Intimations of potassium channel structure from a complete functional map of the molecular surface of charybdotoxin

Stampe, Per; Kolmakova-Partensky, Ludmilla; Miller, Christopher

doi:10.1021/bi00168a008

Cited by 197 publications

(246 citation statements)

References 32 publications

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“…4C). These results demonstrate that synthetic IbTx-LC-biotin is a potent inhibitor of the cloned HSlo BK channel with a slower dissociation rate than charybdotoxin, as originally observed for blocking interactions of the native toxins (Candia et al, 1992;Giangiacomo et al, 1992;Stampe et al, 1994).…”

Section: Electrophysiological Assays Of Bk Channel Block By Ibtx-biotinsupporting

confidence: 70%

Synthesis of a Biotin Derivative of Iberiotoxin: Binding Interactions with Streptavidin and the BK Ca²⁺-Activated K⁺ Channel Expressed in a Human Cell Line

et al. 2006

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Iberiotoxin (IbTx) is a scorpion venom peptide that inhibits BK Ca 2+ -activated K + channels with high affinity and specificity. Automated solid phase synthesis was used to prepare a biotin-labeled derivative (IbTx-LC-biotin) of IbTx by substitution of Asp19 of the native 37-residue peptide with N-ε-(d-biotin-6-amidocaproate)-L-lysine. Both IbTx-LC-biotin and its complex with streptavidin (StrAv) block single BK channels from rat skeletal muscle with nanomolar affinity, indicating that the biotin-labeled residue, either alone or in complex with StrAv, does not obstruct the toxin binding interaction with the BK channel. IbTx-LC-biotin exhibits high affinity (K D = 26 nM) and a slow dissociation rate (k off = 5.4 × 10 -4 s -1 ) in a macroscopic blocking assay of whole-cell current of the cloned human BK channel. Titration of IbTx-LC-biotin with StrAv monitored by high performance size exclusion chromatography is consistent with a stoichiometry of two binding sites for IbTx-LCbiotin per StrAv tetramer, indicating that steric interference hinders simultaneous binding of two toxin molecules on each of the two biotin-binding faces of StrAv. In combination with fluorescent conjugates of StrAv or anti-biotin antibody, IbTx-LC-biotin was used to image the surface distribution of BK channels on a transfected cell line. Fluorescence microscopy revealed a patchlike surface distribution of BK channel protein. The results support the feasibility of using IbTx-LCbiotin and similar biotin-tagged K + channel toxins for diverse applications in cellular neurobiology.

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Section: Electrophysiological Assays Of Bk Channel Block By Ibtx-biotinsupporting

confidence: 70%

Synthesis of a Biotin Derivative of Iberiotoxin: Binding Interactions with Streptavidin and the BK Ca²⁺-Activated K⁺ Channel Expressed in a Human Cell Line

et al. 2006

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“…Tyr-7, Lys-8, Leu-9, and to a lesser extent His-11 therefore seem likely to directly interact with the transporter, whereas Gly-6 probably plays a structural role to allow the correct orientation of the other residues in the loop for better NET binding. The involvement of tyrosine and lysine residues in the high affinity interaction of other peptide toxins with their targets has been reported previously (48,49), warranting further investigation into -MrIA's use of these residues as high affinity binding determinants in experiments with additional analogs. Phenylalanine was found to be able to largely substitute for Tyr-7, indicating that the hydroxyl group of the tyrosine residue is not of critical importance for binding.…”

Section: Discussionmentioning

confidence: 72%

“…8) reveals a highly exposed Lys-8 flanked by three hydrophobic residues to form the pharmacophore. Given its exposed position, it is possible that Lys-8 could direct the binding into a pore, perhaps reminiscent of how toxins such as charybdotoxin block the movement of K ϩ ions through voltage-dependent channels (49). If this is indeed correct as suggested by Bryan-Lluka et al (43), -conopeptides might be useful molecular calipers to probe the size of the norepinephrine permeation pathway.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of the Norepinephrine Transporter by the Venom Peptide χ-MrIA

Sharpe

Palant²,

Schroeder

et al. 2003

Journal of Biological Chemistry

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-Conopeptide MrIA (-MrIA) is a 13-residue peptide contained in the venom of the predatory marine snail Conus marmoreus that has been found to inhibit the norepinephrine transporter (NET). We investigated whether -MrIA targeted the other members of the monoamine transporter family and found no effect of the peptide (100 M) on the activity of the dopamine transporter and the serotonin transporter, indicating a high specificity of action. The binding of the NET inhibitors, [ Because of its poor lipid solubility and degree of ionization at physiological pH, norepinephrine crosses cell membranes poorly by diffusion (1) and so relies on the operation of the norepinephrine transporter (NET) 1 for uptake into cells. Clearance by this integral membrane protein constitutes the major mechanism for the termination of action of this neurotransmitter at noradrenergic synapses (2), and disturbances in the functioning of the NET are associated with pathological states including depression (3), congestive heart failure (4), and orthostatic intolerance, and tachycardia (5). Known inhibitors of the NET include antidepressants (e.g. desipramine and nisoxetine), the appetite suppressant mazindol, and the abused drug cocaine (for review, see Ref. 6). The NET, together with the dopamine transporter (DAT) and the serotonin transporter (SERT), forms a family of Na ϩ -and Cl

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“…6A, these potassium channel toxins isolated from the venoms of snake, scorpion, spider, sea anemone, and cone snail have distinctly different three-dimensional structures. However, these toxins possess a diad, consisting of a lysine and a hydrophobic residue (mostly Phe or Tyr), that is fully exposed from a flat surface and plays a critical role in their interaction with voltage-gated K ϩ channels (31,33,34). Despite their differences in the protein folding, the conserved diads are superimposable, and the average distance between the lysine ␣-carbon and the center of the aromatic ring of the hydrophobic residue was ϳ6.52 Ϯ 0.62 Å (Fig.…”

Section: Discussionmentioning

confidence: 99%

κ-Hefutoxin1, a Novel Toxin from the ScorpionHeterometrus fulvipes with Unique Structure and Function

Srinivasan¹,

Vaithiyalingam²,

Huys³

et al. 2002

Journal of Biological Chemistry

131

112

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An important and exciting challenge in the postgenomic era is to understand the functions of newly discovered proteins based on their structures. The main thrust is to find the common structural motifs that contribute to specific functions. Using this premise, here we report the purification, solution NMR, and functional characterization of a novel class of weak potassium channel toxins from the venom of the scorpion Heterometrus fulvipes. These toxins, -hefutoxin1 and -hefutoxin2, exhibit no homology to any known toxins. NMR studies indicate that -hefutoxin1 adopts a unique three-dimensional fold of two parallel helices linked by two disulfide bridges without any ␤؊sheets. Based on the presence of the functional diad (Tyr 5 /Lys 19 ) at a distance (6.0 ؎ 1.0 Å) comparable with other potassium channel toxins, we hypothesized its function as a potassium channel toxin. -Hefutoxin 1 not only blocks the voltage-gated K ؉ -channels, Kv1.3 and Kv1.2, but also slows the activation kinetics of Kv1.3 currents, a novel feature of -hefutoxin 1, unlike other scorpion toxins, which are considered solely pore blockers. Alanine mutants (Y5A, K19A, and Y5A/K19A) failed to block the channels, indicating the importance of the functional diad.

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Intimations of potassium channel structure from a complete functional map of the molecular surface of charybdotoxin

Cited by 197 publications

References 32 publications

Synthesis of a Biotin Derivative of Iberiotoxin: Binding Interactions with Streptavidin and the BK Ca²⁺-Activated K⁺ Channel Expressed in a Human Cell Line

Synthesis of a Biotin Derivative of Iberiotoxin: Binding Interactions with Streptavidin and the BK Ca²⁺-Activated K⁺ Channel Expressed in a Human Cell Line

Inhibition of the Norepinephrine Transporter by the Venom Peptide χ-MrIA

κ-Hefutoxin1, a Novel Toxin from the ScorpionHeterometrus fulvipes with Unique Structure and Function

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Intimations of potassium channel structure from a complete functional map of the molecular surface of charybdotoxin

Cited by 197 publications

References 32 publications

Synthesis of a Biotin Derivative of Iberiotoxin: Binding Interactions with Streptavidin and the BK Ca2+-Activated K+ Channel Expressed in a Human Cell Line

Synthesis of a Biotin Derivative of Iberiotoxin: Binding Interactions with Streptavidin and the BK Ca2+-Activated K+ Channel Expressed in a Human Cell Line

Inhibition of the Norepinephrine Transporter by the Venom Peptide χ-MrIA

κ-Hefutoxin1, a Novel Toxin from the ScorpionHeterometrus fulvipes with Unique Structure and Function

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Product

Resources

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Synthesis of a Biotin Derivative of Iberiotoxin: Binding Interactions with Streptavidin and the BK Ca²⁺-Activated K⁺ Channel Expressed in a Human Cell Line

Synthesis of a Biotin Derivative of Iberiotoxin: Binding Interactions with Streptavidin and the BK Ca²⁺-Activated K⁺ Channel Expressed in a Human Cell Line