Molecular Determinants of Brevetoxin Binding to Voltage-Gated Sodium Channels.

Publisher: MDPI Country of Publication: Switzerland NLM ID: 101530765 Publication Model: Electronic Cited Medium: Internet ISSN: 2072-6651 (Electronic) Linking ISSN: 20726651 NLM ISO Abbreviation: Toxins (Basel) Subsets: MEDLINE

Original Publication: Basel : MDPI

Marine Toxins/*metabolism ; Oxocins/*metabolism ; Voltage-Gated Sodium Channels/*metabolism; Binding Sites ; Cell Line ; Humans ; Marine Toxins/chemistry ; Mutation ; Oxocins/chemistry ; Protein Isoforms ; Voltage-Gated Sodium Channels/genetics

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- ³ 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 v 1.2, Na v 1.4 and Na v 1.5 channels showed that Na v 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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R01 NS015751 United States NS NINDS NIH HHS; R35 NS111573 United States NS NINDS NIH HHS; U54 HD083091 United States HD NICHD NIH HHS; R-01 N515751 United States NH NIH HHS

Keywords: binding assay; neurotoxic shellfish poisoning; voltage-gated sodium channels

0 (Marine Toxins)
0 (Oxocins)
0 (Protein Isoforms)
0 (Voltage-Gated Sodium Channels)
98225-48-0 (brevetoxin)

Date Created: 20190906 Date Completed: 20201013 Latest Revision: 20201013

20240829

PMC6784055

10.3390/toxins11090513

31484365