Fluorescent protein-scorpion toxin chimera is a convenient molecular tool for studies of potassium channels

Kuzmenkov, Alexey I.; Nekrasova, Oksana V.; Kudryashova, Kseniya S.; Peigneur, Steve; Tytgat, Jan; Stepanov, Alexey V.; Kirpichnikov, Mikhaǐl P.; Grishin, Eugene V.; Feofanov, Аlexey V.; Vassilevski, Alexander A.

doi:10.1038/srep33314

Cited by 32 publications

(53 citation statements)

References 42 publications

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“…The apparent dissociation constants K ap calculated from the titration curves ( Figure 3E,F) using Equation (2) are presented in Table 1 and demonstrate good similarity for two sets of experiments performed with different fluorescent ligands. Moreover, the calculated K ap values (Table 1) are consistent with those reported previously for corresponding peptide blockers, when the competitive binding experiments were performed with R-AgTx2, RFP-L1-AgTx2 or GFP-OSK1 [12,15,18]. These results indicate that both GFP-L2-AgTx2 and His6-GFP-L2-AgTx2 can be successfully used as a component of the KcsA-Kv1.3-based bioengineering system to estimate affinities of different peptide blockers to Kv1.3 binding site.…”

Section: Gfp-tagged Agtx2 As a Component Of The Kcsa-kv13-based Bioesupporting

confidence: 89%

“…KcsA-Kv1.x (x = 1, 3, 6) hybrid channels bear extracellular binding sites of the corresponding eukaryotic Kv1 channels and provide both recognition of Kv1 channels pore blockers and evaluation of their affinity [15][16][17][18][19]. As systematically verified, data obtained with KcsA-Kv1.x (x = 1, 3, 6) for small organic compounds, peptide toxins, as well as fluorescent protein-tagged pore blockers are in agreement with the results of electrophysiological studies [12,16].…”

Section: Design and Properties Of Gfp-l2-agtx2supporting

confidence: 81%

“…Primers used in this study. To construct pET23-GFP-L1-AgTx2 ( Figure 1A), a gene coding for RFP in the previously obtained plasmid pET23-RFP-L1-AgTx2 [12] was replaced by the GFP coding sequence using NcoI/EcoRI sites. For this, the GFP gene was PCR-amplified from pUC-eGFP plasmid using oligonucleotide primers GFP-f1 and GFP-r1.…”

Section: Gene Design Synthesis and Cloningmentioning

confidence: 99%

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N-Terminal Tagging with GFP Enhances Selectivity of Agitoxin 2 to Kv1.3-Channel Binding Site

Nekrasova

Primak

Ignatova

et al. 2020

Toxins

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Recently developed fluorescent protein-scorpion toxin chimeras (FP-Tx) show blocking activities for potassium voltage-gated channels of Kv1 family and retain almost fully pharmacological profiles of the parental peptide toxins (Kuzmenkov et al., Sci Rep. 2016, 6, 33314). Here we report on N-terminally green fluorescent protein (GFP)-tagged agitoxin 2 (GFP-L2-AgTx2) with high affinity and selectivity for the binding site of Kv1.3 channel involved in the pathogenesis of various (primarily of autoimmune origin) diseases. The basis for this selectivity relates to N-terminal location of GFP, since transposition of GFP to the C-terminus of AgTx2 recovered specific interactions with the Kv1.1 and Kv1.6 binding sites. Competitive binding experiments revealed that the binding site of GFP-L2-AgTx2 overlaps that of charybdotoxin, kaliotoxin 1, and agitoxin 2, the known Kv1.3-channel pore blockers. GFP-L2-AgTx2 was demonstrated to be applicable as a fluorescent probe to search for Kv1.3 pore blockers among individual compounds and in complex mixtures, to measure blocker affinities, and to visualize Kv1.3 distribution at the plasma membrane of Kv1.3-expressing HEK293 cells. Our studies show that definite combinations of fluorescent proteins and peptide blockers can result in considerable modulation of the natural blocker-channel binding profile yielding selective fluorescent ligands of certain channels.

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Section: Gfp-tagged Agtx2 As a Component Of The Kcsa-kv13-based Bioesupporting

confidence: 89%

Section: Design and Properties Of Gfp-l2-agtx2supporting

confidence: 81%

Section: Gene Design Synthesis and Cloningmentioning

confidence: 99%

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N-Terminal Tagging with GFP Enhances Selectivity of Agitoxin 2 to Kv1.3-Channel Binding Site

Nekrasova

Primak

Ignatova

et al. 2020

Toxins

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“…Very similar in structure, a component of deathstalker scorpion venom chlorotoxin has high affinity for chloride channels (DeBin et al, 1993). Many other scorpion venom components are used to study channels and receptors and can also be produced as recombinant fluorescent proteins to be used in microscopy (Kuzmenkov et al, 2016). While these toxins provide very high affinity and specificity, working in nano- and picomolar concentrations and being able to distinguish between very similar classes of receptors, their small size often makes it difficult and expensive to label them with fluorescent reporters, thus limiting their use.…”

Section: Methodsmentioning

confidence: 99%

Interrogating Synaptic Architecture: Approaches for Labeling Organelles and Cytoskeleton Components

Reshetniak

Rizzoli

2019

Front. Synaptic Neurosci.

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Synaptic transmission has been studied for decades, as a fundamental step in brain function. The structure of the synapse, and its changes during activity, turned out to be key aspects not only in the transfer of information between neurons, but also in cognitive processes such as learning and memory. The overall synaptic morphology has traditionally been studied by electron microscopy, which enables the visualization of synaptic structure in great detail. The changes in the organization of easily identified structures, such as the presynaptic active zone, or the postsynaptic density, are optimally studied via electron microscopy. However, few reliable methods are available for labeling individual organelles or protein complexes in electron microscopy. For such targets one typically relies either on combination of electron and fluorescence microscopy, or on super-resolution fluorescence microscopy. This review focuses on approaches and techniques used to specifically reveal synaptic organelles and protein complexes, such as cytoskeletal assemblies. We place the strongest emphasis on methods detecting the targets of interest by affinity binding, and we discuss the advantages and limitations of each method.

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“…C and F) Representative traces of early NaV1.6 channel opening caused by Cn2 elicited by pulses to -40 mV. Fluorescently labelled Cn2 as a NaV1.6 specific probeFluorescently labelled probes have proved useful as tools for investigating receptorligand interactions(233,249). To assess whether the fluorescent Cn2 probe had maintained selectivity for NaV1.6, and to compare their performance to a commercially available polyclonal antibody directed against NaV1.6, immunohistochemistry of HEK293 cells with stable NaV expression was performed.…”

mentioning

confidence: 99%

Elucidating the role of NaV1.6 in peripheral sensory neurons

Israel¹

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Chronic pain is estimated to affect 1 in 5 Australians. The condition is debilitating for patients and remains a costly burden on the healthcare system. Analgesics currently in clinical use lack efficacy, have low tolerability and are hampered with wide side-effect profiles. Therefore, the underlying molecular mechanisms of pain sensing must be elucidated with a view to the development of improved therapeutics. Peripheral sensory neurons are the site of pain signal initiation and are critical for the detection of painful stimuli. Neuronal excitability relies on integral membrane bound proteins such as voltage-gated sodium channels (NaV), which allow the influx of sodium and action potential propagation in these cells. Nine different NaV subtypes have been identified (NaV1.1-1.9) of which a subset are expressed in peripheral sensory neurons. The exact role that each NaV isoform plays in the excitability of different fibre types remains unclear. This thesis uses a multidisciplinary approach to assess the role of the NaV subtype NaV1.6 in peripheral sensory neurons.NaV1.6 channels share high sequence homology with other NaV isoforms expressed in peripheral sensory neurons. Scorpion venom is a rich source of bioactive compounds, many of which act on mammalian NaV channels. One such peptide, Cn2, is reported to be a selective NaV1.6 activator. Chapter 2 describes the chemical synthesis of Cn2 using a strategy of native chemical ligation. The pharmacology of the synthetically derived Cn2 is in line with that of the venom derived peptide. This approach allows the development of a number of probes, including a novel NaV1.6 antagonist Cn2[E15R], that retain selectivity for NaV1.6. Therefore, this chapter characterises novel NaV1.6 selective compounds and provides insight into NaV1.6-toxin interactions.Peripheral sensory neurons express a range of different ion channels that are critical for their function. Chapter 3 aimed to assess the contribution of NaV1.6 to Na + currents in mouse isolated dorsal root ganglion cells. High-content calcium imaging and whole-cell patch clamp electrophysiology was performed using the selective Cn2 peptide as a probe for NaV1.6 function. Corresponding to expression of NaV1.6, Cn2 induces early Na + currents in large but not small diameter DRG neurons. The Cn2 effect is absent in NaV1.6 -/neurons confirming the selectivity of the peptide in a neuronal environment. Furthermore, Cn2 enhances excitability of large diameter neurons in vitro.

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Fluorescent protein-scorpion toxin chimera is a convenient molecular tool for studies of potassium channels

Cited by 32 publications

References 42 publications

N-Terminal Tagging with GFP Enhances Selectivity of Agitoxin 2 to Kv1.3-Channel Binding Site

N-Terminal Tagging with GFP Enhances Selectivity of Agitoxin 2 to Kv1.3-Channel Binding Site

Interrogating Synaptic Architecture: Approaches for Labeling Organelles and Cytoskeleton Components

Elucidating the role of NaV1.6 in peripheral sensory neurons

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