Charybdotoxin and Margatoxin Acting on the Human Voltage-Gated Potassium Channel <i>h</i>K<sub>v</sub>1.3 and Its H399N Mutant: An Experimental and Computational Comparison

Nikouee, Azadeh; Khabiri, Morteza; Grissmer, Stephan; Ettrich, Rüdiger

doi:10.1021/jp2102463

Cited by 13 publications

(6 citation statements)

References 63 publications

(93 reference statements)

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“…We in fact confirmed this beneficial effect for Apamin (500 nM) and XE991 (10 lM), which reduced the aggregate burden measured in single neurons by microscopy (Appendix Fig S8C ) and decreased the levels of activated caspase 3 (Appendix Fig S8D). Since Charybdotoxin has a broader selectivity (Garcia et al, 1995;Nikouee et al, 2012), it was not investigated further. The anti-synaptotagmin antibody feeding assay, aimed at quantifying pre-synaptic vesicle release (Catanese et al, 2018), confirmed that both Apamin and XE991 increased neuronal firing in treated cultures (Appendix Fig S8E).…”

Section: Resultsmentioning

confidence: 99%

Synaptic disruption and CREB‐regulated transcription are restored by K ⁺ channel blockers in ALS

et al. 2021

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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, which is still missing effective therapeutic strategies. Although manipulation of neuronal excitability has been tested in murine and human ALS models, it is still under debate whether neuronal activity might represent a valid target for efficient therapies. In this study, we exploited a combination of transcriptomics, proteomics, optogenetics and pharmacological approaches to investigate the activity-related pathological features of iPSC-derived C9orf72-mutant motoneurons (MN). We found that human ALS C9orf72 MN are characterized by accumulation of aberrant aggresomes, reduced expression of synaptic genes, loss of synaptic contacts and a dynamic "malactivation" of the transcription factor CREB. A similar phenotype was also found in TBK1-mutant MN and upon overexpression of poly(GA) aggregates in primary neurons, indicating a strong convergence of pathological phenotypes on synaptic dysregulation. Notably, these alterations, along with neuronal survival, could be rescued by treating ALS-related neurons with the K + channel blockers Apamin and XE991, which, respectively, target the SK and the Kv7 channels. Thus, our study shows that restoring the activitydependent transcriptional programme and synaptic composition exerts a neuroprotective effect on ALS disease progression.

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Section: Resultsmentioning

confidence: 99%

Synaptic disruption and CREB‐regulated transcription are restored by K ⁺ channel blockers in ALS

et al. 2021

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“…Recent docking studies predicted that residue K28 in MgTx, which is conserved in α-KTx scorpion toxins, inserts into the pore of the K V 1.3 channel and makes contact with the channel's selectivity filter [50]. In support of this prediction, we found that mutation of MgTx residue K28 to alanine completely abolished the ability of the toxin to inhibit mK V 1.1 and reduced its activity on mK V 1.3 by more than 2000-fold.…”

Section: Discussionmentioning

confidence: 99%

Recombinant Expression of Margatoxin and Agitoxin-2 in Pichia pastoris: An Efficient Method for Production of KV1.3 Channel Blockers

et al. 2012

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The Kv1.3 voltage-gated potassium channel regulates membrane potential and calcium signaling in human effector memory T cells that are key mediators of autoimmune diseases such as multiple sclerosis, type 1 diabetes, and rheumatoid arthritis. Thus, subtype-specific Kv1.3 blockers have potential for treatment of autoimmune diseases. Several Kv1.3 channel blockers have been characterized from scorpion venom, all of which have an α/β scaffold stabilized by 3–4 intramolecular disulfide bridges. Chemical synthesis is commonly used for producing these disulfide-rich peptides but this approach is time consuming and not cost effective for production of mutants, fusion proteins, fluorescently tagged toxins, or isotopically labelled peptides for NMR studies. Recombinant production of Kv1.3 blockers in the cytoplasm of E. coli generally necessitates oxidative refolding of the peptides in order to form their native disulfide architecture. An alternative approach that avoids the need for refolding is expression of peptides in the periplasm of E. coli but this often produces low yields. Thus, we developed an efficient Pichia pastoris expression system for production of Kv1.3 blockers using margatoxin (MgTx) and agitoxin-2 (AgTx2) as prototypic examples. The Pichia system enabled these toxins to be obtained in high yield (12–18 mg/L). NMR experiments revealed that the recombinant toxins adopt their native fold without the need for refolding, and electrophysiological recordings demonstrated that they are almost equipotent with the native toxins in blocking KV1.3 (IC50 values of 201±39 pM and 97±3 pM for recombinant AgTx2 and MgTx, respectively). Furthermore, both recombinant toxins inhibited T-lymphocyte proliferation. A MgTx mutant in which the key pharmacophore residue K28 was mutated to alanine was ineffective at blocking KV1.3 and it failed to inhibit T-lymphocyte proliferation. Thus, the approach described here provides an efficient method of producing toxin mutants with a view to engineering Kv1.3 blockers with therapeutic potential.

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“…In a molecular dynamics study in which His399 within the pore of hKv1.3 was mutated to Asp, the distance between interacting residues of MgTx and Kv1.3 was altered owing to changed electrostatic interactions, which reduced the strength of block (Nikouee et al . ). The Kv1.3 pore and its four voltage sensors are predicted to be 3 nm in length across the membrane (Long et al .…”

Section: Discussionmentioning

confidence: 97%

Margatoxin‐bound quantum dots as a novel inhibitor of the voltage‐gated ion channel Kv1.3

Schwartz

Kapur

Wang

et al. 2016

Journal of Neurochemistry

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Venom-derived ion channel inhibitors have strong channel selectivity, potency, and stability; however, tracking delivery to their target can be challenging. Herein, we utilized luminescent quantum dots (QDs) conjugated to margatoxin (MgTx) as a traceable vehicle to target a voltage-dependent potassium channel, Kv1.3, which has a select distribution and well characterized role in immunity, glucose metabolism, and sensory ability. We screened both unconjugated (MgTx) and conjugated MgTx (QD-MgTx) for their ability to inhibit Shaker channels Kv1.1 to Kv1.7 using patch-clamp electrophysiology in HEK293 cells. Our data indicate that MgTx inhibits 79% of the outward current in Kv1.3-transfected cells and that the QD-MgTx conjugate is able to achieve a similar level of block, albeit a slightly reduced efficacy (66%) and at a slower time course (50% block by 10.9 ± 1.1 min, MgTx; vs. 15.3 ± 1.2 min, QD-MgTx). Like the unbound peptide, the QD-MgTx conjugate inhibits both Kv1.3 and Kv1.2 at a 1 nM concentration, whereas it does not inhibit other Shaker channels screened. We tested the ability of QD-MgTx to excite native Kv1.3 expressed in mouse olfactory bulb slices and found that the conjugate had a similar ability as MgTx in enhancing action potential firing frequency attributed to decreased intraburst duration rather than interspike interval. Our data demonstrate a retention of known biophysical properties associated with block of the vestibule of Kv1.3 by QD-MgTx conjugate compared to that of MgTx, inferring QDs could provide a useful tool to deliver ion channel inhibitors to targeted tissues in vivo.

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Charybdotoxin and Margatoxin Acting on the Human Voltage-Gated Potassium Channel hK_v1.3 and Its H399N Mutant: An Experimental and Computational Comparison

Cited by 13 publications

References 63 publications

Synaptic disruption and CREB‐regulated transcription are restored by K ⁺ channel blockers in ALS

Synaptic disruption and CREB‐regulated transcription are restored by K ⁺ channel blockers in ALS

Recombinant Expression of Margatoxin and Agitoxin-2 in Pichia pastoris: An Efficient Method for Production of KV1.3 Channel Blockers

Margatoxin‐bound quantum dots as a novel inhibitor of the voltage‐gated ion channel Kv1.3

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Charybdotoxin and Margatoxin Acting on the Human Voltage-Gated Potassium Channel hKv1.3 and Its H399N Mutant: An Experimental and Computational Comparison

Cited by 13 publications

References 63 publications

Synaptic disruption and CREB‐regulated transcription are restored by K + channel blockers in ALS

Synaptic disruption and CREB‐regulated transcription are restored by K + channel blockers in ALS

Recombinant Expression of Margatoxin and Agitoxin-2 in Pichia pastoris: An Efficient Method for Production of KV1.3 Channel Blockers

Margatoxin‐bound quantum dots as a novel inhibitor of the voltage‐gated ion channel Kv1.3

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Charybdotoxin and Margatoxin Acting on the Human Voltage-Gated Potassium Channel hK_v1.3 and Its H399N Mutant: An Experimental and Computational Comparison

Synaptic disruption and CREB‐regulated transcription are restored by K ⁺ channel blockers in ALS

Synaptic disruption and CREB‐regulated transcription are restored by K ⁺ channel blockers in ALS