Abstract
Here we report on the development of the recombinant fluorescent ligand RFP-AgTx2 by fusing tagRFP fluorescent protein through a flexible linker with a peptide blocker of Kv1.x (x = 1, 3) channels, agitoxin 2 (AgTx2). RFP-AgTx2 can be easily produced in E. coli cells and purified in the folded functionally active form. The yield of RFP-AgTx2 achieves 100 mg per 1 L of bacterial culture that makes it the cheapest fluorescently labeled ligand of Kv1 channels. It is shown that RFP-AgTx2 binds to chimeric KcsA-Kv1.1 and KcsA-Kv1.3 channels with low nanomolar dissociation constants and is displaced from these complexes with Kv1.x pore blockers such as tetraethylammonium, AgTx2, kaliotoxin and OSK1. RFP-AgTx2 is an advanced fluorescent molecular tool to image Kv1 channels in cells and tissues. We present and discuss the distribution of Kv1 channels highlighted with RFP-AgTx2 in living rat glioma C6 and pheochromocytoma PC12 cells.
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Acknowledgments
This work was supported by the Russian Science Foundation (grant no. 14-14-00239). We thank A. Kuzmenkov and Dr. A.Vassilevskiy (Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry) for collaboration in the pioneering development of recombinant fluorescent ligands of Kv1 channels.
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Feofanov, A.V., Kudryashova, K.S., Ignatova, A.A., Nekrasova, O.V. (2017). Recombinant Fluorescent Ligand of Potassium Kv1.1 and Kv1.3 Channels: Design, Properties and Applications. In: Oral, A., Bahsi Oral, Z. (eds) 3rd International Multidisciplinary Microscopy and Microanalysis Congress (InterM). Springer Proceedings in Physics, vol 186. Springer, Cham. https://doi.org/10.1007/978-3-319-46601-9_2
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