Kv1 Potassium Channel Ligands Based on Hongotoxin 1 and Red Fluorescent Protein

Primak, Alexandra; Skutel, M. A.; Nekrasova, Oksana V.; Arseniev, Alexander S.; Kirpichnikov, Mikhaǐl P.; Feofanov, Аlexey V.

doi:10.1134/s1068162020060266

Cited by 3 publications

(5 citation statements)

References 18 publications

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“…Data on the properties of AgTx2-GFP and previously studied FP-Txs [ 7 , 45 , 46 , 47 ] have shown that FP-Txs can be considered as a reliable alternative to peptide blockers labeled with organic fluorophores for the fluorescent imaging of K v 1 channels, as well as for a number of analytical applications. FPs can modulate the pharmacological profiles of peptide blockers of K v 1 channels, maintaining a high (nanomolar) affinity to some of the target channels or even enhancing considerably the ligand selectivity for a particular channel.…”

Section: Discussionmentioning

confidence: 99%

“…( a ) Comparison of dissociation constants ( K d , mean ± SEM) of KcsA-K v 1.x (x = 1, 3) complexes with AgTx2-GFP and with previously studied FP-tagged pore blockers. * [ 7 ], # [ 47 ], & [ 46 ], ^ [ 45 ]. ( b ) Displacement of AgTx2-GFP (5 nM) from complexes with KcsA-K v 1.3 (control) by kaliotoxin 1 (KTx1), AgTx2, and the components of crude venoms of Mesobuthus eupeus and Orthochirus scrobiculosus scorpions.…”

Section: Figurementioning

confidence: 99%

“…An alternative strategy to create fluorescently labeled peptide blockers is the production of genetically encoded peptide toxins fused with fluorescent proteins (FPs) [ 7 , 45 , 46 , 47 ]. Currently, FPs used for such constructions are limited to TagRFP (hereinafter RFP) [ 48 ] and enhanced GFP [ 49 ], while a list of peptide blockers includes MgTx, OSK1, hongotoxin 1, and AgTx2.…”

Section: Introductionmentioning

confidence: 99%

“…The relative affinities of FP-Txs to target channels may vary compared to those of a native toxin. Indeed, the affinities of FP-Txs to K v 1 channels are lower than those of free toxins, but the extent of affinity reduction varies in a complicated way for particular combinations of FPs and toxins [ 7 , 45 , 46 , 47 ], for different FPs fused with the same toxin [ 7 , 46 ], and for different modes of tagging (N- or C-terminal) [ 46 , 47 ]. For example, N-terminal tagging of AgTx2 with RFP retained, in general, the relative affinities to the K v 1.1, K v 1.3, and K v 1.6 channels but reduced moderately the relative affinity toward the K v 1.2 channel [ 7 ].…”

Section: Introductionmentioning

confidence: 99%

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AgTx2-GFP, Fluorescent Blocker Targeting Pharmacologically Important Kv1.x (x = 1, 3, 6) Channels

Primak

Orlov

Peigneur

et al. 2023

Toxins

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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 Kv1.x (x = 1, 3, 6) channels. AgTx2-GFP possesses subnanomolar affinities for hybrid KcsA-Kv1.x (x = 3, 6) channels and a low nanomolar affinity to KcsA-Kv1.1 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 Kv1.x (x = 1, 3, 6) channels and at micromolar concentrations for Kv1.2. AgTx2-GFP bound to Kv1.3 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-Kv1.x (x = 1, 3, 6) channels on the membranes of E. coli spheroplasts or with Kv1.3 channels on the membranes of mammalian cells for the search and study of nonlabeled peptide pore blockers, including measurement of their affinity.

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Section: Discussionmentioning

confidence: 99%

Section: Figurementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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AgTx2-GFP, Fluorescent Blocker Targeting Pharmacologically Important Kv1.x (x = 1, 3, 6) Channels

Primak

Orlov

Peigneur

et al. 2023

Toxins

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“…Notably, AgTx-2 with GFP at its N-terminus exhibits high specificity for chimeric K V 1.3 channels over K V 1.1 and K V 1.6 in spheroplasts, and KcsA-K V 1.3 transfected HEK293 cells [ 62 ]. Similar studies with the fusion of HgTx with Tag-RFP showed maintained specificity and utility for targeting KcsA-K V 1.1 [ 63 ] as well as both KcsA-K V 1.1 and KcsA-K V 1.3 [ 64 ]. Finally, GFP-MgTx fusion protein revealed specific labelling of the K V 1.3 subunits of the KcsA-K V 1.3 hybrid channels expressed in E. coli spheroplasts, whilst control experiments with non-transfected material showed no fluorescence signal [ 65 ].…”

Section: Optical Probes Targeting Potassium Channelsmentioning

confidence: 90%

Neurotoxin-Derived Optical Probes for Biological and Medical Imaging

Ergen,

Shorter,

Ntziachristos

et al. 2023

Mol Imaging Biol

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The superb specificity and potency of biological toxins targeting various ion channels and receptors are of major interest for the delivery of therapeutics to distinct cell types and subcellular compartments. Fused with reporter proteins or labelled with fluorophores and nanocomposites, animal toxins and their detoxified variants also offer expanding opportunities for visualisation of a range of molecular processes and functions in preclinical models, as well as clinical studies. This article presents state-of-the-art optical probes derived from neurotoxins targeting ion channels, with discussions of their applications in basic and translational biomedical research. It describes the design and production of probes and reviews their applications with advantages and limitations, with prospects for future improvements. Given the advances in imaging tools and expanding research areas benefiting from the use of optical probes, described here resources should assist the discovery process and facilitate high-precision interrogation and therapeutic interventions.

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