scholarly journals Combining endocannabinoids with retigabine for enhanced M-channel effect and improved KV7 subtype selectivity

2020 ◽  
Vol 152 (8) ◽  
Author(s):  
Johan E. Larsson ◽  
Urban Karlsson ◽  
Xiongyu Wu ◽  
Sara I. Liin
Keyword(s):  
Adverse Effects ◽  
Xenopus Oocytes ◽  
Site Directed Mutagenesis ◽  
Resting Membrane Potential ◽  
Xenopus Laevis Oocytes ◽  
Kv7 Channels ◽  
Subtype Selectivity ◽  
Channel Effect ◽  
Low Concentrations ◽  
Electrode Voltage

Retigabine is unique among anticonvulsant drugs by targeting the neuronal M-channel, which is composed of KV7.2/KV7.3 and contributes to the negative neuronal resting membrane potential. Unfortunately, retigabine causes adverse effects, which limits its clinical use. Adverse effects may be reduced by developing M-channel activators with improved KV7 subtype selectivity. The aim of this study was to evaluate the prospect of endocannabinoids as M-channel activators, either in isolation or combined with retigabine. Human KV7 channels were expressed in Xenopus laevis oocytes. The effect of extracellular application of compounds with different properties was studied using two-electrode voltage clamp electrophysiology. Site-directed mutagenesis was used to construct channels with mutated residues to aid in the mechanistic understanding of these effects. We find that arachidonoyl-L-serine (ARA-S), a weak endocannabinoid, potently activates the human M-channel expressed in Xenopus oocytes. Importantly, we show that ARA-S activates the M-channel via a different mechanism and displays a different KV7 subtype selectivity compared with retigabine. We demonstrate that coapplication of ARA-S and retigabine at low concentrations retains the effect on the M-channel while limiting effects on other KV7 subtypes. Our findings suggest that improved KV7 subtype selectivity of M-channel activators can be achieved through strategically combining compounds with different subtype selectivity.

Properties of the beta 1- and beta 2-adrenergic receptor subtypes revealed by molecular cloning.

1989 ◽  
Vol 35 (5) ◽  
pp. 721-725 ◽  
Author(s):  
T Frielle ◽  
M G Caron ◽  
R J Lefkowitz
Keyword(s):  
Adrenergic Receptor ◽  
Radioligand Binding ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Xenopus Laevis Oocytes ◽  
Receptor Subtypes ◽  
Subtype Selectivity ◽  
Beta 2 ◽  
Transmembrane Regions ◽  
Chimeric Rna

Abstract The beta 1- and beta 2-adrenergic receptor subtypes are biochemically and functionally similar, because both receptors mediate the catecholamine-dependent activation of adenylate cyclase through the GTP-binding protein, Gs. Pharmacologically, the two receptors can be distinguished on the basis of their relative affinities for the agonists epinephrine and norepinephrine as well as their affinities for several selective antagonists. The primary structures of the human beta 1- and beta 2-adrenergic receptors have recently been deduced from the cloning of their genes and (or) cDNAs, revealing high sequence homology and a membrane topography of seven putative transmembrane regions similar to that of rhodopsin. Chimeric beta 1/beta 2-adrenergic receptor cDNAs have been constructed by site-directed mutagenesis and the chimeric RNA transcripts expressed in Xenopus laevis oocytes. The pharmacological properties of the expressed chimeric receptor proteins were assessed by radioligand binding utilizing subtype-selective agonists and antagonists. Apparently, several of the putative transmembrane regions contribute significantly to the determination of subtype selectivity, presumably by formation of a ligand-binding pocket, with determinants for agonist and antagonist binding being distinguishable.

P1601N-glycosylation of TREK-1/hK2P2.1 two-pore-domain (K2P) potassium channels

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
F Wiedmann ◽  
D Schlund ◽  
A Ratte ◽  
H A Katus ◽  
M Kraft ◽  
...  
Keyword(s):  
Potassium Channels ◽  
Posttranslational Modifications ◽  
Enzymatic Digestion ◽  
Resting Membrane Potential ◽  
Xenopus Laevis Oocytes ◽  
Channel Function ◽  
Excitable Tissue ◽  
Electrode Voltage ◽  
Channel Surface ◽  
Pore Domain

Abstract Background and purpose Mechanosensitive hTREK-1 (hK2P2.1) two-pore-domain potassium channels give rise to background currents that control resting membrane potential in excitable tissue. Recently TREK-1 currents have been linked to regulation of cardiac rhythm as well as hypertrophy and fibrosis. Even though pharmacological and biophysical characteristics of hTREK-1 channels have been widely studied, less is known about its posttranslational modifications. This study aims to evaluate whether hTREK-1 channels are N-glycosylated and whether glycosylation may affect channel functionality. Experimental approach Following pharmacological inhibition of N glycosylation, enzymatic digestion or mutagenesis, immunoblots of Xenopus laevis oocytes and HEK-233T cell lysates were used to assess electrophoretic mobility. Two-electrode voltage clamp measurements were employed to study channel function. Key results TREK-1 channels subunits undergo N-glycosylation at asparagine residues 110 and 134. The presence of sugar moieties at these two sites increases channel function. Detection of glycosylation-deficient mutant channels in surface fractions and recordings of macroscopic potassium currents mediated by these subunits demonstrate that non-glycosylated hTREK-1 channels subunits are able to reach the cell surface in general, but seemingly with reduced efficiency. Conclusion and implications hTREK-1 are glycoproteins and N glycosylation at positions 110 and 134 is involved in channel surface trafficking. These findings extend our view on regulation of hTREK-1 currents by posttranslational modifications and provide novel insights into how glycosylation deficiency disorders may promote arrhythmogenesis.

scholarly journals Enhanced Shear Force Responsiveness of Epithelial Na+ Channel’s (ENaC) δ Subunit Following the Insertion of N-Glycosylation Motifs Relies on the Extracellular Matrix

2021 ◽  
Vol 22 (5) ◽  
pp. 2500
Author(s):  
Daniel Barth ◽  
Fenja Knoepp ◽  
Martin Fronius
Keyword(s):  
Shear Force ◽  
Xenopus Oocytes ◽  
Site Directed Mutagenesis ◽  
Na Channel ◽  
Cell Matrix ◽  
Core Feature ◽  
Electrode Voltage ◽  
Ecm Degradation ◽  
Epithelial Na Channel

Members of the Degenerin/epithelial Na+ channel (ENaC) protein family and the extracellular cell matrix (ECM) form a mechanosensitive complex. A core feature of this complex are tethers, which connect the channel with the ECM, however, knowledge about the nature of these tethers is scarce. N-glycans of α ENaC were recently identified as potential tethers but whether N-glycans serve as a ubiquitous feature for mechanosensation processes remains unresolved. The purpose of this study was to reveal whether the addition of N-glycans to δ ENaC—which is less responsive to shear force (SF)—increases its SF-responsiveness and whether this relies on a linkage to the ECM. Therefore, N-glycosylation motifs were introduced via site-directed mutagenesis, the resulting proteins expressed with β and γ ENaC in Xenopus oocytes, and SF-activated currents measured by two-electrode voltage-clamp. The insertion of N-glycosylation motifs increases δ ENaC’s SF responsiveness. The inclusion of a glycosylated asparagine (N) at position 487 did increase the molecular mass and provided a channel whose SF response was abolished following ECM degradation via hyaluronidase. This indicates that the addition of N-glycans improves SF-responsiveness and that this effect relies on an intact ECM. These findings further support the role of N-glycans as tethers for mechanotransduction.

scholarly journals Heterologous Expression of Aedes aegypti Cation Chloride Cotransporter 2 (aeCCC2) in Xenopus laevis Oocytes Induces an Enigmatic Na+/Li+ Conductance

Insects ◽  
2019 ◽  
Vol 10 (3) ◽  
pp. 71 ◽  
Author(s):  
Megha Kalsi ◽  
Christopher Gillen ◽  
Peter Piermarini
Keyword(s):  
Plasma Membrane ◽  
Aedes Aegypti ◽  
Heterologous Expression ◽  
Xenopus Oocytes ◽  
Xenopus Laevis Oocytes ◽  
Conductive Pathway ◽  
Electrode Voltage ◽  
Genes Encoding ◽  
Voltage Clamping ◽  
Cation Chloride Cotransporters

The yellow fever mosquito Aedes aegypti possesses three genes encoding putative Na+-coupled cation chloride cotransporters (CCCs): aeNKCC1, aeCCC2, and aeCCC3. To date, none of the aeCCCs have been functionally characterized. Here we expressed aeCCC2 heterologously in Xenopus oocytes and measured the uptake of Li+ (a tracer for Na+) and Rb+ (a tracer for K+). Compared to control (H2O-injected) oocytes, the aeCCC2-expressing oocytes exhibited significantly greater uptake of Li+, but not Rb+. However, the uptake of Li+ was neither Cl−-dependent nor inhibited by thiazide, loop diuretics, or amiloride, suggesting unconventional CCC activity. To determine if the Li+-uptake was mediated by a conductive pathway, we performed two-electrode voltage clamping (TEVC) on the oocytes. The aeCCC2 oocytes were characterized by an enhanced conductance for Li+ and Na+, but not K+, compared to control oocytes. It remains to be determined whether aeCCC2 directly mediates the Na+/Li+ conductance or whether heterologous expression of aeCCC2 stimulates an endogenous cation channel in the oocyte plasma membrane.

scholarly journals Retigabine holds KV7 channels open and stabilizes the resting potential

2016 ◽  
Vol 147 (3) ◽  
pp. 229-241 ◽  
Author(s):  
Aaron Corbin-Leftwich ◽  
Sayeed M. Mossadeq ◽  
Junghoon Ha ◽  
Iwona Ruchala ◽  
Audrey Han Ngoc Le ◽  
...  
Keyword(s):  
Voltage Dependence ◽  
Resting Potential ◽  
Resting Membrane Potential ◽  
Xenopus Laevis Oocytes ◽  
Channel Activity ◽  
Pore Region ◽  
Threshold Potential ◽  
Kv7 Channels ◽  
Open States ◽  
Pore Domain

The anticonvulsant Retigabine is a KV7 channel agonist used to treat hyperexcitability disorders in humans. Retigabine shifts the voltage dependence for activation of the heteromeric KV7.2/KV7.3 channel to more negative potentials, thus facilitating activation. Although the molecular mechanism underlying Retigabine’s action remains unknown, previous studies have identified the pore region of KV7 channels as the drug’s target. This suggested that the Retigabine-induced shift in voltage dependence likely derives from the stabilization of the pore domain in an open (conducting) conformation. Testing this idea, we show that the heteromeric KV7.2/KV7.3 channel has at least two open states, which we named O1 and O2, with O2 being more stable. The O1 state was reached after short membrane depolarizations, whereas O2 was reached after prolonged depolarization or during steady state at the typical neuronal resting potentials. We also found that activation and deactivation seem to follow distinct pathways, suggesting that the KV7.2/KV7.3 channel activity displays hysteresis. As for the action of Retigabine, we discovered that this agonist discriminates between open states, preferentially acting on the O2 state and further stabilizing it. Based on these findings, we proposed a novel mechanism for the therapeutic effect of Retigabine whereby this drug reduces excitability by enhancing the resting potential open state stability of KV7.2/KV7.3 channels. To address this hypothesis, we used a model for action potential (AP) in Xenopus laevis oocytes and found that the resting membrane potential became more negative as a function of Retigabine concentration, whereas the threshold potential for AP firing remained unaltered.

Blockade and Activation of the Human Neuronal Nicotinic Acetylcholine Receptors by Atracurium and Laudanosine

2001 ◽  
Vol 94 (4) ◽  
pp. 643-651 ◽  
Author(s):  
Florence Chiodini ◽  
Eric Charpantier ◽  
Dominique Muller ◽  
Edomer Tassonyi ◽  
Thomas Fuchs-Buder ◽  
...  
Keyword(s):  
Adverse Effects ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Nicotinic Receptors ◽  
Receptor Subtype ◽  
Xenopus Laevis Oocytes ◽  
Neuronal Nicotinic Acetylcholine Receptors ◽  
Neuronal Nicotinic Receptors ◽  
Arterial Blood ◽  
Low Concentrations

Background Curaremimetic nondepolarizing muscle relaxants are widely used in clinical practice to prevent muscle contraction either during surgery or during intensive care. Although primarily acting at the neuromuscular junction, these compounds can cause adverse effects, including modification of cardiac rhythm, arterial blood pressure, and in the worst cases, triggering of seizures. In this study, we assessed the interaction of atracurium and its metabolite, laudanosine, with neuronal nicotinic receptors. Methods The human neuronal nicotinic receptors alpha4beta2, alpha3beta4, alpha3alpha5beta4, and alpha7 are heterologously expressed in Xenopus laevis oocytes, and the effect of atracurium and its degradation product, laudanosine, were studied on these receptors. Results Atracurium and laudanosine inhibited in the micromolar range the major brain alpha4beta2 receptor and the ganglionic alpha3beta4 or alpha3beta4alpha5 and the homomeric alpha7 receptors. For all four receptors, inhibition was rapid and readily reversible within less than 1 min. Atracurium blockade was competitive at alpha4beta2 and alpha7 receptors but displayed a noncompetitive blockade at the alpha3beta4 receptors. Inhibition at this receptor subtype was not modified by alpha5. Laudanosine was found to have a dual mode of action; first, it competes with acetylcholine and, second, it blocks the ionic pore by steric hindrance. At low concentrations, these two drugs are able to activate both the alpha4beta2 and the alpha3beta4 receptors. Conclusion Adverse effects observed during atracurium administration may be attributed, at least partly, to an interaction with neuronal nicotinic receptors.

scholarly journals Site-directed mutagenesis and expression of PC2 in microinjected Xenopus oocytes.

1991 ◽  
Vol 266 (35) ◽  
pp. 24011-24017
Author(s):  
K.I. Shennan ◽  
A.J. Seal ◽  
S.P. Smeekens ◽  
D.F. Steiner ◽  
K. Docherty
Keyword(s):  
Xenopus Oocytes ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

Characterization of Transport Activity of SLC11 Transporters in Xenopus laevis Oocytes by Fluorophore Quenching

2021 ◽  
pp. 247255522110041
Author(s):  
Raffaella Cinquetti ◽  
Francesca Guia Imperiali ◽  
Salvatore Bozzaro ◽  
Daniele Zanella ◽  
Francesca Vacca ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Voltage Clamp ◽  
Expression System ◽  
Peptide Transporter ◽  
Xenopus Laevis Oocytes ◽  
Substrate Uptake ◽  
Transport Activity ◽  
Experimental Conditions ◽  
Metal Transporters ◽  
Electrode Voltage

Membrane proteins are involved in different physiological functions and are the target of pharmaceutical and abuse drugs. Xenopus laevis oocytes provide a powerful heterologous expression system for functional studies of these proteins. Typical experiments investigate transport using electrophysiology and radiolabeled uptake. A two-electrode voltage clamp is suitable only for electrogenic proteins, and uptake measurements require the existence of radiolabeled substrates and adequate laboratory facilities. Recently, Dictyostelium discoideum Nramp1 and NrampB were characterized using multidisciplinary approaches. NrampB showed no measurable electrogenic activity, and it was investigated in Xenopus oocytes by acquiring confocal images of the quenching of injected fluorophore calcein. This method is adequate to measure the variation in emitted fluorescence, and thus transporter activity indirectly, but requires long experimental procedures to collect statistically consistent data. Considering that optimal expression of heterologous proteins lasts for 48–72 h, a slow acquiring process requires the use of more than one batch of oocytes to complete the experiments. Here, a novel approach to measure substrate uptake is reported. Upon injection of a fluorophore, oocytes were incubated with the substrate and the transport activity measured, evaluating fluorescence quenching in a microplate reader. The technique permits the testing of tens of oocytes in different experimental conditions simultaneously, and thus the collection of significant statistical data for each batch, saving time and animals. The method was tested with different metal transporters (SLC11), DMT1, DdNramp1, and DdNrampB, and verified with the peptide transporter PepT1 (SLC15). Comparison with traditional methods (uptake, two-electrode voltage clamp) and with quenching images acquired by fluorescence microscopy confirmed its efficacy.

scholarly journals The Molecular Physiology of Sodium- and Proton-Coupled Solute Transporters

Physiology ◽  
1998 ◽  
Vol 13 (3) ◽  
pp. 123-131 ◽  
Author(s):  
Angela Steel ◽  
Matthias A. Hediger
Keyword(s):  
Xenopus Laevis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Xenopus Laevis Oocytes ◽  
Missense Mutations ◽  
Molecular Physiology ◽  
Inherited Diseases ◽  
Biophysical Analysis ◽  
Solute Transporters

The expression of cloned Na+- and H+-coupled solute transporters in Xenopus laevis oocytes has permitted detailed molecular and biophysical analysis and illuminated unique mechanistic features. The identification of missense mutations in inherited diseases and site-directed mutagenesis studies have enhanced our understanding of their roles in physiological and pathological processes.

scholarly journals Identification of renal transporters involved in sulfate excretion in marine teleost fish

2009 ◽  
Vol 297 (6) ◽  
pp. R1647-R1659 ◽  
Author(s):  
Akira Kato ◽  
Min-Hwang Chang ◽  
Yukihiro Kurita ◽  
Tsutomu Nakada ◽  
Maho Ogoshi ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
Proximal Tubules ◽  
Border Region ◽  
Marine Teleost ◽  
Renal Transporters ◽  
Electrode Voltage ◽  
Cloned Cdna ◽  
Cdna Fragments ◽  
Immunohistochemical Analyses ◽  
Marine Teleost Fish

Sulfate (SO42−) is the second most abundant anion in seawater (SW), and excretion of excess SO42− from ingested SW is essential for marine fish to survive. Marine teleosts excrete SO42− via the urine produced in the kidney. The SO42− transporter that secretes and concentrates SO42− in the urine has not previously been identified. Here, we have identified and characterized candidates for the long-sought transporters. Using sequences from the fugu database, we have cloned cDNA fragments of all transporters belonging to the Slc13 and Slc26 families from mefugu ( Takifugu obscurus ). We compared Slc13 and Slc26 mRNA expression in the kidney between freshwater (FW) and SW mefugu. Among 14 clones examined, the expression of a Slc26a6 paralog (mfSlc26a6A) was the most upregulated (30-fold) in the kidney of SW mefugu. Electrophysiological analyses of Xenopus oocytes expressing mfSlc26a6A, mfSlc26a6B, and mouse Slc26a6 (mSlc26a6) demonstrated that all transporters mediate electrogenic Cl−/SO42−, Cl−/oxalate2−, and Cl−/ nHCO3− exchanges and electroneutral Cl−/formate− exchange. Two-electrode voltage-clamp experiments demonstrated that the SO42−-elicited currents of mfSlc26a6A is quite large (∼35 μA at +60 mV) and 50- to 200-fold higher than those of mfSlc26a6B and mSlc26a6. Conversely, the currents elicited by oxalate and HCO3− are almost identical among mfSlc26a6A, mfSlc26a6B, and mSlc26a6. Kinetic analysis revealed that mfSlc26a6A has the highest SO42− affinity as well as capacity. Immunohistochemical analyses demonstrated that mfSlc26a6A localizes to the apical (brush-border) region of the proximal tubules. Together, these findings suggest that mfSlc26a6A is the most likely candidate for the major apical SO42− transporter that mediates SO42− secretion in the kidney of marine teleosts.

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