Identification of Three Separate Binding Sites on SHK Toxin, a Potent Inhibitor of Voltage-Dependent Potassium Channels in Human T-Lymphocytes and Rat Brain

Pennington, Michael W.; Mahnir, Vladimir M.; Krafte, Douglas S.; Zaydenberg, I.; Byrnes, M.; Khaytin, Ilya; Crowley, Kelsey; Kem, William R.

doi:10.1006/bbrc.1996.0297

Cited by 66 publications

(76 citation statements)

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“…It has been demonstrated that besides the functional dyad other determinants are involved in the interaction between pore blocker toxins and K V channels [27]. For instance, despite the existence of a dyad (Lys22-Tyr23) in ShK, other residues (Ile7, Lys9, Arg11, Ser20 and Phe27) that are clustered around it interact with residues located in the vicinity of the selectivity filter and turret region of the K V channels [28,29]. The amino acids Arg3, Trp5, Phe6, Lys7, His13, Ser23, Lys25, Tyr26 and Arg27 play a similar binding role in BgK [25,30,31].…”

Section: Discussionmentioning

confidence: 99%

BcsTx3 is a founder of a novel sea anemone toxin family of potassium channel blocker

et al. 2013

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Sea anemone venoms have become a rich source of peptide toxins which are invaluable tools for studying the structure and functions of ion channels. In this work, BcsTx3, a toxin found in the venom of a Bunodosoma caissarum (population captured at the Saint Peter and Saint Paul Archipelago, Brazil) was purified and biochemically and pharmacologically characterized. The pharmacological effects were studied on 12 different subtypes of voltage-gated potassium channels (K V 1.1-K V 1.6; K V 2.1; K V 3.1; K V 4.2; K V 4.3; hERG and Shaker IR) and three cloned voltagegated sodium channel isoforms (Na V 1.2, Na V 1.4 and BgNa V 1.1) expressed in Xenopus laevis oocytes. BcsTx3 shows a high affinity for Drosophila Shaker IR channels over rKv1.2, hKv1.3 and rKv1.6, and is not active on Na V channels. Biochemical characterization reveals that BcsTx3 is a 50 amino acid peptide crosslinked by four disulfide bridges, and sequence comparison allowed BcsTx3 to be classified as a novel type of sea anemone toxin acting on K V channels. Moreover, putative toxins homologous to BcsTx3 from two additional actiniarian species suggest an ancient origin of this newly discovered toxin family.

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Section: Discussionmentioning

confidence: 99%

BcsTx3 is a founder of a novel sea anemone toxin family of potassium channel blocker

et al. 2013

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“…of 3-8 experiments. Experimental points were fitted to the theoretical curve y ϭ 1/(1 ϩ ( [T] n / IC 50 ) n ), in which y is the fraction of unblocked current, [T] is the concentration of toxin, IC 50 is the concentration of toxin inducing 50% of blockade, and n is the Hill coefficient corresponding to the number of molecules of toxin required to block one channel. For each mutant, IC 50 was determined without constraint on n. When n Ϸ 1, as previously determined for the BgK blockade of Kv1.1, Kv1.2, and Kv1.3 (30), IC 50 gave the apparent dissociation constant K D .…”

Section: Data Collection and Analysismentioning

confidence: 99%

“…The binding site of ShK comprises the three basic residues Lys-9, Arg-11, and Lys-22 and probably Tyr-23 (50), that of KTX comprises Lys-27 and probably Arg-24 and Phe-25, and that of MgTX comprises Lys-28, Met-30, Asn-31, His-39, and Tyr-37. Fitting the four diads Lys-22/Tyr-23 (ShK), Lys-25/Tyr-26 (BgK), Lys-28/ Tyr-37 (MgTX), and Lys-27/Phe-25 (KTX) revealed a closed topological location of Arg-11 in ShK, His-13 in BgK and Arg-24 in KTX (Fig.…”

Section: Contribution Of the Diad Lys-25/tyr-26 In The Bgkmentioning

confidence: 99%

Mapping the Functional Anatomy of BgK on Kv1.1, Kv1.2, and Kv1.3

Alessandri‐Haber

Lecoq

Gasparini

et al. 1999

Journal of Biological Chemistry

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BgK is a peptide from the sea anemone Bunodosoma granulifera, which blocks Kv1.1, Kv1.2, and Kv1.3 potassium channels. Using 25 analogs substituted at a single position by an alanine residue, we performed the complete mapping of the BgK binding sites for the three Kv1 channels. These binding sites included three common residues (Ser-23, Lys-25, and Tyr-26) and a variable set of additional residues depending on the particular channel. Shortening the side chain of Lys-25 by taking out the four methylene groups dramatically decreased the BgK affinity to all Kv1 channels tested. However, the analog K25Orn displayed increased potency on Kv1.2, which makes this peptide a selective blocker for Kv1.2 (K D 50-and 300-fold lower than for Kv1.1 and Kv1.3, respectively). BgK analogs with enhanced selectivity could also be made by substituting residues that are differentially involved in the binding to some of the three Kv1 channels. For example, the analog F6A was found to be >500-fold more potent for Kv1.1 than for Kv1.2 and Kv1.3. These results provide new information about the mechanisms by which a channel blocker distinguishes individual channels among closely related isoforms and give clues for designing analogs with enhanced selectivity.

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“…The other residue in SmIIIA that merits investigation is Arg 17 , which replaces Lys in the other -conotoxins. Although the charge is the same, Arg is a bulkier side chain, and in the case of the K ϩ -channel ShK toxin, where Lys 22 inserts into the pore of the channel (53,55), substitution with Arg reduces K ϩ -channel binding significantly (56). Consistent with these inferences from the structure, construction and assay of two chimeras of SmIIIA and PIIIA in which their N-and C-terminal halves were recombined indicated that residues in the C-terminal half of SmIIIA confer affinity for TTX-resistant VGSCs.…”

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confidence: 99%

Structural Basis for Tetrodotoxin-resistant Sodium Channel Binding by μ-Conotoxin SmIIIA

Keizer

West

Lee

et al. 2003

Journal of Biological Chemistry

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SmIIIA is a new -conotoxin isolated recently from Conus stercusmuscarum. Although it shares several biochemical characteristics with other -conotoxins (the arrangement of cysteine residues and a conserved arginine believed to interact with residues near the channel pore), it has several distinctive features, including the absence of hydroxyproline, and is the first specific antagonist of tetrodotoxin-resistant voltage-gated sodium channels to be characterized. It therefore represents a potentially useful tool to investigate the functional roles of these channels. We have determined the three-dimensional structure of SmIIIA in aqueous solution. Consistent with the absence of hydroxyprolines, SmIIIA adopts a single conformation with all peptide bonds in the trans configuration. The spatial orientations of several conserved Arg and Lys side chains, including Arg 14 (using a consensus numbering system), which plays a key role in sodium channel binding, are similar to those in other -conotoxins but the N-terminal regions differ, reflecting the trans conformation for the peptide bond preceding residue 8 in SmIIIA, as opposed to the cis conformation in -conotoxins GIIIA and GIIIB. Comparison of the surfaces of SmIIIA with other -conotoxins suggests that the affinity of SmIIIA for TTX-resistant channels is influenced by the Trp 15 side chain, which is unique to SmIIIA. Arg 17 , which replaces Lys in the other -conotoxins, may also be important. Consistent with these inferences from the structure, assays of two chimeras of SmIIIA and PIIIA in which their N-and Cterminal halves were recombined, indicated that residues in the C-terminal half of SmIIIA confer affinity for tetrodotoxin-resistant sodium channels in the cell bodies of frog sympathetic neurons. SmIIIA and the chimera possessing the C-terminal half of SmIIIA also inhibit tetrodotoxin-resistant sodium channels in the postganglionic axons of sympathetic neurons, as indicated by their inhibition of C-neuron compound action potentials that persist in the presence of tetrodotoxin.Polypeptide toxins typically interact with their target receptors with high potency and exquisite selectivity and as such are valuable tools in elucidating the physiological functions of their targets and in probing the size and shape of their cognate binding sites. Toxins from the genus Conus have been especially valuable in this respect, and, of the various classes of conotoxin that have been characterized to date (1, 2), the -conotoxins represent a particularly good example. Their targets are the voltage-gated sodium channels (VGSCs), 1 which are responsible for the influx of sodium ions during action potentials in excitable tissues.Three families of conotoxins target VGSCs, causing either inhibition (-and O-conotoxins) or delayed inactivation (␦-conotoxins), but to date a detailed understanding of their interactions with the channel has been achieved only in the case of the -conotoxins. These toxins bind to Site 1 on VGSCs, one of several toxin binding sites identified on these channels ...

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Identification of Three Separate Binding Sites on SHK Toxin, a Potent Inhibitor of Voltage-Dependent Potassium Channels in Human T-Lymphocytes and Rat Brain

Cited by 66 publications

References 1 publication

BcsTx3 is a founder of a novel sea anemone toxin family of potassium channel blocker

BcsTx3 is a founder of a novel sea anemone toxin family of potassium channel blocker

Mapping the Functional Anatomy of BgK on Kv1.1, Kv1.2, and Kv1.3

Structural Basis for Tetrodotoxin-resistant Sodium Channel Binding by μ-Conotoxin SmIIIA

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