Molecular modeling of interactions of agitoxin 2 with Kv1.3 voltage-gated potassium channel

Volyntseva, A. D.; Novoseletsky, Valery; Шайтан, К. В.; Feofanov, Аlexey V.

doi:10.3103/s0096392517010060

Cited by 5 publications

(5 citation statements)

References 22 publications

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“…While high structural and topological homology of the outer vestibule of the pore is inherent to Kv1 channels, a turret, the most protruding part of the outer binding site of the pore domain, comprises a stretch of anionic amino acid (a.a.) residues in each of four subunits, which varies among Kv1 channels: EEAE (Kv1.1), DDPT (Kv1.3), and DDDD (Kv1.6). A negatively charged array formed at the top of the channel pore is supposed to navigate a cationic peptide blocker during diffusion-limited stage of its binding to the channel, and some of these anionic residues are involved in the formation of complexes with AgTx2 [ 17 , 23 ]. Total net charge of the Kv1.3 binding site is less negative (−16) compared to the binding sites of Kv1.1 (−20) and Kv1.6 (−24) [ 17 ].…”

Section: Discussionmentioning

confidence: 99%

“…The latter conclusion is consistent with the fact that transposition of GFP from N- to C-terminus of AgTx2 restores formation of complexes with the Kv1.1 and Kv1.6 binding sites, suggesting that changes in the spatial distribution of interacting a.a. residues of GFP, AgTx2 and a channel binding site determine the binding mode of GFP-L2-AgTx2 ligand. According to molecular modeling data an AgTx2-channel complex is stabilized by a complex network of bonds involving 10–11 channel residues and 16–18 peptide residues [ 17 , 23 ] that hamper prediction of the key interactions in AgTx2-channel complexes that are affected by GFP.…”

Section: Discussionmentioning

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: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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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“…This state is characterized by a conductive conformation of the selectivity filter and a closed activation gate of the channel, which is formed by the bundle-crossing of S6 transmembrane helices of four α-subunits [ 43 ]. The binding of the peptide blocker is accompanied by occlusion of the channel pore by the lysine side chain and the formation of multiple bonds with the residues of the P-loop connecting the transmembrane helices S5 and S6 of the channel ( Figure 2 b,c) [ 40 , 44 , 45 ]. Voltage-dependent activation leads to structural transitions in the channel, starting with the movement of S4 helices, which sense the transmembrane potential, followed by alterations in the conformation of the S6 helices and the opening of the inner activation gate.…”

Section: Resultsmentioning

confidence: 99%

Interactions of the Kv1.1 Channel with Peptide Pore Blockers: A Fluorescent Analysis on Mammalian Cells

et al. 2023

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The voltage-gated potassium channel Kv1.1, which is abundant in the CNS and peripheral nervous system, controls neuronal excitability and neuromuscular transmission and mediates a number of physiological functions in non-excitable cells. The development of some diseases is accompanied by changes in the expression level and/or activity of the channels in particular types of cells. To meet the requirements of studies related to the expression and localization of the Kv1.1 channels, we report on the subnanomolar affinity of hongotoxin 1 N-terminally labeled with Atto 488 fluorophore (A-HgTx) for the Kv1.1 channel and its applicability for fluorescent imaging of the channel in living cells. Taking into consideration the pharmacological potential of the Kv1.1 channel, a fluorescence-based analytical system was developed for the study of peptide ligands that block the ion conductivity of Kv1.1 and are potentially able to correct abnormal activity of the channel. The system is based on analysis of the competitive binding of the studied compounds and A-HgTx to the mKate2-tagged human Kv1.1 (S369T) channel, expressed in the plasma membrane of Neuro2a cells. The system was validated by measuring the affinities of the known Kv1.1-channel peptide blockers, such as agitoxin 2, kaliotoxin 1, hongotoxin 1, and margatoxin. Peptide pore blocker Ce1, from the venom of the scorpion Centruroides elegans, was shown to possess a nanomolar affinity for the Kv1.1 channel. It is reported that interactions of the Kv1.1 channel with the studied peptide blockers are not affected by the transition of the channel from the closed to open state. The conclusion is made that the structural rearrangements accompanying the channel transition into the open state do not change the conformation of the P-loop (including the selectivity filter) involved in the formation of the binding site of the peptide pore blockers.

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“…In order to estimate the possibilities and limitations of the assay, the molecular details of blocker interaction with a natural and chimeric channel should be considered. The common feature of the peptide blocker binding is an occlusion of the pore with the side chain of a lysine residue, which is accompanied by the formation of multiple bonds between the peptide and amino acid residues surrounding the pore [28,29]. Since the pore occlusion is the essential feature of any Kv1-channel blocker interacting with a channel at the extracellular side, the binding sites of peptide blockers and small organic molecule blockers overlap.…”

Section: Discussionmentioning

confidence: 99%

“…Many other residues besides lysine are involved in the interaction of peptide blockers with the Kv1 channel. For example, 16 residues of AgTx2 interact with 11 residues of KcsA-Kv1.3, according to a molecular modeling study [29]. Thus, while a secondary structure of peptide blockers from scorpion venoms, such as AgTx2, ChTx and KTx, is similar, their differences in amino acid composition change a peptide-channel interaction pattern in a complex way and define the observed variations in affinity (Table 1).…”

Section: Discussionmentioning

confidence: 99%

Bioengineered System for High Throughput Screening of Kv1 Ion Channel Blockers

et al. 2021

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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.

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Molecular modeling of interactions of agitoxin 2 with Kv1.3 voltage-gated potassium channel

Cited by 5 publications

References 22 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

Interactions of the Kv1.1 Channel with Peptide Pore Blockers: A Fluorescent Analysis on Mammalian Cells

Bioengineered System for High Throughput Screening of Kv1 Ion Channel Blockers

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