scholarly journals Expression and characterization of the bacterial mechanosensitive channel MscS in Xenopus laevis oocytes

2011 ◽  
Vol 138 (6) ◽  
pp. 641-649 ◽  
Author(s):  
Grigory Maksaev ◽  
Elizabeth S. Haswell
Keyword(s):  
Xenopus Laevis ◽  
Single Channel ◽  
Ease Of Use ◽  
Mechanosensitive Channel ◽  
Xenopus Laevis Oocytes ◽  
E Coli ◽  
African Clawed Frog ◽  
Clawed Frog ◽  
Small Conductance

We have successfully expressed and characterized mechanosensitive channel of small conductance (MscS) from Escherichia coli in oocytes of the African clawed frog, Xenopus laevis. MscS expressed in oocytes has the same single-channel conductance and voltage dependence as the channel in its native environment. Two hallmarks of MscS activity, the presence of conducting substates at high potentials and reversible adaptation to a sustained stimulus, are also exhibited by oocyte-expressed MscS. In addition to its ease of use, the oocyte system allows the user to work with relatively large patches, which could be an advantage for the visualization of membrane deformation. Furthermore, MscS can now be compared directly to its eukaryotic homologues or to other mechanosensitive channels that are not easily studied in E. coli.

scholarly journals Cyclodextrins increase membrane tension and are universal activators of mechanosensitive channels

2021 ◽  
Author(s):  
Charles D Cox ◽  
Yixiao Zhang ◽  
Zijing Zhou ◽  
Thomas Walz ◽  
Boris Martinac
Keyword(s):  
Conformational Changes ◽  
Functional Characterization ◽  
Mechanosensitive Channel ◽  
Mechanical Forces ◽  
Mechanosensitive Channels ◽  
Membrane Tension ◽  
E Coli ◽  
Lipid Environment ◽  
Small Conductance

AbstractThe bacterial mechanosensitive channel of small conductance, MscS, has been extensively studied to understand how mechanical forces are converted into the conformational changes that underlie mechanosensitive (MS) channel gating. We showed that lipid removal by β-cyclodextrin can mimic membrane tension. Here, we show that all cyclodextrins (CDs) can activate reconstituted E. coli MscS, that MscS activation by CDs depends on CD-mediated lipid removal, and that the CD amount required to gate MscS scales with the channel’s sensitivity to membrane tension. CD-mediated lipid removal ultimately causes MscS desensitization, which we show is affected by the lipid environment. CDs can also activate the structurally unrelated MscL. While many MS channels respond to membrane forces, generalized by the ‘force-from-lipids’ principle, their different molecular architectures suggest that they use unique ways to convert mechanical forces into conformational changes. CDs emerge as a universal tool for the structural and functional characterization of unrelated MS channels.

scholarly journals Functional Characterization of the Oxantel-Sensitive Acetylcholine Receptor from Trichuris muris

2021 ◽  
Vol 14 (7) ◽  
pp. 698
Author(s):  
Tina V. A. Hansen ◽  
Richard K. Grencis ◽  
Mohamed Issouf ◽  
Cédric Neveu ◽  
Claude L. Charvet
Keyword(s):  
Single Dose ◽  
Mouse Model ◽  
Xenopus Laevis ◽  
Acetylcholine Receptor ◽  
Drug Target ◽  
Functional Characterization ◽  
Xenopus Laevis Oocytes ◽  
Trichuris Muris ◽  
Electrode Voltage

The human whipworm, Trichuris trichiura, is estimated to infect 289.6 million people globally. Control of human trichuriasis is a particular challenge, as most anthelmintics have a limited single-dose efficacy, with the striking exception of the narrow-spectrum anthelmintic, oxantel. We recently identified a novel ACR-16-like subunit from the pig whipworm, T. suis which gave rise to a functional acetylcholine receptor (nAChR) preferentially activated by oxantel. However, there is no ion channel described in the mouse model parasite T. muris so far. Here, we have identified the ACR-16-like and ACR-19 subunits from T. muris, and performed the functional characterization of the receptors in Xenopus laevis oocytes using two-electrode voltage-clamp electrophysiology. We found that the ACR-16-like subunit from T. muris formed a homomeric receptor gated by acetylcholine whereas the ACR-19 failed to create a functional channel. The subsequent pharmacological analysis of the Tmu-ACR-16-like receptor revealed that acetylcholine and oxantel were equally potent. The Tmu-ACR-16-like was more responsive to the toxic agonist epibatidine, but insensitive to pyrantel, in contrast to the Tsu-ACR-16-like receptor. These findings confirm that the ACR-16-like nAChR from Trichuris spp. is a preferential drug target for oxantel, and highlights the pharmacological difference between Trichuris species.

scholarly journals Developmental changes of purinergic control of intestinal motor activity during metamorphosis in the African clawed frog, Xenopus laevis

2007 ◽  
Vol 292 (5) ◽  
pp. R1916-R1925 ◽  
Author(s):  
Monika Sundqvist
Keyword(s):  
Xenopus Laevis ◽  
Motor Activity ◽  
Receptor Antagonist ◽  
Circular Muscle ◽  
Developmental Changes ◽  
Disulfonic Acid ◽  
Developmental Phase ◽  
African Clawed Frog ◽  
Low Concentrations ◽  
Clawed Frog

Little is known about the purinergic regulation of intestinal motor activity in amphibians. Purinergic control of intestinal motility is subject to changes during development in mammals. The aim of this study was to investigate purinergic control of intestinal smooth muscle in the amphibian Xenopus laevis and explore possible changes in this system during the developmental phase of metamorphosis. Effects of purinergic compounds on mean force and contraction frequency in intestinal circular muscle strips from prometamorphic, metamorphic, and juvenile animals were investigated. Before metamorphosis, low concentrations of ATP reduced motor activity, whereas the effects were reversed at higher concentrations. ATP-induced relaxation was not inhibited by the P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) but was blocked by the ecto-nucleotidase inhibitor 6- N, N-diethyl-d-β,γ-dibromomethylene ATP ( ARL67256 ), indicating that an ATP-derived metabolite mediated the relaxation response at this stage. Adenosine induced relaxation before, during, and after metamorphosis, which was blocked by the A1-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). The stable ATP-analog adenosine 5′-[γ-thio]-triphosphate (ATPγS) and 2-methylthioATP (2-MeSATP) elicited contractions in the circular muscle strips in prometamorphic tadpoles. However, in juvenile froglets, 2-MeSATP caused relaxation, as did ATPγS at low concentrations. The P2Y11/P2X1-receptor antagonist NF157 antagonized the ATPγS-induced relaxation. The P2X-preferring agonist α-β-methyleneadenosine 5′-triphosphate (α-β-MeATP) evoked PPADS-sensitive increases in mean force at all stages investigated. This study demonstrates the existence of an adenosine A1-like receptor mediating relaxation and a P2X-like receptor mediating contraction in the X. laevis gut before, during, and after metamorphosis. Furthermore, the development of a P2Y11-like receptor-mediated relaxation during metamorphosis is shown.

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