Ivorra I et al. (2022), Xenopus Oocytes as a Powerful Cellular Model to...

XB-ART-59332

Membranes (Basel) 2022 Oct 11;1210:. doi: 10.3390/membranes12100986.

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Xenopus Oocytes as a Powerful Cellular Model to Study Foreign Fully-Processed Membrane Proteins.

Ivorra I , Alberola-Die A , Cobo R , González-Ros JM , Morales A .

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The use of Xenopus oocytes in electrophysiological and biophysical research constitutes a long and successful story, providing major advances to the knowledge of the function and modulation of membrane proteins, mostly receptors, ion channels, and transporters. Earlier reports showed that these cells are capable of correctly expressing heterologous proteins after injecting the corresponding mRNA or cDNA. More recently, the Xenopus oocyte has become an outstanding host-cell model to carry out detailed studies on the function of fully-processed foreign membrane proteins after their microtransplantation to the oocyte. This review focused on the latter overall process of transplanting foreign membrane proteins to the oocyte after injecting plasma membranes or purified and reconstituted proteins. This experimental approach allows for the study of both the function of mature proteins, with their native stoichiometry and post-translational modifications, and their putative modulation by surrounding lipids, mostly when the protein is purified and reconstituted in lipid matrices of defined composition. Remarkably, this methodology enables functional microtransplantation to the oocyte of membrane receptors, ion channels, and transporters from different sources including human post-mortem tissue banks. Despite the large progress achieved over the last decades on the structure, function, and modulation of neuroreceptors and ion channels in healthy and pathological tissues, many unanswered questions remain and, most likely, Xenopus oocytes will continue to help provide valuable responses.

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Species referenced: Xenopus laevis
GO keywords: ion channel activity [+]

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	Figure 1. Micrographs of Xenopus oocytes. (A) Image of an opened and extended fragment of the Xenopus ovary lobule, showing oocytes at different stages of development. (B) Full-grown, immature oocyte, and the plasma membrane (together with its vitelline envelope) from another oocyte, manually isolated and stained with ink for better observation.
	Figure 2. Methodology followed to microtransplant foreign membrane proteins to oocytes. Scheme of the steps to microtransplant the purified and reconstituted nicotinic acetylcholine (ACh) receptors (nAChRs) to the Xenopus oocyte membrane to carry out detailed functional studies.
	Figure 3. The microtransplantation of proteoliposomes to the Xenopus oocyte membrane does not depend on the increase in the intracellular calcium concentration ([Ca2+]i). ACh-elicited currents in oocytes microinjected with proteolipomes bearing nAChRs in the control oocytes (left, control) and in oocytes previously loaded with ca. 5 nM EGTA to chelate [Ca2+]i (right). Note the slower desensitization on nAChRs in the EGTA loaded cell.
	Figure 4. Patchy distribution of microtransplanted nAChRs on the oocyte surface. (A) Scheme showing patches of microtransplanted nAChRs on the oocyte surface (red spots) to be activated by the ACh solution superfused from a nearby tube. (B) Two superimposed IAChs showing at least two sizeable “humps” (more evident in the derivatives of IACh records shown below), which were most likely due to the subsequent activation of large patches of nAChRs.
	Figure 5. Protein function depends on its adequate orientation in the membrane after microtransplantation. (A) Scheme of the putative orientation in the plasma membrane of the microtransplanted nAChRs after intracellular injection of proteoliposomes bearing this protein. Note that the orientation of nAChRs in the injected proteoliposomes was mostly outside-out (left; ACh-binding sites shown as green spheres), whereas it turned to the outside-in orientation after the proteoliposome fused with the plasma membrane (right-side panel). nAChRs adopting a “wrong” orientation have been crossed out; Ec and Ic indicate extracellular and intracellular sides of the membrane, respectively. (B) nAChRs with an outside-in orientation lack functional activity, as evidenced by the lack of response when ACh was injected intracellularly (right panel). In contrast, focal ACh pulses over the oocyte surface, applied to nAChRs with the outside-out orientation, elicited IAChs (left).
	Figure 6. Modulation of nAChR function by specific lipids. (A) IAChs elicited in oocytes injected with proteoliposomes bearing nAChRs reconstituted in either asolectin (R-Aso), phosphatidylcholine (PC) and Chol (R-PC+Chol), or phosphatidic acid (PA), PC, and Chol (R-PA+PC+Chol). Notice the larger IACh in the R-PA+PC+Chol oocyte. (B) Effect of pre-injecting the oocyte with liposomes of different composition (Aso, PA, or PA+PC+Chol) 6 h before microinjecting proteoliposomes bearing nAChRs reconstituted in Aso (R-Aso). Left, the scheme shows the experimental procedure. Right, column bars comparing the relative amplitude of the IAChs of the indicated groups with respect to the control values (in oocytes pre-injected with Aso liposomes).

References [+] :

Alberola-Die, Muscle-Type Nicotinic Receptor Blockade by Diethylamine, the Hydrophilic Moiety of Lidocaine. 2016, Pubmed, Xenbase