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Open AccessArticle

Molecular Determinants of Brevetoxin Binding to Voltage-Gated Sodium Channels

Department of Pharmacology, Box 357280, University of Washington, Seattle, WA 98195-7280, USA
Center for Marine Science, University of North Carolina, Wilmington, NC 28409, USA
Authors to whom correspondence should be addressed.
Current address is Graduate School of Agricultural Science, Tohoku University, 468-1 Aramaki-Aza-Aoba, Aoba-ku, Sendai 980-8572, Japan.
Toxins 2019, 11(9), 513;
Received: 31 July 2019 / Revised: 30 August 2019 / Accepted: 30 August 2019 / Published: 3 September 2019
(This article belongs to the Special Issue Marine Toxins Detection)
Brevetoxins are produced by dinoflagellates such as Karenia brevis in warm-water red tides and cause neurotoxic shellfish poisoning. They bind to voltage-gated sodium channels at neurotoxin receptor 5, making the channels more active by shifting the voltage-dependence of activation to more negative potentials and by slowing the inactivation process. Previous work using photoaffinity labeling identified binding to the IS6 and IVS5 transmembrane segments of the channel α subunit. We used alanine-scanning mutagenesis to identify molecular determinants for brevetoxin binding in these regions as well as adjacent regions IVS5-SS1 and IVS6. Most of the mutant channels containing single alanine substitutions expressed functional protein in tsA-201 cells and bound to the radioligand [42-3H]-PbTx3. Binding affinity for the great majority of mutant channels was indistinguishable from wild type. However, transmembrane segments IS6, IVS5 and IVS6 each contained 2 to 4 amino acid positions where alanine substitution resulted in a 2–3-fold reduction in brevetoxin affinity, and additional mutations caused a similar increase in brevetoxin affinity. These findings are consistent with a model in which brevetoxin binds to a protein cleft comprising transmembrane segments IS6, IVS5 and IVS6 and makes multiple distributed interactions with these α helices. Determination of brevetoxin affinity for Nav1.2, Nav1.4 and Nav1.5 channels showed that Nav1.5 channels had a characteristic 5-fold reduction in affinity for brevetoxin relative to the other channel isoforms, suggesting the interaction with sodium channels is specific despite the distributed binding determinants. View Full-Text
Keywords: neurotoxic shellfish poisoning; voltage-gated sodium channels; binding assay neurotoxic shellfish poisoning; voltage-gated sodium channels; binding assay
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MDPI and ACS Style

Konoki, K.; Baden, D.G.; Scheuer, T.; Catterall, W.A. Molecular Determinants of Brevetoxin Binding to Voltage-Gated Sodium Channels. Toxins 2019, 11, 513.

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