Systematic mapping of the voltage-gated sodium channel outer pore using modified saxitoxins
Abstract/Contents
- Abstract
- Modulation of action potentials in electrically excitable cells is controlled by tight regulation of ion channel expression and distribution. Voltage-gated sodium channels (NaVs) represent one such class of obligatory membrane proteins, comprised of nine mammalian isoforms (NaV1.1--NaV1.9) differentially expressed in excitable tissues. Our ambition to understand the role of individual NaV subtypes in neuronal signaling motivates development of isoform-selective small molecule modulators of NaV function. The paralytic shellfish poison saxitoxin (STX) has historically been used as a critical tool for interrogating NaV structure and function and, as a result, represents an excellent template for creating such probes. However, given the marked sequence similarity between members of this protein family, designing NaV selective agents requires a comprehensive understanding of channel structure. Accordingly, we endeavored to gain insight into the three-dimensional architecture of the outer vestibule of NaV (Site 1) through a systematic structure-activity relationship (SAR) study employing saxitoxin, synthetically modified saxitoxins, and protein mutagenesis. The preparation and electrophysiological characterization of natural and non-natural STX analogues modified at six positions (N7, N9, C10, C11, C13, and N21) is described. A single synthetic route adapted from a previously disclosed strategy toward gonyautoxin-3 facilitates access to the bis-guanidinium core of STX and enables late-stage introduction of substituent groups. Mutant cycle analysis, a systematic method for mapping non-covalent ligand-receptor interactions, has been conducted with modified saxitoxins and point mutant NaVs. These studies have culminated in a revised toxin-receptor binding model, which is consistent with the large body of SAR data described in Chapters 3 and 4. During the course of this work, an acetylated variant of STX was identified with unprecedented affinity for human NaV1.7 (hNaV1.7), a channel subtype that has been implicated in pain perception. We have leveraged bis-guanidinium toxin analogues modified at positions C10/C13, C11, and C12 in supplemental studies aimed to examine a possible two-state toxin binding model. Although a two-state model cannot be completely discounted at this time, the large majority of results with STX-based probes and mutant NaVs support our revised one-state binding model. Insight gained from mutant cycle analysis has been used to rationally develop a cysteine mutant NaV that is irreversibly inhibited by nanomolar concentrations of a STX-maleimide conjugate. These findings provide a proof-of-concept for engineering NaV-mutant specific covalent inhibitors around the Site 1 locus. Future efforts to design selective, non-covalent NaV inhibitors should also be facilitated with the availability of our proposed STX-NaV binding model.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2016 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Thomas-Tran, Rhiannon |
|---|---|
| Associated with | Stanford University, Department of Chemistry. |
| Primary advisor | Du Bois, Justin |
| Thesis advisor | Du Bois, Justin |
| Thesis advisor | Wandless, Thomas |
| Thesis advisor | Wender, Paul A |
| Advisor | Wandless, Thomas |
| Advisor | Wender, Paul A |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Rhiannon Thomas-Tran. |
|---|---|
| Note | Submitted to the Department of Chemistry. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2016. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2016 by Rhiannon Thomas-Tran
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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