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Volume 271, Number 39, Issue of September 27, 1996 pp. 23828-23835
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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The Role of Exposed Tryptophan Residues in the Activity of the Cardiotonic Polypeptide Anthopleurin B

(Received for publication, May 15, 1996, and in revised form, June 25, 1996)

Belinda L. Dias-Kadambi , Kelly A. Combs , Chester L. Drum ^§ , Dorothy A. Hanck ^§ and Kenneth M. Blumenthal

From the  Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267-0524 and ^§ Departments of Medicine and of the Pharmacological and Physiological Sciences, University of Chicago, Chicago, Illinois 60637

Scorpion and sea anemone venoms contain several polypeptides that delay inactivation of voltage-sensitive sodium channels via interaction with a common site. In this report, we target exposed hydrophobic residues at positions 33 and 45 of anthopleurin B (ApB) by polymerase chain reaction mutagenesis to ascertain their contribution to toxin activity. Nonconservative replacements are not permitted at position 33, indicating that Trp-33 may play an important structural role. Strikingly, the relatively conservative substitution of Trp-33 by phenylalanine results in major reductions in binding affinity for both the cardiac and neuronal channel isoforms as measured by ion flux, whereas substitution with tyrosine is tolerated and exhibits near wild-type affinities, suggesting that either the ability to form a hydrogen bond or the amphiphilic nature of the side chain are important at this position. Electrophysiological analysis of W33F indicates that its diminished affinity is primarily due to a decreased association rate. Analysis of a panel of mutants at Trp-45 shows only modest changes in apparent binding affinity for both channel isoforms but significant effects on V_max. In neuronal channels, the maximal levels of uptake for W45A/S/F are about 50% those seen with ApB. This effect is also observed for W45A and W45S in the cardiac model, wherein W45F is normal. These results suggest that a hydrophobic contact is involved in toxin-induced stabilization of the open conformation of the cardiac sodium channel. We conclude that Trp-33 contributes significantly to apparent affinity, whereas Trp-45 does not appear to affect binding per se. Furthermore, W33F is the first ApB mutant that displays a significantly altered association rate and may prove to be a useful probe of the channel binding site.

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