دورية أكاديمية
Genetically engineered human embryonic kidney cells as a novel vehicle for dual patch clamp study of human gap junction channels.
| العنوان: | Genetically engineered human embryonic kidney cells as a novel vehicle for dual patch clamp study of human gap junction channels. |
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| المؤلفون: | Chen H; Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada N6A 5C1., Li YX; Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada N6A 5C1., Wong RS; Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada N6A 5C1., Esseltine JL; Division of BioMedical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland and Labrador, Canada A1B 3V6., Bai D; Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada N6A 5C1. |
| المصدر: | The Biochemical journal [Biochem J] 2024 Jun 19; Vol. 481 (12), pp. 741-758. |
| نوع المنشور: | Journal Article |
| اللغة: | English |
| بيانات الدورية: | Publisher: Published by Portland Press on behalf of the Biochemical Society Country of Publication: England NLM ID: 2984726R Publication Model: Print Cited Medium: Internet ISSN: 1470-8728 (Electronic) Linking ISSN: 02646021 NLM ISO Abbreviation: Biochem J Subsets: MEDLINE |
| أسماء مطبوعة: | Original Publication: London, UK : Published by Portland Press on behalf of the Biochemical Society |
| مواضيع طبية MeSH: | Connexins*/genetics , Connexins*/metabolism , Gap Junctions*/metabolism , Gap Junctions*/genetics , Patch-Clamp Techniques*, Humans ; HEK293 Cells ; Connexin 43/genetics ; Connexin 43/metabolism ; CRISPR-Cas Systems ; Genetic Engineering/methods ; Gene Knockout Techniques/methods |
| مستخلص: | Mutations in more than half of human connexin genes encoding gap junction (GJ) subunits have been linked to inherited human diseases. Functional studies of human GJ channels are essential for revealing mechanistic insights into the etiology of disease-linked connexin mutants. However, the commonly used Xenopus oocytes, N2A, HeLa, and other model cells for recombinant expression of human connexins have different and significant limitations. Here we developed a human cell line (HEK293) with each of the endogenous connexins (Cx43 and Cx45) knocked out using the CRISPR-Cas9 system. Double knockout HEK293 cells showed no background GJ coupling, were easily transfected with several human connexin genes (such as those encoding Cx46, Cx50, Cx37, Cx45, Cx26, and Cx36) which successfully formed functional GJs and were readily accessible for dual patch clamp analysis. Single knockout Cx43 or Cx45 HEK cell lines could also be used to characterize human GJ channels formed by Cx45 or Cx43, respectively, with an expression level suitable for studying macroscopic and single channel GJ channel properties. A cardiac arrhythmia linked Cx45 mutant R184G failed to form functional GJs in DKO HEK293 cells with impaired localizations. These genetically engineered HEK293 cells are well suited for patch clamp study of human GJ channels. (© 2024 The Author(s).) |
| فهرسة مساهمة: | Keywords: Cx43; HEK-293 cells; gap junction channel; patch clamp; single channel analysis |
| المشرفين على المادة: | 0 (Connexins) 0 (Connexin 43) 0 (connexin 45) 0 (GJA1 protein, human) |
| تواريخ الأحداث: | Date Created: 20240516 Date Completed: 20240610 Latest Revision: 20240610 |
| رمز التحديث: | 20240610 |
| DOI: | 10.1042/BCJ20240016 |
| PMID: | 38752978 |
| قاعدة البيانات: | MEDLINE |
| تدمد: | 1470-8728 |
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| DOI: | 10.1042/BCJ20240016 |