Molecular Determinants of Brevetoxin Binding to Voltage-Gated Sodium Channels
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 activatio...
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| Format: | Article |
| Language: | English |
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MDPI AG
2019-09-01
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| Series: | Toxins |
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| Online Access: | https://www.mdpi.com/2072-6651/11/9/513 |
| _version_ | 1811301833145057280 |
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| author | Keiichi Konoki Daniel G. Baden Todd Scheuer William A. Catterall |
| author_facet | Keiichi Konoki Daniel G. Baden Todd Scheuer William A. Catterall |
| author_sort | Keiichi Konoki |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-13T07:17:05Z |
| format | Article |
| id | doaj.art-affbf8b639ca4f539e9ef2120f956c94 |
| institution | Directory Open Access Journal |
| issn | 2072-6651 |
| language | English |
| last_indexed | 2024-04-13T07:17:05Z |
| publishDate | 2019-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Toxins |
| spelling | doaj.art-affbf8b639ca4f539e9ef2120f956c942022-12-22T02:56:44ZengMDPI AGToxins2072-66512019-09-0111951310.3390/toxins11090513toxins11090513Molecular Determinants of Brevetoxin Binding to Voltage-Gated Sodium ChannelsKeiichi Konoki0Daniel G. Baden1Todd Scheuer2William A. Catterall3Department of Pharmacology, Box 357280, University of Washington, Seattle, WA 98195-7280, USACenter for Marine Science, University of North Carolina, Wilmington, NC 28409, USADepartment of Pharmacology, Box 357280, University of Washington, Seattle, WA 98195-7280, USADepartment of Pharmacology, Box 357280, University of Washington, Seattle, WA 98195-7280, USABrevetoxins 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.https://www.mdpi.com/2072-6651/11/9/513neurotoxic shellfish poisoningvoltage-gated sodium channelsbinding assay |
| spellingShingle | Keiichi Konoki Daniel G. Baden Todd Scheuer William A. Catterall Molecular Determinants of Brevetoxin Binding to Voltage-Gated Sodium Channels Toxins neurotoxic shellfish poisoning voltage-gated sodium channels binding assay |
| title | Molecular Determinants of Brevetoxin Binding to Voltage-Gated Sodium Channels |
| title_full | Molecular Determinants of Brevetoxin Binding to Voltage-Gated Sodium Channels |
| title_fullStr | Molecular Determinants of Brevetoxin Binding to Voltage-Gated Sodium Channels |
| title_full_unstemmed | Molecular Determinants of Brevetoxin Binding to Voltage-Gated Sodium Channels |
| title_short | Molecular Determinants of Brevetoxin Binding to Voltage-Gated Sodium Channels |
| title_sort | molecular determinants of brevetoxin binding to voltage gated sodium channels |
| topic | neurotoxic shellfish poisoning voltage-gated sodium channels binding assay |
| url | https://www.mdpi.com/2072-6651/11/9/513 |
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