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.
Footnotes
Competing interests
The authors declare no competing or financial interests.
Author contributions
Conceptualization: R.R.G., R.M.-A.; Methodology: J.K., O.B.-S., R.R.G., R.M.-A.; Formal analysis: R.R.G., R.M.-A.; Investigation: J.K., R.R.G., R.M.-A.; Writing - original draft: J.K., R.R.G., R.M.-A.; Writing - review & editing: J.K., R.R.G., R.M.-A.; Supervision: R.R.G., R.M.-A.; Project administration: R.R.G., R.M.-A.; Funding acquisition: R.M.-A.
Funding
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (2013/11364-3 and 2013/23087-4 to R.M-A.; 2013/10780-3 to R.R.G) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (147216/2014-1 to J.K.).
- Received December 11, 2017.
- Accepted March 1, 2018.
- © 2018. Published by The Company of Biologists Ltd
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
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