Fluorescence Approaches for Characterizing Ion Channels in Synthetic Bilayers

Islam, Md. Sirajul; Gaston, James P.; Baker, Matthew A. B.

doi:10.3390/membranes11110857

Cited by 5 publications

(3 citation statements)

References 160 publications

(174 reference statements)

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“…The binding is specific, as well-known Kv1.3 channel blockers completely inhibited the staining of spheroplasts with GFP-MgTx (Figure 3A), and no binding of GFP-MgTx was detected at the membrane of spheroplasts prepared from non-expressing E. coli BL21(DE3) cells, or cells expressing KcsA channel (Figure 2C). As shown earlier, GFP itself is unable to bind to KcsA-Kv1.3 or KcsA, as well as to spheroplasts without heterologously expressed channels [14].…”

Section: Gfp-mgtx Interaction With the Kv13 Binding Sitesupporting

confidence: 56%

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GFP–Margatoxin, a Genetically Encoded Fluorescent Ligand to Probe Affinity of Kv1.3 Channel Blockers

Denisova

Orlov

Yakimov

et al. 2022

IJMS

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Peptide pore blockers and their fluorescent derivatives are useful molecular probes to study the structure and functions of the voltage-gated potassium Kv1.3 channel, which is considered as a pharmacological target in the treatment of autoimmune and neurological disorders. We present Kv1.3 fluorescent ligand, GFP–MgTx, constructed on the basis of green fluorescent protein (GFP) and margatoxin (MgTx), the peptide, which is widely used in physiological studies of Kv1.3. Expression of the fluorescent ligand in E. coli cells resulted in correctly folded and functionally active GFP–MgTx with a yield of 30 mg per 1 L of culture. Complex of GFP–MgTx with the Kv1.3 binding site is reported to have the dissociation constant of 11 ± 2 nM. GFP–MgTx as a component of an analytical system based on the hybrid KcsA–Kv1.3 channel is shown to be applicable to recognize Kv1.3 pore blockers of peptide origin and to evaluate their affinities to Kv1.3. GFP–MgTx can be used in screening and pre-selection of Kv1.3 channel blockers as potential drug candidates.

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Section: Gfp-mgtx Interaction With the Kv13 Binding Sitesupporting

confidence: 56%

“…Although these methods are based on indirect measurement of ion-channel activity, they do not require complex equipment and are easier to implement. Noticeably, one major limitation of fluorescence approaches is their low temporal resolution [14].…”

Section: Introductionmentioning

confidence: 99%

GFP–Margatoxin, a Genetically Encoded Fluorescent Ligand to Probe Affinity of Kv1.3 Channel Blockers

Denisova

Orlov

Yakimov

et al. 2022

IJMS

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“…Fluorescent proteins, e.g. , the green fluorescent protein (GFP), are frequently fused to proteins to monitor their expression levels, folding, functional state, and cellular localization. − Furthermore, pH-sensitive GFP mutants, so-called pHluorins, have been developed, which can be fused to proteins of interest to report local pH changes in cells or vesicles with greater sensitivity than commonly used pH-sensitive dyes such as ACMA (9-amino-6-chloro-2-methoxyacridine) or pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid). − Even though the level of sensitivity might not have reached its full potential, these tools are especially valuable for assessing the correct reconstitution and function of energizing and transport modules (see sections and ) in synthetic vesicles, a majority of which create or depend on established proton gradients. In addition, the fusion of soluble protein domains to integral membrane proteins has been proposed as an approach to create more water-soluble constructs and thus facilitate their expression and purification .…”

Section: Engineering Of Protein and Scaffold Modulesmentioning

confidence: 99%

Synthetic Biology: Bottom-Up Assembly of Molecular Systems

et al. 2022

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The bottom-up assembly of biological and chemical components opens exciting opportunities to engineer artificial vesicular systems for applications with previously unmet requirements. The modular combination of scaffolds and functional building blocks enables the engineering of complex systems with biomimetic or new-to-nature functionalities. Inspired by the compartmentalized organization of cells and organelles, lipid or polymer vesicles are widely used as model membrane systems to investigate the translocation of solutes and the transduction of signals by membrane proteins. The bottom-up assembly and functionalization of such artificial compartments enables full control over their composition and can thus provide specifically optimized environments for synthetic biological processes. This review aims to inspire future endeavors by providing a diverse toolbox of molecular modules, engineering methodologies, and different approaches to assemble artificial vesicular systems. Important technical and practical aspects are addressed and selected applications are presented, highlighting particular achievements and limitations of the bottom-up approach. Complementing the cutting-edge technological achievements, fundamental aspects are also discussed to cater to the inherently diverse background of the target audience, which results from the interdisciplinary nature of synthetic biology. The engineering of proteins as functional modules and the use of lipids and block copolymers as scaffold modules for the assembly of functionalized vesicular systems are explored in detail. Particular emphasis is placed on ensuring the controlled assembly of these components into increasingly complex vesicular systems. Finally, all descriptions are presented in the greater context of engineering valuable synthetic biological systems for applications in biocatalysis, biosensing, bioremediation, or targeted drug delivery.

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Fluorescence‐Based HTS Assays for Ion Channel Modulation in Drug Discovery Pipelines

Voldřich,

Matoušová,

Šmídková

et al. 2024

ChemMedChem

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Ion channels represent a druggable family of transmembrane pore‐forming proteins with important (patho)physiological functions. While electrophysiological measurement (manual patch clamp) remains the only direct method for detection of ion currents, it is a labor‐intensive technique. Although automated patch clamp instruments have become available to date, their high costs limit their use to large pharma companies or commercial screening facilities. Therefore, fluorescence‐based assays are particularly important for initial screening of compound libraries. Despite their numerous disadvantages, they are highly amenable to high‐throughput screening and in many cases, no sophisticated instrumentation or materials are required. These features predispose them for implementation in early phases of drug discovery pipelines (hit identification), even in an academic environment. This review summarizes the advantages and pitfalls of individual methodological approaches for identification of ion channel modulators employing fluorescent probes (i. e., membrane potential and ion flux assays) with emphasis on practical aspects of their adaptation to high‐throughput format.

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Fluorescence Approaches for Characterizing Ion Channels in Synthetic Bilayers

Cited by 5 publications

References 160 publications

GFP–Margatoxin, a Genetically Encoded Fluorescent Ligand to Probe Affinity of Kv1.3 Channel Blockers

GFP–Margatoxin, a Genetically Encoded Fluorescent Ligand to Probe Affinity of Kv1.3 Channel Blockers

Synthetic Biology: Bottom-Up Assembly of Molecular Systems

Fluorescence‐Based HTS Assays for Ion Channel Modulation in Drug Discovery Pipelines

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