S3b amino acid substitutions and ancillary subunits alter the affinity of Heteropoda venatoria toxin 2 for Kv4.3.
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 K(d) value of 2.3 microM. Two alanine mutants in Kv4.3 increased K(d) values to 6.4 microM for V276A and 25 microM 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 K(d) value to 0.54 microM, 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 K(d) value for Kv4.3 [N280A] was 0.26 microM. 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 K(d) value for Kv4.3 + KChIP2b was 0.95 microM. 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.
DeSimone
Christopher V
CV
Department of Physiology and Biophysics, University at Buffalo, The State University of New York, Buffalo, NY 14214, USA.
Lu
YiChun
Y
Bondarenko
Vladimir E
VE
Morales
Michael J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D005260:Female; D008969:Molecular Sequence Data; D021122:Protein Subunits; D051669:Shab Potassium Channels / Q000037:antagonists & inhibitors; D051672:Shal Potassium Channels / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D013111:Spider Venoms / Q000378:metabolism / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014982:Xenopus laevis
C524386:HpTx-2 protein, Heteropoda venatoria; D021122:Protein Subunits; D051669:Shab Potassium Channels; D051672:Shal Potassium Channels; D013111:Spider Venoms