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16 pages, 2774 KiB

Open AccessFeature PaperArticle

AgTx2-GFP, Fluorescent Blocker Targeting Pharmacologically Important K_v1.x (x = 1, 3, 6) Channels

by Alexandra L. Primak, Nikita A. Orlov, Steve Peigneur, Jan Tytgat, Anastasia A. Ignatova, Kristina R. Denisova, Sergey A. Yakimov, Mikhail P. Kirpichnikov, Oksana V. Nekrasova and Alexey V. Feofanov

Toxins 2023, 15(3), 229; https://doi.org/10.3390/toxins15030229 - 18 Mar 2023

Cited by 4 | Viewed by 2544

Abstract

The growing interest in potassium channels as pharmacological targets has stimulated the development of their fluorescent ligands (including genetically encoded peptide toxins fused with fluorescent proteins) for analytical and imaging applications. We report on the properties of agitoxin 2 C-terminally fused with enhanced GFP (AgTx2-GFP) as one of the most active genetically encoded fluorescent ligands of potassium voltage-gated K_v1.x (x = 1, 3, 6) channels. AgTx2-GFP possesses subnanomolar affinities for hybrid KcsA-K_v1.x (x = 3, 6) channels and a low nanomolar affinity to KcsA-K_v1.1 with moderate dependence on pH in the 7.0–8.0 range. Electrophysiological studies on oocytes showed a pore-blocking activity of AgTx2-GFP at low nanomolar concentrations for K_v1.x (x = 1, 3, 6) channels and at micromolar concentrations for K_v1.2. AgTx2-GFP bound to K_v1.3 at the membranes of mammalian cells with a dissociation constant of 3.4 ± 0.8 nM, providing fluorescent imaging of the channel membranous distribution, and this binding depended weakly on the channel state (open or closed). AgTx2-GFP can be used in combination with hybrid KcsA-K_v1.x (x = 1, 3, 6) channels on the membranes of E. coli spheroplasts or with K_v1.3 channels on the membranes of mammalian cells for the search and study of nonlabeled peptide pore blockers, including measurement of their affinity. Full article

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9 pages, 1304 KiB

Open AccessArticle

Atto488-Agitoxin 2—A Fluorescent Ligand with Increased Selectivity for Kv1.3 Channel Binding Site

by Kristina R. Denisova, Nikita A. Orlov, Sergey A. Yakimov, Mikhail P. Kirpichnikov, Alexey V. Feofanov and Oksana V. Nekrasova

Bioengineering 2022, 9(7), 295; https://doi.org/10.3390/bioengineering9070295 - 1 Jul 2022

Cited by 3 | Viewed by 1981

Abstract

Fluorescently labeled peptide blockers of ion channels are useful probes in studying the localization and functioning of the channels and in the performance of a search for new channel ligands with bioengineering screening systems. Here, we report on the properties of Atto488-agitoxin 2 (A-AgTx2), a derivative of the Kv1 channel blocker agitoxin 2 (AgTx2), which was N-terminally labeled with Atto 488 fluorophore. The interactions of A-AgTx2 with the outer binding sites of the potassium voltage-gated Kv1.x (x = 1, 3, 6) channels were studied using bioengineered hybrid KcsA–Kv1.x (x = 1, 3, 6) channels. In contrast to AgTx2, A-AgTx2 was shown to lose affinity for the Kv1.1 and Kv1.6 binding sites but to preserve it for the Kv1.3 site. Thus, Atto488 introduces two new functionalities to AgTx2: fluorescence and the selective targeting of the Kv1.3 channel, which is known for its pharmacological significance. In the case of A-AgTx2, fluorescent labeling served as an alternative to site-directed mutagenesis in modulating the pharmacological profile of the channel blocker. Although the affinity of A-AgTx2 for the Kv1.3 binding site was decreased as compared to the unlabeled AgTx2, its dissociation constant value was within a low nanomolar range (4.0 nM). The properties of A-AgTx2 allow one to use it for the search and study of Kv1.3 channel blockers as well as to consider it for the imaging of the Kv1.3 channel in cells and tissues. Full article

(This article belongs to the Section Nanobiotechnology and Biofabrication)

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15 pages, 3234 KiB

Open AccessArticle

AsKC11, a Kunitz Peptide from Anemonia sulcata, Is a Novel Activator of G Protein-Coupled Inward-Rectifier Potassium Channels

by Dongchen An, Ernesto Lopes Pinheiro-Junior, László Béress, Irina Gladkikh, Elena Leychenko, Eivind A. B. Undheim, Steve Peigneur and Jan Tytgat

Mar. Drugs 2022, 20(2), 140; https://doi.org/10.3390/md20020140 - 15 Feb 2022

Cited by 8 | Viewed by 4357

Abstract

(1) Background: G protein-coupled inward-rectifier potassium (GIRK) channels, especially neuronal GIRK1/2 channels, have been the focus of intense research interest for developing drugs against brain diseases. In this context, venom peptides that selectively activate GIRK channels can be seen as a new source for drug development. Here, we report on the identification and electrophysiological characterization of a novel activator of GIRK1/2 channels, AsKC11, found in the venom of the sea anemone Anemonia sulcata. (2) Methods: AsKC11 was purified from the sea anemone venom by reverse-phase chromatography and the sequence was identified by mass spectrometry. Using the two-electrode voltage-clamp technique, the activity of AsKC11 on GIRK1/2 channels was studied and its selectivity for other potassium channels was investigated. (3) Results: AsKC11, a Kunitz peptide found in the venom of A. sulcata, is the first peptide shown to directly activate neuronal GIRK1/2 channels independent from Gi/o protein activity, without affecting the inward-rectifier potassium channel (IRK1) and with only a minor effect on K_V1.6 channels. Thus, AsKC11 is a novel activator of GIRK channels resulting in larger K⁺ currents because of an increased chord conductance. (4) Conclusions: These discoveries provide new insights into a novel class of GIRK activators. Full article

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11 pages, 1451 KiB

Open AccessArticle

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

by Kristina R. Denisova, Nikita A. Orlov, Sergey A. Yakimov, Elena A. Kryukova, Dmitry A. Dolgikh, Mikhail P. Kirpichnikov, Alexey V. Feofanov and Oksana V. Nekrasova

Int. J. Mol. Sci. 2022, 23(3), 1724; https://doi.org/10.3390/ijms23031724 - 2 Feb 2022

Cited by 9 | Viewed by 2567

Abstract

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. Full article

(This article belongs to the Special Issue Molecular Pathogenesis of Neurodegenerative Diseases Targeting Diagnosis and Treatment)

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14 pages, 1961 KiB

Open AccessArticle

Bioengineered System for High Throughput Screening of Kv1 Ion Channel Blockers

by George V. Sharonov, Oksana V. Nekrasova, Ksenia S. Kudryashova, Mikhail P. Kirpichnikov and Alexey V. Feofanov

Bioengineering 2021, 8(11), 187; https://doi.org/10.3390/bioengineering8110187 - 16 Nov 2021

Cited by 2 | Viewed by 3455

Abstract

Screening drug candidates for their affinity and selectivity for a certain binding site is a crucial step in developing targeted therapy. Here, we created a screening assay for receptor binding that can be easily scaled up and automated for the high throughput screening of Kv channel blockers. It is based on the expression of the KcsA-Kv1 hybrid channel tagged with a fluorescent protein in the E. coli membrane. In order to make this channel accessible for the soluble compounds, E. coli were transformed into spheroplasts by disruption of the cellular peptidoglycan envelope. The assay was evaluated using a hybrid KcsA-Kv1.3 potassium channel tagged with a red fluorescent protein (TagRFP). The binding of Kv1.3 channel blockers was measured by flow cytometry either by using their fluorescent conjugates or by determining the ability of unconjugated compounds to displace fluorescently labeled blockers with a known affinity. A fraction of the occupied receptor was calculated with a dedicated pipeline available as a Jupyter notebook. Measured binding constants for agitoxin-2, charybdotoxin and kaliotoxin were in firm agreement with the earlier published data. By using a mid-range flow cytometer with manual sample handling, we measured and analyzed up to ten titration curves (eight data points each) in one day. Finally, we considered possibilities for multiplexing, scaling and automation of the assay. Full article

(This article belongs to the Special Issue Biomedical Applications of Ionic Liquids and Deep Eutectic Solvents)

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1 pages, 199 KiB

Open AccessAbstract

Docking into Symmetric Binding Sites: Problems and Solutions Exemplified on the hERG Potassium Channel

by A. SCHIESARO, L. RICHTER, A. WINDISCH, E. TIMIN, S. HERING and G. F. ECKER

Sci. Pharm. 2009, 77(7), 206; https://doi.org/10.3797/scipharm.oephg.21.PO-07 - 16 Apr 2009

Viewed by 776

Abstract

The hERG potassium channel is highly related to severe cardiac side effects. With the technique of homology modelling it is possible to model the homo tetrameric subunits of the hERG channel in the closed and open state, using KcsA and KirBac1.1 as well as MthK and KvAP as templates. Usually only the S5, P, and S6 segments of one subunit are modelled, while the other three subunits are just copies of the first one. This operation results in an artificial symmetry of the hERG channel, so the binding site in the internal part of the channel is also symmetric. This symmetry renders ligand-docking into hERG channel a quite challenging task. In order to investigate the molecular basis of drug trapping/non trapping in the hERG channel we docked a series of propafenone derivatives in a hERG channel homology model of the closed state. Drug trapping was experimentally determined in voltage clamp experiments on Xenopus oocytes. Full article

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