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.

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.

We report on a compact two-electrode voltage clamping system composed of microfabricated electrodes and a fluidic device for Xenopus laevis oocytes. The device was fabricated by assembling Si-based electrode chips and acrylic frames to form fluidic channels. After the installation of Xenopus oocytes into the fluidic channels, the device can be separated in order to measure changes in oocyte plasma membrane potential in each channel using an external amplifier. Using fluid simulations and experiments, we investigated the success rates of Xenopus oocyte arrays and electrode insertion with respect to the flow rate. We successfully located each oocyte in the array and detected oocyte responses to chemical stimuli using our device.

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.

Functional investigations of enzymes involving cellular expression systems are important for pharmacological studies. The precise control of expression is challenging in transiently transfected mammalian cell lines. Here, we explored the ability of Xenopus laevis oocytes to express a membrane-bound enzyme for functional characterization using standard 96-well plates and a fluorescence-based plate reader assay. We microinjected oocytes with cRNA encoding the angiotensin converting enzyme 2 (ACE2) and measured the enzymatic activity in single oocytes using a commercial fluorescence-based assay. The injected oocytes showed up to a 50-fold increase in fluorescence compared to uninjected oocytes. This fluorescence intensity was dose-dependent on the amount of ACE2 cRNA. These results suggest that Xenopus oocytes can be used for the functional evaluation of membrane-bound enzymes, decreasing the experimental workload.

Avenanthramides are phenolic compounds unique to oats and may contribute to health-promoting properties associated with oat consumption. This study used Xenopus laevis oocytes expressing the glucose transporters, glucose transporter 2 (GLUT2) or sodium-glucose transport protein 1 (SGLT1) and human Caco-2 cells models to investigate the effect of oat avenanthramides on human intestinal glucose transporters. The presence of avenanthramide reduced the glucose uptake in a dose-dependent manner in Caco-2 cells. Glucose uptake in oocytes expressing either GLUT2 or SGLT1 was nullified by oat avenanthramide. There was no significant difference between the inhibition potencies of avenanthramides C and B. Thus, our results suggest that avenanthramides may contribute to the antidiabetic properties of oats. PRACTICAL APPLICATIONS: The present research focus on the antidiabetic properties of avenanthramides, which are unique phenolic compounds found in oats. Inhibiting the activities of the glucose transport proteins expressed in the small intestine is a known strategy to improve the control of postprandial glucose level. We therefore examined the inhibitory effects of avenanthramides on two glucose transporters, glucose transporter 2 and sodium-glucose transport protein 1, predominantly found in the small intestine using the human small intestinal cell model Caco-2 cell line and by heterologously expressing these two transporters in the Xenopus laevis oocytes. Based on our results, we have confirmed for the first time that the glucose uptake is indeed inhibited by the presence of avenanthramides, suggesting the possibility of incorporating avenanthramides in foods to enhance postprandial glucose response, and ultimately improve the management of diabetes. Therefore, future research could consider utilizing this evidence in the development of diabetic-friendly functional foods or nutraceuticals containing avenanthramides.

Rhodopsins with enzymatic activity were found in microbes, in 2004 hypothetically from sequence data and since 2014 by experimental proof. So far three different types are known: light-activated guanylyl cyclase opsins (Cyclop) in fungi, light-inhibited two-component guanylyl cyclase opsins (2c-Cyclop) in green algae, and rhodopsin phosphodiesterases (RhoPDE) in choanoflagellates. They are integral membrane proteins with eight transmembrane helices (TM), different to the other microbial (type I) rhodopsins with 7 TM. Therefore, we propose a classification as type Ib rhodopsins for opsins with 8 TM and type Ia for the ones with 7 TM. To characterize those rhodopsins or their mutants, the expression in Xenopus laevis oocytes proved to be an efficient strategy. Functional analysis was initially performed \"in oocyte\" (in vivo), but more detailed characterization can be obtained with an in vitro assay. In this chapter, we describe procedures how to extract membranes from oocytes after cRNA microinjection and heterologous protein expression. Enzymatic activity of these membranes is then analyzed under different illumination conditions. In addition, fluorescent labeling of the rhodopsins is employed to quantify the expression level and the absolute activity of designed mutants. We discuss strengths and pitfalls, associated with this expression system, and strategies for selecting potentially useful optogenetic tools.

BACKGROUND: The clinical use of calcitonin gene-related peptide receptor (CGRP-R) antagonists and monoclonal antibodies against CGRP and CGRP-R has offered new treatment possibilities for migraine patients. CGRP activates both the CGRP-R and structurally related amylin 1 receptor (AMY1-R). The relative effect of erenumab and the small-molecule CGRP-R antagonist, rimegepant, towards the CGRP-R and AMY-R needs to be further characterized.
METHODS: The effect of CGRP and two CGRP-R antagonists were examined in Xenopus laevis oocytes expressing human CGRP-R, human AMY1-R and their subunits.
RESULTS: CGRP administered to receptor expressing oocytes induced a concentration-dependent increase in current with the order of potency CGRP-R> > AMY1-R > calcitonin receptor (CTR). There was no effect on single components of the CGRP-R; calcitonin receptor-like receptor and receptor activity-modifying protein 1. Amylin was only effective on AMY1-R and CTR. Inhibition potencies (pIC50 values) for erenumab on CGRP induced currents were 10.86 and 9.35 for CGRP-R and AMY1-R, respectively. Rimegepant inhibited CGRP induced currents with pIC50 values of 11.30 and 9.91 for CGRP-R and AMY1-R, respectively.
CONCLUSIONS: Our results demonstrate that erenumab and rimegepant are potent antagonists of CGRP-R and AMY1-R with 32- and 25-times preference for the CGRP-R over the AMY1-R, respectively. It is discussed if this difference in affinity between the two receptors is the likely reason why constipation is a common and serious adverse effect during CGRP-R antagonism but less so with CGRP binding antibodies.

Heterologous expression systems (e.g., Xenopus laevis oocytes) are useful to study the biophysical properties and pharmacology of ionotropic receptors such as ionotropic glutamate (iGLuRs) and nicotinic acetylcholine (nAChRs) receptors. However, insect receptors often require the co-expression of chaperone proteins to be functional. Only few iGluRs and nAChRs have been successfully expressed in such systems. Here, we compared the efficiency of chaperone proteins to promote the functional expression of one Apis mellifera iGluR and several nAChR subunit combinations (α1α8β1, α7, α2α8β1 and α2α7α8β1) in Xenopus oocytes. To this end, we cloned a new iGluR (GluR-1) and potential chaperone proteins (e.g., SOL-1, Neto, NACHO) and tested more than 40 combinations of human, nematode and honeybee proteins. We obtained robust expression of GluR-1 and α1α8β1 when co-expressed with honeybee chaperone proteins and found that nAChR expression critically depended on the α1 subunit N-terminal sequence. We recorded small ACh-gated currents in few oocytes when the α7 subunit was co-expressed with Caenorhabditis elegans RIC-3, but none of the chaperone proteins allowed efficient expression of α2α8β1 or α2α7α8β1. Our results show that only some protein combinations can reconstitute functional receptors in Xenopus oocytes and that protein combination efficient in one species is not always efficient in another species.

How phosphorylation of the epithelial sodium channel (ENaC) contributes to its regulation is incompletely understood. Previously, we demonstrated that in outside-out patches ENaC activation by serum- and glucocorticoid-inducible kinase isoform 1 (SGK1) was abolished by mutating a serine residue in a putative SGK1 consensus motif RXRXX(S/T) in the channel\'s α-subunit (S621 in rat). Interestingly, this serine residue is followed by a highly conserved proline residue rather than by a hydrophobic amino acid thought to be required for a functional SGK1 consensus motif according to in vitro data. This suggests that this serine residue is a potential phosphorylation site for the dual-specificity tyrosine phosphorylated and regulated kinase 2 (DYRK2), a prototypical proline-directed kinase. Its phosphorylation may prime a highly conserved preceding serine residue (S617 in rat) to be phosphorylated by glycogen synthase kinase 3 β (GSK3β). Therefore, we investigated the effect of DYRK2 on ENaC activity in outside-out patches of Xenopus laevis oocytes heterologously expressing rat ENaC. DYRK2 included in the pipette solution significantly increased ENaC activity. In contrast, GSK3β had an inhibitory effect. Replacing S621 in αENaC with alanine (S621A) abolished the effects of both kinases. A S617A mutation reduced the inhibitory effect of GKS3β but did not prevent ENaC activation by DYRK2. Our findings suggest that phosphorylation of S621 activates ENaC and primes S617 for subsequent phosphorylation by GSK3β resulting in channel inhibition. In proof-of-concept experiments, we demonstrated that DYRK2 can also stimulate ENaC currents in microdissected mouse distal nephron, whereas GSK3β inhibits the currents.