Editorial

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3 pages, 229 KiB

Open AccessEditorial

The Xenopus Oocyte: A Tool for Membrane Biology

by Agenor Limon and César Mattei

Membranes 2023, 13(10), 831; https://doi.org/10.3390/membranes13100831 - 15 Oct 2023

Viewed by 1171

Abstract

The Xenopus is a special study model in experimental research [...] Full article

(This article belongs to the Special Issue The Xenopus Oocyte: A Tool for Membrane Biology)

Research

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15 pages, 3451 KiB

Open AccessArticle

Asbestos Fibers Enhance the TMEM16A Channel Activity in Xenopus Oocytes

by Annalisa Bernareggi, Martina Zangari, Andrew Constanti, Paola Zacchi, Violetta Borelli, Alessandro Mangogna, Paola Lorenzon and Giuliano Zabucchi

Membranes 2023, 13(2), 180; https://doi.org/10.3390/membranes13020180 - 01 Feb 2023

Cited by 2 | Viewed by 1463

Abstract

Background: The interaction of asbestos fibers with target cell membranes is still poorly investigated. Here, we detected and characterized an enhancement of chloride conductance in Xenopus oocyte cell membranes induced by exposure to crocidolite (Croc) asbestos fibers. Methods: A two-microelectrode voltage clamp technique [...] Read more.

Background: The interaction of asbestos fibers with target cell membranes is still poorly investigated. Here, we detected and characterized an enhancement of chloride conductance in Xenopus oocyte cell membranes induced by exposure to crocidolite (Croc) asbestos fibers. Methods: A two-microelectrode voltage clamp technique was used to test the effect of Croc fiber suspensions on outward chloride currents evoked by step membrane depolarization. Calcium imaging experiments were also performed to investigate the variation of ‘resting’ oocyte [Ca²⁺]_i following asbestos exposure. Results: The increase in chloride current after asbestos treatment, was sensitive to [Ca²⁺]_e, and to specific blockers of TMEM16A Ca²⁺-activated chloride channels, MONNA and Ani9. Furthermore, asbestos treatment elevated the ‘resting’ [Ca²⁺]_i likelihood by increasing the cell membrane permeability to Ca² in favor of a tonic activation of TMEME16A channels_. Western blot analysis confirmed that TMEME16A protein was endogenously present in the oocyte cell membrane and absorbed by Croc. Conclusion: the TMEM16A channels endogenously expressed by Xenopus oocytes are targets for asbestos fibers and represent a powerful tool for asbestos–membrane interaction studies. Interestingly, TMEM16A channels are highly expressed in many types of tumors, including some asbestos-related cancers, suggesting them, for the first time, as a possible early target of crocidolite-mediated tumorigenic effects on target cell membranes. Full article

(This article belongs to the Special Issue The Xenopus Oocyte: A Tool for Membrane Biology)

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12 pages, 1361 KiB

Open AccessArticle

Methylene Blue Inhibits Cromakalim-Activated K⁺ Currents in Follicle-Enclosed Oocytes

by Dmytro Isaev, Keun-Hang Susan Yang, Georg Petroianu, Dietrich Ernst Lorke and Murat Oz

Membranes 2023, 13(2), 121; https://doi.org/10.3390/membranes13020121 - 18 Jan 2023

Cited by 1 | Viewed by 1238

Abstract

The effects of methylene blue (MB) on cromakalim-induced K⁺ currents were investigated in follicle-enclosed Xenopus oocytes. In concentrations ranging from 3–300 μM, MB inhibited K⁺ currents (IC₅₀: 22.4 μM) activated by cromakalim, which activates K_ATP channels. MB inhibited [...] Read more.

The effects of methylene blue (MB) on cromakalim-induced K⁺ currents were investigated in follicle-enclosed Xenopus oocytes. In concentrations ranging from 3–300 μM, MB inhibited K⁺ currents (IC₅₀: 22.4 μM) activated by cromakalim, which activates K_ATP channels. MB inhibited cromakalim-activated K⁺ currents in a noncompetitive and voltage-independent manner. The respective EC₅₀ and slope values for cromakalim-activation of K⁺ currents were 194 ± 21 µM and 0.91 for controls, and 206 ± 24 µM and 0.87 in the presence of 30 μM MB. The inhibition of cromakalim-induced K⁺ currents by MB was not altered by pretreatment with the Ca²⁺ chelator BAPTA, which suggests that MB does not influence Ca²⁺-activated second messenger pathways. K⁺ currents mediated through a C-terminally deleted form of Kir6.2 (KirΔC26), which does not contain the sulfonylurea receptor, were still inhibited by MB, indicating direct interaction of MB with the channel-forming Kir6.2 subunit. The binding characteristics of the K_ATP ligand [³H]glibenclamide are not altered by MB in a concentration range between 1 μM-1 mM, as suggested by radioligand binding assay. The presence of a membrane permeable cGMP analogue (8-Br-cGMP, 100 µM) and a guanylate cyclase activator (BAY 58-2667, 3 µM) did not affect the inhibitory effects of MB, suggesting that MB does not inhibit cromakalim-activated K⁺ currents through guanylate cyclase. Collectively, these results suggest that MB directly inhibits cromakalim-activated K⁺ currents in follicular cells of Xenopus oocytes. Full article

(This article belongs to the Special Issue The Xenopus Oocyte: A Tool for Membrane Biology)

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23 pages, 5515 KiB

Open AccessArticle

Functional Characterization of Four Known Cav2.1 Variants Associated with Neurodevelopmental Disorders

by Mathilde Folacci, Sébastien Estaran, Claudine Ménard, Anaïs Bertaud, Matthieu Rousset, Julien Roussel, Jean-Baptiste Thibaud, Michel Vignes, Alain Chavanieu, Pierre Charnet and Thierry Cens

Membranes 2023, 13(1), 96; https://doi.org/10.3390/membranes13010096 - 11 Jan 2023

Cited by 2 | Viewed by 1889

Abstract

Cav2.1 channels are expressed throughout the brain and are the predominant Ca²⁺ channels in the Purkinje cells. These cerebellar neurons fire spontaneously, and Cav2.1 channels are involved in the regular pacemaking activity. The loss of precision of the firing pattern of Purkinje [...] Read more.

Cav2.1 channels are expressed throughout the brain and are the predominant Ca²⁺ channels in the Purkinje cells. These cerebellar neurons fire spontaneously, and Cav2.1 channels are involved in the regular pacemaking activity. The loss of precision of the firing pattern of Purkinje cells leads to ataxia, a disorder characterized by poor balance and difficulties in performing coordinated movements. In this study, we aimed at characterizing functional and structural consequences of four variations (p.A405T in I-II loop and p.R1359W, p.R1667W and p.S1799L in IIIS4, IVS4, and IVS6 helices, respectively) identified in patients exhibiting a wide spectrum of disorders including ataxia symptoms. Functional analysis using two major Cav2.1 splice variants (Cav2.1+e47 and Cav2.1−e47) in Xenopus laevis oocytes, revealed a lack of effect upon A405T substitution and a significant loss-of-function caused by R1359W, whereas R1667W and S1799L caused both channel gain-of-function and loss-of-function, in a splice variant-dependent manner. Structural analysis revealed the loss of interactions with S1, S2, and S3 helices upon R1359W and R1667W substitutions, but a lack of obvious structural changes with S1799L. Computational modeling suggests that biophysical changes induced by Cav2.1 pathogenic mutations might affect action potential frequency in Purkinje cells. Full article

(This article belongs to the Special Issue The Xenopus Oocyte: A Tool for Membrane Biology)

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12 pages, 3082 KiB

Open AccessArticle

Expression of Mutant Glycine Receptors in Xenopus Oocytes Using Canonical and Non-Canonical Amino Acids Reveals Distinct Roles of Conserved Proline Residues

by Sarah C. R. Lummis and Dennis A. Dougherty

Membranes 2022, 12(10), 1012; https://doi.org/10.3390/membranes12101012 - 19 Oct 2022

Cited by 2 | Viewed by 1113

Abstract

Pentameric ligand-gated ion channels (pLGIC) play important roles in fast neuronal signal transmission. Functional receptors are pentamers, with each subunit having an extracellular domain (ECD), a transmembrane domain (TMD) and an intracellular domain. The binding of the agonist to the ECD induces a [...] Read more.

Pentameric ligand-gated ion channels (pLGIC) play important roles in fast neuronal signal transmission. Functional receptors are pentamers, with each subunit having an extracellular domain (ECD), a transmembrane domain (TMD) and an intracellular domain. The binding of the agonist to the ECD induces a structural change that is transduced to the TMD to open the channel. Molecular details of this process are emerging, but a comprehensive understanding is still lacking. Proline (Pro) is one amino acid that has attracted much interest; its unusual features generate bends in loops and kinks and bulges in helices, which can be essential for function in some pLGICs. Here, we explore the roles of four conserved Pros in the glycine receptor (GlyR), creating substitutions with canonical and noncanonical amino acids, characterizing them using two electrode voltage clamp electrophysiology in Xenopus oocytes, and interpreting changes in receptor parameters using structural data from the open and closed states of the receptor. The data reveal that for efficient function, the Pro in the α1β1 loop is needed to create a turn and to be the correct size and shape to interact with nearby residues; the peptide bond of the Pro in the Cys-loop requires the cis conformation; and the Pros in loop A and M1 allow efficient function because of their reduced hydrogen bonding capacity. These data are broadly consistent with data from other pLGICs, and therefore likely represent the important features of these Pros in all members of the family. Full article

(This article belongs to the Special Issue The Xenopus Oocyte: A Tool for Membrane Biology)

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12 pages, 2290 KiB

Open AccessArticle

Microtransplantation of Postmortem Native Synaptic mGluRs Receptors into Xenopus Oocytes for Their Functional Analysis

by Brice Miller, Naomi Moreno, Berenice A. Gutierrez and Agenor Limon

Membranes 2022, 12(10), 931; https://doi.org/10.3390/membranes12100931 - 26 Sep 2022

Cited by 4 | Viewed by 1537

Abstract

Metabotropic glutamate receptors (mGluRs) are membrane receptors that play a central role in the modulation of synaptic transmission and neuronal excitability and whose dysregulation is implicated in diverse neurological disorders. Most current understanding about the electrophysiological properties of such receptors has been determined [...] Read more.

Metabotropic glutamate receptors (mGluRs) are membrane receptors that play a central role in the modulation of synaptic transmission and neuronal excitability and whose dysregulation is implicated in diverse neurological disorders. Most current understanding about the electrophysiological properties of such receptors has been determined using recombinant proteins. However, recombinant receptors do not necessarily recapitulate the properties of native receptors due to the lack of obligated accessory proteins, some of which are differentially expressed as function of developmental stage and brain region. To overcome this limitation, we sought to microtransplant entire synaptosome membranes from frozen rat cortex into Xenopus oocytes, and directly analyze the responses elicited by native mGluRs. We recorded ion currents elicited by 1 mM glutamate using two electrodes voltage clamp. Glutamate produced a fast ionotropic response (6 ± 0.3 nA) in all microtransplanted oocytes (n = 218 oocytes) and a delayed oscillatory response (52 ± 7 nA) in 73% of them. The participation of Group 1 mGluRs was confirmed by the presence of metabotropic oscillations during the administration of (±)-1-Aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD; Group 1 mGluR agonist), and the absence of oscillations during co-administration of N-(1-adamantyl)quinoxaline-2-carboxamide (NPS 2390; Group 1 mGluR antagonist). Since both mGluR1 and mGluR5 belong to Group 1 mGluRs, further investigation revealed that mGluR1 antagonism with LY 456236 has little effect on metabotropic oscillations, while mGluR5 antagonism with 100 µM AZD 9272 has significant reduction of metabotropic currents elicited by ACPD and glutamate. We confirmed the expression of mGluR1 and mGluR5 in native synaptosomes by immunoblots, both of which are enhanced when compared to their counterpart proteins in rat cortex tissue lysates. Finally, these results demonstrate the merit of using microtransplantation of native synaptosomes for the study of mGluRs and the contribution of mGluR5 to the metabotropic glutamate signaling, providing a better tool for the understanding of the role of these receptors in neurological disorders. Full article

(This article belongs to the Special Issue The Xenopus Oocyte: A Tool for Membrane Biology)

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20 pages, 2726 KiB

Open AccessArticle

Coupling the Cardiac Voltage-Gated Sodium Channel to Channelrhodopsin-2 Generates Novel Optical Switches for Action Potential Studies

by Christian vom Dahl, Christoph Emanuel Müller, Xhevat Berisha, Georg Nagel and Thomas Zimmer

Membranes 2022, 12(10), 907; https://doi.org/10.3390/membranes12100907 - 20 Sep 2022

Cited by 2 | Viewed by 1375

Abstract

Voltage-gated sodium (Na⁺) channels respond to short membrane depolarization with conformational changes leading to pore opening, Na⁺ influx, and action potential (AP) upstroke. In the present study, we coupled channelrhodopsin-2 (ChR2), the key ion channel in optogenetics, directly to the [...] Read more.

Voltage-gated sodium (Na⁺) channels respond to short membrane depolarization with conformational changes leading to pore opening, Na⁺ influx, and action potential (AP) upstroke. In the present study, we coupled channelrhodopsin-2 (ChR2), the key ion channel in optogenetics, directly to the cardiac voltage-gated Na⁺ channel (Na_v1.5). Fusion constructs were expressed in Xenopus laevis oocytes, and electrophysiological recordings were performed by the two-microelectrode technique. Heteromeric channels retained both typical Na_v1.5 kinetics and light-sensitive ChR2 properties. Switching to the current-clamp mode and applying short blue-light pulses resulted either in subthreshold depolarization or in a rapid change of membrane polarity typically seen in APs of excitable cells. To study the effect of individual K⁺ channels on the AP shape, we co-expressed either K_v1.2 or hERG with one of the Na_v1.5-ChR2 fusions. As expected, both delayed rectifier K⁺ channels shortened AP duration significantly. K_v1.2 currents remarkably accelerated initial repolarization, whereas hERG channel activity efficiently restored the resting membrane potential. Finally, we investigated the effect of the LQT3 deletion mutant ΔKPQ on the AP shape and noticed an extremely prolonged AP duration that was directly correlated to the size of the non-inactivating Na⁺ current fraction. In conclusion, coupling of ChR2 to a voltage-gated Na⁺ channel generates optical switches that are useful for studying the effect of individual ion channels on the AP shape. Moreover, our novel optogenetic approach provides the potential for an application in pharmacology and optogenetic tissue-engineering. Full article

(This article belongs to the Special Issue The Xenopus Oocyte: A Tool for Membrane Biology)

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10 pages, 2150 KiB

Open AccessCommunication

3D Media Stabilizes Membrane and Prolongs Lifespan of Defolliculated Xenopus laevis Oocytes

by Nikhil D. Aggarwal, Steven L. Zeng, Ryan J. Lashgari, Leland C. Sudlow and Mikhail Y. Berezin

Membranes 2022, 12(8), 754; https://doi.org/10.3390/membranes12080754 - 31 Jul 2022

Cited by 1 | Viewed by 1378

Abstract

Xenopus laevis oocytes are commonly used in many fundamental biological studies. One of the major limitations of X. laevis oocytes is their short storage lifespan with most defolliculated oocytes physically deteriorating in 10 days or less. Herein, we identified a 3D Cultrex-based storage [...] Read more.

Xenopus laevis oocytes are commonly used in many fundamental biological studies. One of the major limitations of X. laevis oocytes is their short storage lifespan with most defolliculated oocytes physically deteriorating in 10 days or less. Herein, we identified a 3D Cultrex-based storage media that incorporates extracellular membrane-based hydrogels to maintain oocyte integrity. Under these treatments, the lifespan of the oocytes increased to more than 20 days compared to standard conditions. The treatment preserved the oocytes membrane integrity and did not interfere with mRNA- or cDNA-derived protein expression. Full article

(This article belongs to the Special Issue The Xenopus Oocyte: A Tool for Membrane Biology)

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11 pages, 1493 KiB

Open AccessArticle

Functional Analysis of Gastric Tight Junction Proteins in Xenopus laevis Oocytes

by Laura Stein, Nora Brunner and Salah Amasheh

Membranes 2022, 12(8), 731; https://doi.org/10.3390/membranes12080731 - 23 Jul 2022

Cited by 5 | Viewed by 1677

Abstract

The epithelial barrier is crucial for proper gastrointestinal function, preventing the unwanted passage of solutes and therefore representing a prerequisite for vectorial transport. Claudin-4 and claudin-18.2, two critical tight junction proteins of the gastric epithelium, seal neighboring cells in a physically and mechanically [...] Read more.

The epithelial barrier is crucial for proper gastrointestinal function, preventing the unwanted passage of solutes and therefore representing a prerequisite for vectorial transport. Claudin-4 and claudin-18.2, two critical tight junction proteins of the gastric epithelium, seal neighboring cells in a physically and mechanically challenging environment. As the Xenopus laevis oocyte allows the functional and molecular analyses of claudin interaction, we have addressed the hypothesis that this interaction is not only dependent on mechanical force but also on pH. We expressed human claudin-4 and claudin-18 in Xenopus oocytes, and analyzed them in a two-cell model approach. Cells were clustered in pairs to form contact areas expressing CLDN18 + CLDN18, CLDN4/18 + CLDN4/18, and compared to controls, respectively. Contact areas in cells incubated in medium at pH 5.5 and 7.4 were quantified by employing transmitted light microscopy. After 24 h at pH 5.5, clustering of CLDN18 + CLDN18 and CLDN4/18 + CLDN4/18-expressing oocytes revealed a contact area reduced by 45% and 32%, compared with controls, respectively. A further approach, high-pressure impulse assay, revealed a stronger tight junction interaction at pH 5.5 in oocyte pairs expressing CLDN18 + CLDN18 or CLDN4/18 + CLDN4/18 indicating a protective role of claudin-18 for tight junction integrity during pH challenge. Thus, our current analysis of gastric tight junction proteins further establishes oocytes as an expression and two-cell screening model for tight junction integrity analysis of organ- and tissue-specific claudins by the characterization of homo- and heterophilic trans-interaction dependent on barrier effectors. Full article

(This article belongs to the Special Issue The Xenopus Oocyte: A Tool for Membrane Biology)

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11 pages, 1304 KiB

Open AccessCommunication

Mammalian Brain Ca²⁺ Channel Activity Transplanted into Xenopus laevis Oocytes

by Matthieu Rousset, Sandrine Humez, Cyril Laurent, Luc Buée, David Blum, Thierry Cens, Michel Vignes and Pierre Charnet

Membranes 2022, 12(5), 496; https://doi.org/10.3390/membranes12050496 - 02 May 2022

Cited by 3 | Viewed by 1613

Abstract

Several mutations on neuronal voltage-gated Ca²⁺ channels (VGCC) have been shown to cause neurological disorders and contribute to the initiation of epileptic seizures, migraines, or cerebellar degeneration. Analysis of the functional consequences of these mutations mainly uses heterologously expressed mutated channels or [...] Read more.

Several mutations on neuronal voltage-gated Ca²⁺ channels (VGCC) have been shown to cause neurological disorders and contribute to the initiation of epileptic seizures, migraines, or cerebellar degeneration. Analysis of the functional consequences of these mutations mainly uses heterologously expressed mutated channels or transgenic mice which mimic these pathologies, since direct electrophysiological approaches on brain samples are not easily feasible. We demonstrate that mammalian voltage-gated Ca²⁺ channels from membrane preparation can be microtransplanted into Xenopus oocytes and can conserve their activity. This method, originally described to study the alteration of GABA receptors in human brain samples, allows the recording of the activity of membrane receptors and channels with their native post-translational processing, membrane environment, and regulatory subunits. The use of hippocampal, cerebellar, or cardiac membrane preparation displayed different efficacy for transplanted Ca²⁺ channel activity. This technique, now extended to the recording of Ca²⁺ channel activity, may therefore be useful in order to analyze the calcium signature of membrane preparations from unfixed human brain samples or normal and transgenic mice. Full article

(This article belongs to the Special Issue The Xenopus Oocyte: A Tool for Membrane Biology)

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16 pages, 2713 KiB

Open AccessArticle

Xenopus Oocytes: A Tool to Decipher Molecular Specificity of Insecticides towards Mammalian and Insect GABA—A Receptors

by Anaïs Bertaud, Thierry Cens, Rosanna Mary, Matthieu Rousset, Elodie Arel, Jean-Baptiste Thibaud, Michel Vignes, Claudine Ménard, Sébastien Dutertre, Claude Collet and Pierre Charnet

Membranes 2022, 12(5), 440; https://doi.org/10.3390/membranes12050440 - 19 Apr 2022

Cited by 4 | Viewed by 2137

Abstract

The number of insect GABA receptors (GABAr) available for expression studies has been recently increased by the cloning of the Acyrthosiphon pisum (pea aphid) RDL subunits. This large number of cloned RDL subunits from pest and beneficial insects opens the door to parallel [...] Read more.

The number of insect GABA receptors (GABAr) available for expression studies has been recently increased by the cloning of the Acyrthosiphon pisum (pea aphid) RDL subunits. This large number of cloned RDL subunits from pest and beneficial insects opens the door to parallel pharmacological studies on the sensitivity of these different insect GABAr to various agonists or antagonists. The resulting analysis of the molecular basis of the species-specific GABAr responses to insecticides is necessary not only to depict and understand species toxicity, but also to help at the early identification of unacceptable toxicity of insecticides toward beneficial insects such as Apis mellifera (honeybees). Using heterologous expression in Xenopus laevis oocytes, and two-electrode voltage-clamp recording to assess the properties of the GABAr, we performed a comparative analysis of the pharmacological sensitivity of RDL subunits from A. pisum, A. mellifera and Varroa destructor GABAr to three pesticides (fipronil, picrotoxin and dieldrin). These data were compared to similar characterizations performed on two Homo sapiens GABA-A receptors (α₂β₂γ₂ and α₂β₂γ₂). Our results underline a global conservation of the pharmacological profiles of these receptors, with some interesting species specificities, nonetheless, and suggest that this approach can be useful for the early identification of poorly specific molecules. Full article

(This article belongs to the Special Issue The Xenopus Oocyte: A Tool for Membrane Biology)

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Review

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18 pages, 1743 KiB

Open AccessReview

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

by Isabel Ivorra, Armando Alberola-Die, Raúl Cobo, José Manuel González-Ros and Andrés Morales

Membranes 2022, 12(10), 986; https://doi.org/10.3390/membranes12100986 - 11 Oct 2022

Cited by 8 | Viewed by 2514

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue The Xenopus Oocyte: A Tool for Membrane Biology)

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19 pages, 1806 KiB

Open AccessReview

The “www” of Xenopus laevis Oocytes: The Why, When, What of Xenopus laevis Oocytes in Membrane Transporters Research

by Manan Bhatt, Angela Di Iacovo, Tiziana Romanazzi, Cristina Roseti, Raffaella Cinquetti and Elena Bossi

Membranes 2022, 12(10), 927; https://doi.org/10.3390/membranes12100927 - 25 Sep 2022

Cited by 8 | Viewed by 2967

Abstract

After 50 years, the heterologous expression of proteins in Xenopus laevis oocytes is still essential in many research fields. New approaches and revised protocols, but also classical methods, such as the two-electrode voltage clamp, are applied in studying membrane transporters. New and old [...] Read more.

After 50 years, the heterologous expression of proteins in Xenopus laevis oocytes is still essential in many research fields. New approaches and revised protocols, but also classical methods, such as the two-electrode voltage clamp, are applied in studying membrane transporters. New and old methods for investigating the activity and the expression of Solute Carriers (SLC) are reviewed, and the kinds of experiment that are still useful to perform with this kind of cell are reported. Xenopus laevis oocytes at the full-grown stage have a highly efficient biosynthetic apparatus that correctly targets functional proteins at the defined compartment. This small protein factory can produce, fold, and localize almost any kind of wild-type or recombinant protein; some tricks are required to obtain high expression and to verify the functionality. The methodologies examined here are mainly related to research in the field of membrane transporters. This work is certainly not exhaustive; it has been carried out to be helpful to researchers who want to quickly find suggestions and detailed indications when investigating the functionality and expression of the different members of the solute carrier families. Full article

(This article belongs to the Special Issue The Xenopus Oocyte: A Tool for Membrane Biology)

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Other

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11 pages, 1159 KiB

Open AccessEssay

Arterial Blood Pressure, Neuronal Excitability, Mineral Metabolism and Cell Volume Regulation Mechanisms Revealed by Xenopus laevis oocytes

by Gerardo Gamba

Membranes 2022, 12(10), 911; https://doi.org/10.3390/membranes12100911 - 21 Sep 2022

Cited by 3 | Viewed by 1268

Abstract

Xenopus laevis oocytes have been an invaluable tool to discover and explore the molecular mechanisms and characteristics of many proteins, in particular integral membrane proteins. The oocytes were fundamental in many projects designed to identify the cDNA encoding a diversity of membrane proteins [...] Read more.

Xenopus laevis oocytes have been an invaluable tool to discover and explore the molecular mechanisms and characteristics of many proteins, in particular integral membrane proteins. The oocytes were fundamental in many projects designed to identify the cDNA encoding a diversity of membrane proteins including receptors, transporters, channels and pores. In addition to being a powerful tool for cloning, oocytes were later used to experiment with the functional characterization of many of the identified proteins. In this review I present an overview of my personal 30-year experience using Xenopus laevis oocytes and the impact this had on a variety of fields such as arterial blood pressure, neuronal excitability, mineral metabolism and cell volume regulation. Full article

(This article belongs to the Special Issue The Xenopus Oocyte: A Tool for Membrane Biology)

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