Reconstitution and Electrophysiological Characterization of Ion Channels in Lipid Bilayers

Klærke, Dan A.; Tejada, Maria de los Angeles; Christensen, Vibeke Grøsfjeld; Lassen, Mette; Pedersen, Per Amstrup; Calloe, Kirstine

doi:10.1002/cpph.37

Cited by 7 publications

(5 citation statements)

References 27 publications

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“…Single channel ion conductance was measured in lipid bilayers using an Orbit Mini workstation (Nanion Technologies, München, Germany) with 50 μm MECA4 recording chips with 4 microelectrode cavities (Ionera Technologies, Freiburg, Germany) as previously described [46]. Voltage was controlled by the EDR 3 software (Elements, Cesena, Italy).…”

Section: Methodsmentioning

confidence: 99%

Purification of Functional Human TRP Channels Recombinantly Produced in Yeast

Zhang

Wang

Klærke

et al. 2019

Cells

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(1) Background: Human transient receptor potential (TRP) channels constitute a large family of ion-conducting membrane proteins that allow the sensation of environmental cues. As the dysfunction of TRP channels contributes to the pathogenesis of many widespread diseases, including cardiac disorders, these proteins also represent important pharmacological targets. TRP channels are typically produced using expensive and laborious mammalian or insect cell-based systems. (2) Methods: We demonstrate an alternative platform exploiting the yeast Saccharomyces cerevisiae capable of delivering high yields of functional human TRP channels. We produce 11 full-length human TRP members originating from four different subfamilies, purify a selected subset of these to a high homogeneity and confirm retained functionality using TRPM8 as a model target. (3) Results: Our findings demonstrate the potential of the described production system for future functional, structural and pharmacological studies of human TRP channels.

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Section: Methodsmentioning

confidence: 99%

Purification of Functional Human TRP Channels Recombinantly Produced in Yeast

Zhang

Wang

Klærke

et al. 2019

Cells

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“…In the conventional artificial lipid bilayer recording technique, it takes several tens of minutes or several hours to measure channel currents because it takes a long time to both make the lipid bilayer membrane (several minutes or more) and incorporate the ion channels into the membrane (from approximately ten minutes to several hours) although it depends on the type of channel. In addition, for a commercially available system to measure channel current in the artificial membrane using giant unilamellar vesicles, it has been reported that average recording times are 1-3 times per day 33 . Our system provides much higher measurement efficiency than the other techniques.…”

Section: Use Of Gold Probes Comprising a Hydrophobic Layer Results In...mentioning

confidence: 99%

Development of an automated system to measure ion channel currents using a surface-modified gold probe

Hirano

Tomita²,

Takahashi

et al. 2021

Sci Rep

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Artificial lipid bilayer single-channel recording technique has been employed to determine the biophysical and pharmacological properties of various ion channels. However, its measurement efficiency is very low, as it requires two time-consuming processes: preparation of lipid bilayer membranes and incorporation of ion channels into the membranes. In order to address these problems, we previously developed a technique based on hydrophilically modified gold probes on which are immobilized ion channels that can be promptly incorporated into the bilayer membrane at the same time as the membrane is formed on the probes’ hydrophilic area. Here, we improved further this technique by optimizing the gold probe and developed an automated channel current measurement system. We found that use of probes with rounded tips enhanced the efficiency of channel current measurements, and introducing a hydrophobic area on the probe surface, beside the hydrophilic one, further increased measurement efficiency by boosting membrane stability. Moreover, we developed an automated measurement system using the optimized probes; it enabled us to automatically measure channel currents and analyze the effects of a blocker on channel activity. Our study will contribute to the development of high-throughput devices to identify drug candidates affecting ion channel activity.

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“…GUVs made of POPC:POPG (1:1) lipids were prepared by the electroformation method [ 26 , 27 ]. Fluorescently labelled lipids (FLLs) were used to stain the membrane of GUVs to perform the vesicle fusion assays.…”

Section: Methodsmentioning

confidence: 99%

The Unusual Aggregation and Fusion Activity of the Antimicrobial Peptide W-BP100 in Anionic Vesicles

Ferreira

Nunes

et al. 2023

Membranes

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Cationic antimicrobial peptides (CAMPs) offer a promising strategy to counteract bacterial resistance, mostly due to their membrane-targeting activity. W-BP100 is a potent broad-spectrum cecropin-melittin CAMP bearing a single N-terminal Trp, which was previously found to improve its antibacterial activity. W-BP100 has high affinity toward anionic membranes, inducing membrane saturation at low peptide-to-lipid (P/L) ratios and membrane permeabilization, with the unique property of promoting the aggregation of anionic vesicles only at specific P/L ratios. Herein, we aimed to investigate this unusual behavior of W-BP100 by studying its aggregation and fusion properties with negatively-charged large (LUVs) or giant (GUVs) unilamellar vesicles using biophysical tools. Circular dichroism (CD) showed that W-BP100 adopted an α-helical conformation in anionic LUVs, neutralizing its surface charge at the aggregation P/L ratio. Its fusion activity, assessed by Förster resonance energy transfer (FRET) using steady-state fluorescence spectroscopy, occurred mainly at the membrane saturation/aggregation P/L ratio. Confocal microscopy studies confirmed that W-BP100 displays aggregation and detergent-like effects at a critical P/L ratio, above which it induces the formation of new lipid aggregates. Our data suggest that W-BP100 promotes the aggregation and fusion of anionic vesicles at specific P/L ratios, being able to reshape the morphology of GUVs into new lipid structures.

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Reconstitution and Electrophysiological Characterization of Ion Channels in Lipid Bilayers

Cited by 7 publications

References 27 publications

Purification of Functional Human TRP Channels Recombinantly Produced in Yeast

Purification of Functional Human TRP Channels Recombinantly Produced in Yeast

Development of an automated system to measure ion channel currents using a surface-modified gold probe

The Unusual Aggregation and Fusion Activity of the Antimicrobial Peptide W-BP100 in Anionic Vesicles

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