| Summary: | Brevetoxins are produced by dinoflagellates such as <i>Karenia brevis</i> 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-<sup>3</sup>H]-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 Na<sub>v</sub>1.2, Na<sub>v</sub>1.4 and Na<sub>v</sub>1.5 channels showed that Na<sub>v</sub>1.5 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.
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