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Biol Open. 2018 Mar 29;7(4). pii: bio031880. doi: 10.1242/bio.031880.

Lithobates catesbeianus (American Bullfrog) oocytes: a novel heterologous expression system for aquaporins.

Author information

1
Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo 05508-900, Brazil.
2
Department of Health Sciences, Paulista University (UNIP), Sao Paulo 06542-001, Brazil.
3
Department of Biochemistry, Institute of Chemistry, University of Sao Paulo, Sao Paulo 05513-970, Brazil.
4
Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo 05508-900, Brazil raifaziz@icb.usp.br.

Abstract

Xenopus laevis oocytes are a valuable tool for investigating the function of membrane proteins. However, regulations around the world, specifically in Brazil, render the import of Xenopus laevis frogs impractical, and, in some cases, impossible. Here, as an alternative, we evaluate the usefulness of the North American aquatic bullfrog Lithobates catesebeianus, which is commercially available in Brazil, for the heterologous expression of aquaporin (AQP) proteins. We have developed a method that combines a brief collagenase treatment and mechanical defolliculation for isolating individual oocytes from Lithobates ovaries. We find that they have a similar size, shape, and appearance to Xenopus oocytes and can tolerate and survive following injections with cRNA or water. Furthermore, surface biotinylation, western blot analysis, and measurements of osmotic water permeability (Pf) show that Lithobates oocytes can express AQPs to the plasma membrane and significantly increase the Pf of the oocytes. In fact, the Pf values are similar to historical values gathered from Xenopus oocytes. Due to the presence of a mercury sensitive cysteine (Cys or C) in the throat of the water channel, the Pf of oocytes expressing human (h) AQP1, hAQP1FLAG [FLAG, short protein tag (DYKDDDDK) added to the N-terminus of AQP1], hAQP8, and rat (r) AQP9 was inhibited with the mercurial compound p-chloromercuribenzene sulfonate (pCMBS), whereas AQPs lacking this Cys - hAQP1C189S mutant [residue Cys 189 was replaced by a serine (Ser or S)] and hAQP7 - were mercury insensitive. Contrary to previous studies with Xenopus oocytes, rAQP3 was also found to be insensitive to mercury, which is consistent with the mercury-sensitive Cys (Cys 11) being located intracellularly. Thus, we consider Lithobates oocytes to be a readily accessible system for the functional expression and study of membrane proteins for international researchers who do not currently have access to Xenopus oocytes.

KEYWORDS:

American Bullfrog; Aquaporins; Heterologous expression system; Lithobates catesbeianus; Oocytes; Water permeability; Xenopus laevis

Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

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