Generating a High Affinity Scorpion Toxin Receptor in KcsA-Kv1.3 Chimeric Potassium Channels

Legros, Christian; Pollmann, Verena; Knaus, Hans−Günther; Farrell, Angela; Darbon, Hervé; Bougis, Pierre E.; Martin‐Eauclaire, Marie‐France; Pongs, Olaf

doi:10.1074/jbc.275.22.16918

Cited by 70 publications

(77 citation statements)

References 29 publications

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“…Although KcsA was an orphan receptor without known ligands, we judged it feasible to isolate peptides that bound with high affinity from the SAK1 library because mutation of KcsA had previously allowed pore binding of α-KTx scorpion toxins, demonstrating its conserved structural relationship with eukaryotic K + channels (8,9). KcsA channels were synthesized as before (16) and used as the target for library screening after adherence in plastic wells.…”

Section: Resultsmentioning

confidence: 99%

“…The 3D structure of KcsA confirmed explanations for selective ion permeation and conduction pathway gating deduced in the period before crystallization and its continued interrogation has been key to delineating the mechanistic bases for channel function (4)(5)(6). Although described 20 y ago (7), KcsA remains an orphan target so that studies with peptide toxins have required production of mutant channels with multiple mutations in the pore domain (8) or chimeras such as Kv1.3-KcsA, where the entire KcsA pore domain is replaced by the one in Kv1.3 (9).…”

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confidence: 99%

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Designer and natural peptide toxin blockers of the KcsA potassium channel identified by phage display

Zhao

Dai

Mendelman

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Peptide neurotoxins are powerful tools for research, diagnosis, and treatment of disease. Limiting broader use, most receptors lack an identified toxin that binds with high affinity and specificity. This paper describes isolation of toxins for one such orphan target, KcsA, a potassium channel that has been fundamental to delineating the structural basis for ion channel function. A phage-display strategy is presented whereby ∼1.5 million novel and natural peptides are fabricated on the scaffold present in ShK, a sea anemone type I (SAK1) toxin stabilized by three disulfide bonds. We describe two toxins selected by sorting on purified KcsA, one novel (Hui1, 34 residues) and one natural (HmK, 35 residues). Hui1 is potent, blocking single KcsA channels in planar lipid bilayers half-maximally (K i ) at 1 nM. Hui1 is also specific, inhibiting KcsA-Shaker channels in Xenopus oocytes with a K i of 0.5 nM whereas Shaker, Kv1.2, and Kv1.3 channels are blocked over 200-fold less well. HmK is potent but promiscuous, blocking KcsA-Shaker, Shaker, Kv1.2, and Kv1.3 channels with K i of 1-4 nM. As anticipated, one Hui1 blocks the KcsA pore and two conserved toxin residues, Lys 21 and Tyr 22 , are essential for highaffinity binding. Unexpectedly, potassium ions traversing the channel from the inside confer voltage sensitivity to the Hui1 off-rate via Arg 23 , indicating that Lys 21 is not in the pore. The 3D structure of Hui1 reveals a SAK1 fold, rationalizes KcsA inhibition, and validates the scaffold-based approach for isolation of highaffinity toxins for orphan receptors.Hui1 toxin | ShK toxin | HmK toxin | sea anemone | NMR

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Designer and natural peptide toxin blockers of the KcsA potassium channel identified by phage display

Zhao

Dai

Mendelman

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…The ability of maurotoxin to inhibit 125 I-apamin binding to SK channels (current study) and 125 I-kaliotoxin binding to Kv channels (Kharrat et al, 1996 indicates that the toxin binds to or near the external vestibule of the pore because amino acid residues critical for the binding of apamin and kaliotoxin and other peptide toxins are found in this region of the channel (Aiyar et al, 1996;Ishii et al, 1997a;Legros et al, 2000;Rauer et al, 2000;Shakkottai et al, 2001). Maurotoxin's potent inhibition of both IK1 and Kv1.2 potassium channels suggests that the external vestibules around the pore in both channels exhibit structural similarities.…”

Section: Discussionmentioning

confidence: 99%

Maurotoxin: A Potent Inhibitor of Intermediate Conductance Ca²⁺-Activated Potassium Channels

Castle¹,

London²,

Creech³

et al. 2003

Mol Pharmacol

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Maurotoxin, a 34-amino acid toxin from Scorpio maurus scorpion venom, was examined for its ability to inhibit cloned human SK (SK1, SK2, and SK3), IK1, and Slo1 calcium-activated potassium (K Ca ) channels. Maurotoxin was found to produce a potent inhibition of Ca 2ϩ -activated 86 Rb efflux (IC 50 , 1.4 nM) and inwardly rectifying potassium currents (IC 50 , 1 nM) in CHO cells stably expressing IK1. In contrast, maurotoxin produced no inhibition of SK1, SK2, and SK3 small-conductance or Slo1 large-conductance K Ca channels at up to 1 M in physiologically relevant ionic strength buffers. Maurotoxin did inhibit 86 Rb efflux (IC 50 , 45 nM) through, and 125

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“…In contrast, R-AgTx2 binding to KcsA-Kv1.1 has a maximum at ϳ4 mM KCl. Low concentrations of potassium ions enhance ligand binding to both natural and artificial Kv1 channels (36,41,42), whereas high KCl concentrations produce a trivial effect of ionic strength. Our experiments revealed that sucrose (up to 0.25 M) and BSA (0.1%) do not affect interactions between R-AgTx2 and KcsA-Kv1.1 (data not shown).…”

Section: P-loop (mentioning

confidence: 99%

Variability of Potassium Channel Blockers in Mesobuthus eupeus Scorpion Venom with Focus on Kv1.1

Kuzmenkov

Vassilevski

Kudryashova

et al. 2015

Journal of Biological Chemistry

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Background: Scorpion venoms are an ample source of toxins targeting potassium channels. Results: A comprehensive search for new toxins was performed by combining transcriptomics and peptidomics with a fluorescent test system. Conclusion: We identified five new high affinity potassium channel blockers in the venom of Mesobuthus eupeus. Significance: The proposed integrated approach is of general utility for potassium channel pharmacology.

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Generating a High Affinity Scorpion Toxin Receptor in KcsA-Kv1.3 Chimeric Potassium Channels

Cited by 70 publications

References 29 publications

Designer and natural peptide toxin blockers of the KcsA potassium channel identified by phage display

Designer and natural peptide toxin blockers of the KcsA potassium channel identified by phage display

Maurotoxin: A Potent Inhibitor of Intermediate Conductance Ca²⁺-Activated Potassium Channels

Variability of Potassium Channel Blockers in Mesobuthus eupeus Scorpion Venom with Focus on Kv1.1

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Generating a High Affinity Scorpion Toxin Receptor in KcsA-Kv1.3 Chimeric Potassium Channels

Cited by 70 publications

References 29 publications

Designer and natural peptide toxin blockers of the KcsA potassium channel identified by phage display

Designer and natural peptide toxin blockers of the KcsA potassium channel identified by phage display

Maurotoxin: A Potent Inhibitor of Intermediate Conductance Ca2+-Activated Potassium Channels

Variability of Potassium Channel Blockers in Mesobuthus eupeus Scorpion Venom with Focus on Kv1.1

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Maurotoxin: A Potent Inhibitor of Intermediate Conductance Ca²⁺-Activated Potassium Channels