein expression and current amplitude in patch-clamp experiments. We used the improved expression of codon-optimized channel subunits to perform Ca<sup>2+</sup> imaging and to demonstrate their glycosylation pattern. In summary, we established efficient expression of a cnidarian ion channel in mammalian cell lines.</p>\",\"PeriodicalId\":72555,\"journal\":{\"name\":\"Channels (Austin, Tex.)\",\"volume\":\" \",\"pages\":\"273-283\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2021-12-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7889164/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Channels (Austin, Tex.)\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1080/19336950.2021.1882762\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Channels (Austin, Tex.)","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1080/19336950.2021.1882762","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
Efficient expression of a cnidarian peptide-gated ion channel in mammalian cells.
Hydra Na+ channels (HyNaCs) are peptide-gated ion channels of the DEG/ENaC gene family that are directly activated by neuropeptides of the Hydra nervous system. They have previously been successfully characterized in Xenopus oocytes. To establish their expression in mammalian cells, we transiently expressed heteromeric HyNaC2/3/5 in human HEK 293 and monkey COS-7 cells. We found that the expression of HyNaC2/3/5 using native cDNAs was inefficient and that codon optimization strongly increased protein expression and current amplitude in patch-clamp experiments. We used the improved expression of codon-optimized channel subunits to perform Ca2+ imaging and to demonstrate their glycosylation pattern. In summary, we established efficient expression of a cnidarian ion channel in mammalian cell lines.