Abstract
Heteropoda venatoria toxin 2 (HpTx2) is an inhibitor cystine knot (ICK)-gating modifier toxin that selectively inhibits Kv4 channels. To characterize the molecular determinants of interaction, we performed alanine scanning of the Kv4.3 S3b region. HpTx2-Kv4.3 interaction had an apparent Kd value of 2.3 μM. Two alanine mutants in Kv4.3 increased Kd values to 6.4 μM for V276A and 25 μM for L275A. Simultaneous mutation of both amino acids to alanine nearly eliminated toxin interaction. Unlike Hanatoxin and other well characterized ICK toxins, HpTx2 binding does not require a charged amino acid for interaction. To determine whether the identity of the S3b binding site amino acids altered HpTx2 specificity, we constructed Kv4.3 [LV275IF]. This mutation decreased the Kd value to 0.54 μM, suggesting that the hydrophobic character of the putative binding site is the most important property for interaction with HpTx2. One mutant, N280A, caused stronger interaction of HpTx2 with Kv4.3; the Kd value for Kv4.3 [N280A] was 0.26 μM. To understand Kv4.3-based transient outward currents in native tissues, we tested the affinity of HpTx2 for Kv4.3 coexpressed with KChIP2b. The toxin's Kd value for Kv4.3 + KChIP2b was 0.95 μM. KChIP2b stabilizes the closed state of Kv4.3, suggesting that the increased toxin affinity is due to increased stabilization of the closed state. These data show that HpTx2 binding to Kv4.3 has aspects in common with other ICK gating modifier toxins but that the interventions that increase toxin affinity suggest flexibility toward channel binding that belies its unusual specificity for Kv4 channels.
Footnotes
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This work was supported by a Scientist Development Grant and Predoctoral Fellowship from the Founders Affiliate of the American Heart Association [Grants 0235500T, 0615662T]; the John R. Oishei Foundation; and the National Institutes of Health National Heart, Lung, and Blood Institute [Grant HL52874].
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Parts of this work were presented at the Joint Meeting of the Biophysical Society 52nd Annual Meeting and 16th International Biophysics Congress, 2-6 Feb 2008, Long Beach, CA; and in thesis form at the University at Buffalo, the State University of New York.
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ABBREVIATIONS: KChIP, Kv4 channel-interacting protein; HpTx2, Heteropoda venatoria toxin 2; HaTx, hanatoxin; WT, wild type; ICK, inhibitor cystine knot; BSA, bovine serum albumin; SGTx, Scodra griseipes toxin.
- Accepted April 8, 2009.
- Received February 16, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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