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Open AccessArticle

Molecular Determinants of Brevetoxin Binding to Voltage-Gated Sodium Channels

1
Department of Pharmacology, Box 357280, University of Washington, Seattle, WA 98195-7280, USA
2
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; https://doi.org/10.3390/toxins11090513
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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