Evaluation of the Ion Channel Assembly in a Eukaryotic Cell-Free System Focusing on Two-Pore Domain Potassium Channels K[sub.2P]

• Published in: International journal of molecular sciences 2023-03, Vol.24 (7)
• Main Authors: Ullrich, Jessica, Ohlhoff, Carsten, Dondapati, Srujan Kumar, Zemella, Anne, Kubick, Stefan
• Format: Article
• Language: English
• Subjects: Analysis; Oligomers; Pharmaceutical industry; Potassium channels
• ISSN: 1422-0067
• DOI: 10.3390/ijms24076299

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 (K[sub.2P]) 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 K[sub.2P] 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 K[sub.2P] 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.