Abstract
The Xenopus oocyte is largely used as a cell expression system for studying both structure and function of transmitter receptors and ion channels. Messenger RNA extracted from the brain and injected into oocytes leads to the synthesis and membrane incorporation of many types of functional ion channels. A new method was developed further to transplant neurotransmitter receptors from human brain or cultured cell lines to the membrane of Xenopus oocytes. This method represents a modification of the method used many years ago of injecting into oocytes membrane vesicles from Torpedo electroplaques, yielding the expression of functional Torpedo acetylcholine receptors. We describe this approach by extracting membrane vesicles from human hippocampus or temporal neocortex and from mammalian cell lines stably expressing glutamate or neuronal nicotinic receptors. Because the human neurotransmitter receptors are “microtransplanted” with their native cell membranes, this method extends the usefulness of Xenopus oocytes as an expression system for addressing issues in many fields, including channelopathies.
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References
Gundersen, C. B., Miledi, R., and Parker, I. (1983) Voltage-operated channels induced by foreign messenger RNA in Xenopus oocytes. Proc. R. Soc. Lond. BBiol. Sci. 220, 131–140
Gundersen, C. B., Miledi, R., and Parker, I. (1984) Messenger RNA from human brain induces drug-and voltage-operated channels in Xenopus oocytes. Nature 308, 421–424.
Miledi, R., Parler, I., and Sumikawa, K. (1989) Transplanting receptors from brain into oocytes, in Fidia Award Lectures, Vol. 3 (Smith, J., ed.), Raven Press, New York, pp. 57–90.
Arellano, R. O., Woodward, R. M., and Miledi, R. (1996) Ion channels and membrane receptors in follicle-enclosed Xenopus oocytes, in Ion Channels, Vol. 4 (Narahashi, T., ed.), Plenum Press, New York, pp. 203–259.
Bertrand D., Cooper, E., Valera, S., Rungger, D., and Ballivet, M. (1991) Electrophysiology of neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes following nuclear injection of genes or cDNAs, in Methods in Neurosciences, Vol. 4, edited by P. Michael Conn, Academic Press, NY, pp. 174–193.
Quick, M. W. and Lester, H. A. (1994) Methods for expression of excitability proteins in Xenopus oocytes, in Methods in Neurosciences, Vol. 19, edited by P. Michael Conn, Academic Press, NY, pp. 261–279.
Palma, E., Mileo, A. M., Eusebi, F., and Miledi, R. (1996) Threonine-for-leucine mutation within domain M2 of the neuronal α7 nicotinic receptor converts 5-hydroxytryptamine from antagonist to agonist. Proc. Natl. Acad. Sci. USA 93, 11,231–11,235.
Marsal, J., Tigyi, G., and Miledi, R. (1995) Incorporation of acetylcholine receptors and Cl-channels in Xenopus oocytes injected with Torpedo electroplaque membranes. Proc. Natl. Acad. Sci. USA 92, 5224–5228.
Morales, A., Aleu, J., Ivorra, I., Ferragut, J. A., Gonzalez-Ros, J. M., and Miledi, R. (1995) Incorporation of reconstituted acetylcholine receptors from Torpedo into Xenopus oocyte membrane. Proc. Natl. Acad. Sci. USA 92, 8468–8472.
Miledi, R., Eusebi, F., Martinez-Torres, A., Palma, E., and Trettel, F. (2002) Expression of functional neurotransmitter receptors in Xenopus oocytes after injection of human brain membranes. Proc. Natl. Acad. Sci. USA 99, 13,238–13,242.
Palma, E., Trettel, F., Fucile, S., Renzi, M., Miledi R., and Eusebi, F. (2003) Microtransplantation of membranes from cultured cells to Xenopus oocytes: a method to study neurotransmitter receptors embedded in native lipids, Proc. Natl. Acad. Sci. USA 100, 2896–2900.
Barnard. E. A., Miledi, R., and Sumikawa, K. (1982) Transplantation of exogenous messenger RNA coding for nicotinic acetylcholine receptors produces functional receptors in Xenopus oocytes. Proc. R. Soc. Lond. B Biol. Sci. 215, 241–246.
Sumikawa, K., Parker, I., and Miledi, R. (1984) Partial purification and functional expression. Proc. Natl. head. Sci. USA 81, 7994–7998.
Smart, T. G. and Krishek, B. J. (1995) Xenopus oocyte microinjection and ion-channel expression, in Patch-Clamp Applications and Protocols (Boulton, A. A., Baker, G. B., and Walz, W., eds.), Humana Press, Totowa, NJ.
Miledi, R. (1982) A calcium-dependent transient outward current in Xenopus laevis oocytes. Proc. R. Soc. Lond. B Biol. Sci. 215, 491–497.
Miledi, R. and Woodward, R. M. (1989) Effects of defolliculation on membrane current responses of Xenopus oocytes. J. Physiol. 416, 601–621.
Palma, E., Esposito, V., Mileo, A. M., et al. (2002) Expression of human temporal lobe neurotransmitter receptors in Xenopus oocytes: an innovative approach to study epilepsy. Proc. Natl. Acad. Sci. USA 99, 15,078–15,083.
Kusano, K., Miledi, R., and Stinnakre, J. (1982) Cholinergic and catecholaminergic receptors in the Xenopus oocyte membrane. J. Physiol. 328, 143–170.
Miledi, R. (1980) Intracellular calcium and desensitization of acetylcholine receptors. Proc. R. Soc. Lond. B Biol. Sci. 209. 447–452.
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Miledi, R., Palma, E., Eusebi, F. (2006). Microtransplantation of Neurotransmitter Receptors From Cells to Xenopus Oocyte Membranes. In: Liu, X.J. (eds) Xenopus Protocols. Methods in Molecular Biology™, vol 322. Humana Press. https://doi.org/10.1007/978-1-59745-000-3_24
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DOI: https://doi.org/10.1007/978-1-59745-000-3_24
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