Evaluation of the Ion Channel Assembly in a Eukaryotic Cell-Free System Focusing on Two-Pore Domain Potassium Channels K2P
| Autor: | Jessica Ullrich, Carsten Ohlhoff, Srujan Kumar Dondapati, Anne Zemella, Stefan Kubick |
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| Rok vydání: | 2023 |
| Předmět: |
TREK-2
cell-free synthesis K2P heterodimerization Organic Chemistry TWIK-1 protein assembly General Medicine Catalysis oligomerization Computer Science Applications eukaryotic cell-free protein synthesis (CFPS) Inorganic Chemistry membrane protein synthesis ion channel cell-free synthesis (CFS) eukaryotic cell-free protein synthesis Physical and Theoretical Chemistry 500 Naturwissenschaften und Mathematik::540 Chemie::540 Chemie und zugeordnete Wissenschaften Molecular Biology Spectroscopy |
| Zdroj: | International Journal of Molecular Sciences; Volume 24; Issue 7; Pages: 6299 |
| DOI: | 10.17169/refubium-39325 |
| Popis: | Oligomeric ion channels are abundant in nature. However, the recombinant expression in cell culture-based systems remains tedious and challenging due to negative side effects, limiting the understanding of their role in health and disease. Accordingly, in this work, we demonstrate the cell-free synthesis (CFS) as an alternative platform to study the assembly of two-pore domain potassium channels (K2P) within endogenous endoplasmic reticulum-derived microsomes. Exploiting the open nature of CFS, we investigate the cotranslational translocation of TREK-2 into the microsomes and suggest a cotranslational assembly with typical single-channel behavior in planar lipid-bilayer electrophysiology. The heteromeric assembly of K2P channels is a contentious matter, accordingly we prove the successful assembly of TREK-2 with TWIK-1 using a biomolecular fluorescence complementation assay, Western blot analysis and autoradiography. The results demonstrate that TREK-2 homodimer assembly is the initial step, followed by heterodimer formation with the nascent TWIK-1, providing evidence of the intergroup heterodimerization of TREK-2 and TWIK-1 in eukaryotic CFS. Since K2P channels are involved in various pathophysiological conditions, including pain and nociception, CFS paves the way for in-depth functional studies and related pharmacological interventions. This study highlights the versatility of the eukaryotic CFS platform for investigating ion channel assembly in a native-like environment. |
| Databáze: | OpenAIRE |
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