The EGFP/hERG fusion protein alter the electrophysiological properties of hERG channels in HEK293 cells
| Autor: | Jiang‑Fang Lian, Xi Yang, Li Ying Liu, Jian‑Qing Zhou, Zongfang Li, Tu Sheng Song, Chen Huang, Na Huang, Lei Ni, Jian‑Hua Huo |
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| Rok vydání: | 2010 |
| Předmět: |
congenital
hereditary and neonatal diseases and abnormalities ERG1 Potassium Channel Patch-Clamp Techniques Calnexin Recombinant Fusion Proteins hERG Green Fluorescent Proteins Green fluorescent protein Humans cardiovascular diseases Patch clamp Cellular localization Microscopy Confocal biology Chemistry HEK 293 cells Cell Biology General Medicine Fusion protein Molecular biology Ether-A-Go-Go Potassium Channels Cell biology Electrophysiological Phenomena Electrophysiology HEK293 Cells Lipofectamine biology.protein |
| Zdroj: | Cell biology international. 35(3) |
| ISSN: | 1095-8355 |
| Popis: | EGFP (enhanced green fluorescent protein) tagged to either the N (amino)-terminus [EGFP/hERG (human ether-a-go-go-related gene)] or C (carboxyl)-terminus (hERG/EGFP) of hERG channel is used to study mutant channel protein trafficking for several years. However, it has been reported that the process can alter hERG channel properties. The aim of the study was to determine whether EGFP tagged to N-terminus of hERG channels would alter the cellular localizations and the electrophysiological properties of hERG channels compared with untagged hERG channels. The hERG channels tagged with or without EGFP were transiently expressed in HEK (human embryonic kidney) 293 cells using a lipofectamine method. HEK 293 cells expressing pCDNA3-hERG or pEGFP-hERG were double immunolabelled with anti-hERG and anti-calnexin (an ER marker protein) followed with FITC- and TRITC (tetramethylrhodamine β-isothiocyanate)-labelled secondary antibodies, respectively. Confocal laser scanning microscope was used to observe the cellular localization of EGFP-tagged hERG channels and untagged hERG channels. Patch-clamp technique was used to record whole cell currents. We found that the EGFP/hERG fusion protein and untagged hERG channels were both expressed not only on the cell surface membrane but also in the cytoplasm of HEK293 cells. The EGFP/hERG appeared to influence the hERG channel gating properties, including reduction of the peak tail current density, more rapid inactivation process, faster recovery from inactivation and faster deactivation kinetics compared with untagged hERG channels. Our results suggest that the EGFP/hERG channel alter the electrophysiological properties of hERG channel, but it does not seem to alter the cellular location of hERG channels. Thus, EGFP tagging to N-terminus might be used for research of subcellular location of hERG channels but not for the channel electrophysiological properties. |
| Databáze: | OpenAIRE |
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