Abstract
The Xenopus laevis oocyte is a widely used system for heterologous expression of exogenous ion channel proteins. They are easy to obtain, mechanically and electrically stable, have a large size, enabling multiple types of electrophysiological recordings: two-electrode voltage clamp, single cell-attached or cell-free patch-clamp and macropatch recordings. The size of an oocyte (1 mm in diameter) also allows for the use of additional electrodes (from 1 to 3) for injection of diverse materials (Ca2+ chelators, peptides, chemicals, antibodies, proteic-partners, etc.) before or during the course of the electrophysiological experiment.
We have successfully used this system to analyze the biophysical properties of pore-forming peptides. Simple extracellular perfusion of these peptides induced the formation of channels in the oocyte plasma membrane; these channels can then be studied and characterized in diverse ionic conditions. The ease of the perfusion and the stability of the voltage-clamped oocyte make it a powerful tool for such analyses. Compared to artificial bilayers, oocytes offer a real animal plasma membrane where biophysical properties and toxicity can be studied in the stable environment.
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References
Hille B (2001) Ion channels of excitable membranes, 3rd edn. Sinauer Associates Inc., Sunderland, MA
Charnet P, Molle G, Marion D, Rousset M, Lullien-Pellerin V (2003) Puroindolines form ion channels in biological membranes. Biophys J 84:2416–2426
Chaloin L, De E, Charnet P, Molle G, Heitz F (1998) Ionic channels formed by a primary amphipathic peptide containing a signal peptide and a nuclear localization sequence. Biochim Biophys Acta 1375:52–60
Mangoni ME, Cens T, Dalle C, Nargeot J, Charnet P (1997) Characterisation of alpha1A Ba2+, Sr2+ and Ca2+ currents recorded with ancillary beta 1–4 subunits. Receptor Channel 5:1–14
Cens T, Rousset M, Kajava A, Charnet P (2007) Molecular determinant for specific Ca/Ba selectivity profiles of low and high threshold Ca2+ channels. J Gen Physiol 130:415–425
Neher E (1992) Correction for liquid junction potentials in patch clamp experiments. Methods Enzymol 207:123–131
Rousset M, Cens T, Vanmau N, Charnet P (2004) Ca2+-dependent interaction of BAPTA with phospholipids. FEBS Lett 576:41–45
Green WN, Andersen OS (1991) Surface charges and ion channel function. Annu Rev Physiol 53:341–359
Begenisich TB, Cahalan MD (1980) Sodium channel permeation in squid axons. I: reversal potential experiments. J Physiol 307:217–242
Campbell DL, Rasmusson RL, Strauss HC (1988) Theoretical study of the voltage and concentration dependence of the anomalous mole fraction effect in single calcium channels. New insights into the characterization of multi-ion channels. Biophys J 54:945–954
McCleskey EW (1999) Calcium channel permeation: a field in flux. J Gen Physiol 113: 765–772
Pellegrin P, Menard C, Mery J, Lory P, Charnet P, Bennes R (1997) Cell cycle dependent toxicity of an amphiphilic synthetic peptide. FEBS Lett 418:101–105
Cens T, Charnet P (2007) Use of Xenopus oocytes to measure ionic selectivity of pore-forming peptides and ion channels. In: Molnar P, Hickman JJ (eds) Patch-clamp methods and protocols. Series: methods in molecular biology, 1st edn. Humana, Totowa, NJ, pp 195–209
Acknowledgements
This work was supported by CNRS, INSERM, and ANR. The authors would like to thank Dr. I. Lefevre for critical reading of the manuscript and Jean-Marc Donnay for oocyte preparation.
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Cens, T., Charnet, P. (2014). Ion Selectivity of Pore-Forming Peptides and Ion Channels Measured in Xenopus Oocytes. In: Martina, M., Taverna, S. (eds) Patch-Clamp Methods and Protocols. Methods in Molecular Biology, vol 1183. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1096-0_22
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DOI: https://doi.org/10.1007/978-1-4939-1096-0_22
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