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Membranes
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The Xenopus Oocyte: A Tool for Membrane Biology, Second Edition
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The Xenopus Oocyte: A Tool for Membrane Biology, Second Edition

A special issue of Membranes (ISSN 2077-0375). This special issue belongs to the section "Biological Membrane Functions".

Deadline for manuscript submissions: closed (31 October 2023) | Viewed by 9406

Special Issue Editors

Dr. Cesar Mattei

E-Mail Website
Guest Editor
UMR CNRS 6214, INSERM U1083, Mitovasc Institute, Angers University, 49045 Angers, France
Interests: cellular and molecular pharmacology of membrane receptors and ion channels; neurotoxins acting on their targets and disturbing physiological processes; pharmacology of GABA receptors; Na+ channels and nicotinic acetylcholine receptors; imaging on in vitro models
Special Issues, Collections and Topics in MDPI journals
Dr. Agenor Limon

E-Mail Website
Guest Editor
Department of Neurology, Mitchel Center for Neurodegenerative Diseases, School of Medicine, University of Texas Medical Branch, Galveston, TX 77555-1045, USA
Interests: molecular mechanisms of excitation-inhibition imbalance in neurodegenerative diseases and mental disorders; Microtransplantation of Synaptic Membranes (MSM); identification and validation of therapeutic targets identified by multidimensional analysis of molecular physiology data
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

This Special Issue is the continuation of the previous one recently published in Membranes with the same title "The Xenopus Oocyte: A Tool for Membrane Biology" https://www.mdpi.com/journal/membranes/special_issues/xenopus_oocyte).

The use of heterologous systems in pharmacology and cell biology is ancient, and there are countless cellular models allowing the characterization of membrane proteins or intracellular actors. Among these methods, the Xenopus oocyte has established itself by its ease of use, its large size, and the multiplicity of uses that can be made of it. This Special Issue, entitled “The Xenopus Oocyte: A Tool for Membrane Biology” and published by the journal Membranes, suggests that authors using the Xenopus oocyte as a study model contribute their work to provide a summary of the state-of-the-art research as well as current and future developments in the field. Topics include, but are not limited to, the heterologous expression of proteins (receptors and ion channels), membrane microtransplantation, ligand–receptor interaction, intracellular Ca2+ signaling, intracellular trafficking/membrane targeting, and pharmacological aspects of ligand–receptor interactions. Authors are invited to submit their latest results; both original papers and reviews are welcome.

Dr. Cesar Mattei
Dr. Agenor Limon
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Membranes is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • Xenopus oocyte
  • two-electrode voltage clamp
  • membrane transplantation
  • DNA and RNA microinjection
  • membrane receptors
  • ion channels
  • toxins
  • anesthetics
  • anxiolytics
  • insecticides

Published Papers (7 papers)

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Research

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9 pages, 953 KiB  
Communication
Membranes and Synaptosomes Used to Investigate Synaptic GABAergic Currents in Epileptic Patients
by Alessandro Gaeta, Lilian Juliana Lissner, Veronica Alfano, Pierangelo Cifelli, Alessandra Morano, Cristina Roseti, Angela Di Iacovo, Eleonora Aronica, Eleonora Palma and Gabriele Ruffolo
Membranes 2024, 14(3), 64; https://doi.org/10.3390/membranes14030064 - 02 Mar 2024
Viewed by 926
Abstract
Among the most prevalent neurological disorders, epilepsy affects about 1% of the population worldwide. We previously found, using human epileptic tissues, that GABAergic neurotransmission impairment is a key mechanism that drives the pathological phenomena that ultimately lead to generation and recurrence of seizures. [...] Read more.
Among the most prevalent neurological disorders, epilepsy affects about 1% of the population worldwide. We previously found, using human epileptic tissues, that GABAergic neurotransmission impairment is a key mechanism that drives the pathological phenomena that ultimately lead to generation and recurrence of seizures. Using both a “microtransplantation technique” and synaptosomes preparations from drug-resistant temporal lobe epilepsies (TLEs), we used the technique of two-electrode voltage clamp to record GABA-evoked currents, focusing selectively on the synaptic “fast inhibition” mediated by low-affinity GABAA receptors. Here, we report that the use-dependent GABA current desensitization (i.e., GABA rundown, which is evoked by applying to the cells consecutive pulses of GABA, at high concentration), which is a distinguishing mark of TLE, is mainly dependent on a dysfunction that affects synaptic GABAA receptors. In addition, using the same approaches, we recorded a depolarized GABA reversal potential in synaptosomes samples from the human epileptic subicula of TLE patients. These results, which confirm previous experiments using total membranes, suggest an altered chloride homeostasis in the synaptic area. Finally, the lack of a Zn2+ block of GABA-evoked currents using the synaptosomes supports the enrichment of “synaptic fast inhibitory” GABAA receptors in this preparation. Altogether, our findings suggest a pathophysiological role of low-affinity GABAA receptors at the synapse, especially during the fast and repetitive GABA release underlying recurrent seizures. Full article
(This article belongs to the Special Issue The Xenopus Oocyte: A Tool for Membrane Biology, Second Edition)
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10 pages, 4356 KiB  
Article
Cinnamic Acid and Caffeic Acid Effects on Gastric Tight Junction Proteins Analyzed in Xenopus laevis Oocytes
by Marie-Luise Vollstädt, Laura Stein, Nora Brunner and Salah Amasheh
Membranes 2024, 14(2), 40; https://doi.org/10.3390/membranes14020040 - 01 Feb 2024
Viewed by 1207
Abstract
Analysis of secondary plant compounds for the development of novel therapies is a common focus of experimental biomedicine. Currently, multiple health-supporting properties of plant-derived molecules are known but still information on many mechanisms is scarce. Cinnamic acid and caffeic acid are two of [...] Read more.
Analysis of secondary plant compounds for the development of novel therapies is a common focus of experimental biomedicine. Currently, multiple health-supporting properties of plant-derived molecules are known but still information on many mechanisms is scarce. Cinnamic acid and caffeic acid are two of the most abundant polyphenols in human dietary fruits and vegetables. In this study, we investigated cinnamic acid and caffeic acid effects on the gastric barrier, which is primarily provided by members of the transmembrane tight junction protein family of claudins. The Xenopus laevis oocyte has been established, in recent years, as a heterologous expression system for analysis of transmembrane tight junction protein interactions, by performing paired oocyte experiments to identify an effect on protein–protein interactions, in vitro. In our current study, human gastric claudin-4, -5, and -18.2. were expressed and detected in the oocyte plasma membrane by freeze fracture electron microscopy and immunoblotting. Oocytes were paired and incubated with 100 µM or 200 µM cinnamic acid or caffeic acid, or Ringer’s solution, respectively. Caffeic acid showed no effect on the contact area strength of paired oocytes but led to an increased contact area size. In contrast, cinnamic acid-incubated paired oocytes revealed a reduced contact area and a strengthening effect on the contact area was identified. These results may indicate that caffeic acid and cinnamic acid both show an effect on gastric barrier integrity via direct effects on tight junction proteins. Full article
(This article belongs to the Special Issue The Xenopus Oocyte: A Tool for Membrane Biology, Second Edition)
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11 pages, 1444 KiB  
Article
Inverse pH Gradient-Assay for Facile Characterization of Proton-Antiporters in Xenopus Oocytes
by Zeinu Mussa Belew, Christa Kanstrup, Chengyao Hua, Christoph Crocoll and Hussam Hassan Nour-Eldin
Membranes 2024, 14(2), 39; https://doi.org/10.3390/membranes14020039 - 01 Feb 2024
Viewed by 1295
Abstract
Xenopus oocytes represent one of the most versatile model systems for characterizing the properties of membrane transporters. However, for studying proton-coupled antiporters, the use of Xenopus oocytes has so far been limited to so-called injection-based transport assays. In such assays, where the compound [...] Read more.
Xenopus oocytes represent one of the most versatile model systems for characterizing the properties of membrane transporters. However, for studying proton-coupled antiporters, the use of Xenopus oocytes has so far been limited to so-called injection-based transport assays. In such assays, where the compound is injected directly into the oocytes’ cytosol and transport is detected by monitoring substrate efflux, poor control over internal diffusion and concentration are incompatible with mechanistic characterizations. In this study, we present an inverse pH-gradient transport assay. Herein, an outward-facing proton gradient enables the characterization of proton antiporters via facile import-based transport assays. We describe two approaches for establishing sustained outward-facing proton gradients across the oocyte membrane, namely by applying alkaline external conditions or through surprisingly stable carbonyl cyanide m-chlorophenyl-hydrazone (CCCP)-mediated acidification of the cytosol. Previously, genetic evidence has shown that DTX18 from Arabidopsis thaliana is essential for the deposition of the hydroxycinnamic acid amide p-coumaroylagmatine (coumaroylagmatine) defence compound on the leaf surface. However, direct evidence for its ability to transport coumarol-agmatine has not been provided. Here, using Xenopus oocytes as expression hosts, we demonstrate DTX18’s ability to transport coumaroyl-agmatine via both injection-based and inverse pH-gradient transport assays. Notably, by showing that DTX18 is capable of accumulating its substrate against its concentration gradient, we showcase the compatibility of the latter with mechanistic investigations. Full article
(This article belongs to the Special Issue The Xenopus Oocyte: A Tool for Membrane Biology, Second Edition)
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13 pages, 2453 KiB  
Article
Cannabidiol Strengthening of Gastric Tight Junction Complexes Analyzed in an Improved Xenopus Oocyte Assay
by Laura Stein, Marie-Luise Vollstaedt and Salah Amasheh
Membranes 2024, 14(1), 18; https://doi.org/10.3390/membranes14010018 - 08 Jan 2024
Viewed by 1558
Abstract
Cannabidiol (CBD), the non-psychoactive compound derived from the cannabis plant, has gained attention in recent years as a remedy against gastrointestinal disorders ranging from nausea and inflammation to abdominal pain. Recent advances demonstrated an effect on inflammatory pathways and barrier proteins. However, information [...] Read more.
Cannabidiol (CBD), the non-psychoactive compound derived from the cannabis plant, has gained attention in recent years as a remedy against gastrointestinal disorders ranging from nausea and inflammation to abdominal pain. Recent advances demonstrated an effect on inflammatory pathways and barrier proteins. However, information on possible direct effects is scarce and needs to be addressed, as applications are currently increasing in popularity. To accomplish this, we have employed Xenopus laevis oocytes as a heterologous expression system for analysis of the direct effects on stomach-specific claudins and further developed tight junction (TJ) protein interaction assays. Human claudin-4, claudin-5, and claudin-18.2 were expressed in Xenopus oocytes, clustered in pairs to form contact areas, and analyzed in a two-cell model approach, including measurement of the contact area and contact strength. CLDN4/5/18 + CLDN4/5/18 oocyte pairs were incubated with 20 µM CBD or with 40 µM CBD and were compared to cells without CBD treatment (ctrl). For interaction analysis, the contact area was measured after 24 h and 48 h. Whereas CBD did not affect the size of the protein interaction area, Double Orbital Challenge experiments revealed an increased contact strength after 24 h incubation with CBD. In addition, the Xenopus oocyte experiments were accompanied by an analysis of claudin-4, -5, and -18 expression in gastric epithelium by immunoblotting and immunohistochemistry. Claudin-4, -5, and -18 were strongly expressed, indicating a major role for gastric epithelial barrier function. In summary, our study shows direct effects of 40 µM CBD on Xenopus oocytes heterologously expressing a stomach-specific claudin combination, indicating a supportive and beneficial effect of CBD on gastric TJ proteins. Full article
(This article belongs to the Special Issue The Xenopus Oocyte: A Tool for Membrane Biology, Second Edition)
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17 pages, 3313 KiB  
Article
Histamine Receptors: Ex Vivo Functional Studies Enabling the Discovery of Hits and Pathways
by Andrea Seldeslachts, Steve Peigneur and Jan Tytgat
Membranes 2023, 13(12), 897; https://doi.org/10.3390/membranes13120897 - 02 Dec 2023
Cited by 1 | Viewed by 1755
Abstract
Histamine receptors (HRs) are G-protein-coupled receptors involved in diverse responses triggered by histamine release during inflammation or by encounters with venomous creatures. Four histamine receptors (H1R–H4R) have been cloned and extensively characterized. These receptors are distributed throughout the body and their activation is [...] Read more.
Histamine receptors (HRs) are G-protein-coupled receptors involved in diverse responses triggered by histamine release during inflammation or by encounters with venomous creatures. Four histamine receptors (H1R–H4R) have been cloned and extensively characterized. These receptors are distributed throughout the body and their activation is associated with clinical manifestations such as urticaria (H1R), gastric acid stimulation (H2R), regulation of neurotransmitters in neuronal diseases (H3R), and immune responses (H4R). Despite significant homologous overlap between H3R and H4R, much remains unknown about their precise roles. Even though some drugs have been developed for H1R, H2R, and H3R, not a single H4R antagonist has been approved for clinical use. To enhance our understanding and advance innovative therapeutic targeting of H1R, H2R, H3R, and H4R, we established a robust ex vivo functional platform. This platform features the successful heterologous expression of H1R–H4R in Xenopus laevis oocytes, utilizing an electrophysiological readout. Our findings contribute to a deeper understanding of the function and pharmacological properties of the histamine receptors. Researchers can benefit from the utility of this platform when investigating the effects of histamine receptors and exploring potential therapeutic targets. In doing so, it broadens the horizon of drug discovery, offering new perspectives for therapeutic interventions. Full article
(This article belongs to the Special Issue The Xenopus Oocyte: A Tool for Membrane Biology, Second Edition)
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11 pages, 3685 KiB  
Article
Recording Sodium Self-Inhibition of Epithelial Sodium Channels Using Automated Electrophysiology in Xenopus Oocytes
by Rene Y. Lawong, Fabian May, Etang C. Etang, Philipp Vorrat, Jonas George, Julia Weder, Dagmar Kockler, Matthias Preller and Mike Althaus
Membranes 2023, 13(5), 529; https://doi.org/10.3390/membranes13050529 - 19 May 2023
Cited by 1 | Viewed by 1227
Abstract
The epithelial sodium channel (ENaC) is a key regulator of sodium homeostasis that contributes to blood pressure control. ENaC open probability is adjusted by extracellular sodium ions, a mechanism referred to as sodium self-inhibition (SSI). With a growing number of identified ENaC gene [...] Read more.
The epithelial sodium channel (ENaC) is a key regulator of sodium homeostasis that contributes to blood pressure control. ENaC open probability is adjusted by extracellular sodium ions, a mechanism referred to as sodium self-inhibition (SSI). With a growing number of identified ENaC gene variants associated with hypertension, there is an increasing demand for medium- to high-throughput assays allowing the detection of alterations in ENaC activity and SSI. We evaluated a commercially available automated two-electrode voltage-clamp (TEVC) system that records transmembrane currents of ENaC-expressing Xenopus oocytes in 96-well microtiter plates. We employed guinea pig, human and Xenopus laevis ENaC orthologs that display specific magnitudes of SSI. While demonstrating some limitations over traditional TEVC systems with customized perfusion chambers, the automated TEVC system was able to detect the established SSI characteristics of the employed ENaC orthologs. We were able to confirm a reduced SSI in a gene variant, leading to C479R substitution in the human α-ENaC subunit that has been reported in Liddle syndrome. In conclusion, automated TEVC in Xenopus oocytes can detect SSI of ENaC orthologs and variants associated with hypertension. For precise mechanistic and kinetic analyses of SSI, optimization for faster solution exchange rates is recommended. Full article
(This article belongs to the Special Issue The Xenopus Oocyte: A Tool for Membrane Biology, Second Edition)
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Review

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16 pages, 3483 KiB  
Review
The Conventional and Breakthrough Tool for the Study of L-Glutamate Transporters
by Kanako Takahashi and Kaoru Sato
Membranes 2024, 14(4), 77; https://doi.org/10.3390/membranes14040077 - 27 Mar 2024
Viewed by 601
Abstract
In our recent report, we clarified the direct interaction between the excitatory amino acid transporter (EAAT) 1/2 and polyunsaturated fatty acids (PUFAs) by applying electrophysiological and molecular biological techniques to Xenopus oocytes. Xenopus oocytes have a long history of use in the scientific [...] Read more.
In our recent report, we clarified the direct interaction between the excitatory amino acid transporter (EAAT) 1/2 and polyunsaturated fatty acids (PUFAs) by applying electrophysiological and molecular biological techniques to Xenopus oocytes. Xenopus oocytes have a long history of use in the scientific field, but they are still attractive experimental systems for neuropharmacological studies. We will therefore summarize the pharmacological significance, advantages (especially in the study of EAAT2), and experimental techniques that can be applied to Xenopus oocytes; our new findings concerning L-glutamate (L-Glu) transporters and PUFAs; and the significant outcomes of our data. The data obtained from electrophysiological and molecular biological studies of Xenopus oocytes have provided us with further important questions, such as whether or not some PUFAs can modulate EAATs as allosteric modulators and to what extent docosahexaenoic acid (DHA) affects neurotransmission and thereby affects brain functions. Xenopus oocytes have great advantages in the studies about the interactions between molecules and functional proteins, especially in the case when the expression levels of the proteins are small in cell culture systems without transfections. These are also proper to study the mechanisms underlying the interactions. Based on the data collected in Xenopus oocyte experiments, we can proceed to the next step, i.e., the physiological roles of the compounds and their significances. In the case of EAAT2, the effects on the neurotransmission should be examined by electrophysiological approach using acute brain slices. For new drug development, pharmacokinetics pharmacodynamics (PKPD) data and blood brain barrier (BBB) penetration data are also necessary. In order not to miss the promising candidate compounds at the primary stages of drug development, we should reconsider using Xenopus oocytes in the early phase of drug development. Full article
(This article belongs to the Special Issue The Xenopus Oocyte: A Tool for Membrane Biology, Second Edition)
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