A synthetic batrachotoxin derivative as a novel molecular probe to study slow inactivation in voltage-gated sodium channels
Abstract/Contents
- Abstract
- Voltage-gated sodium channels (NaVs) are obligatory transmembrane proteins responsible for electrical signaling in biology. By transitioning between different conformational states, NaVs initiate the rising phase of the action potential. A large array of neurotoxins target specific conformational states of the channel or disrupt transitions between them. Among the most powerful of these pharmacological tools is batrachotoxin (BTX), the active ingredient in poison-tipped darts used by the indigenous people of Colombia. Every measurable aspect of channel function is disrupted when this potent small molecule binds in the NaV central pore. Using de novo synthesis, we sought to perform structure-activity relationship (SAR) studies to uncover the molecular underpinnings of the action of this potent toxin. SAR studies focused on derivatizing the C20-ester of BTX. Synthetic toxin analogues were characterized using whole-cell voltage clamp electrophysiology. Using this technique, the efficacy of BTX against a recombinantly expressed single channel isoform was measured for the first time. The ester derivatives show comparable ability to the natural product in their ability to hyperpolarize NaV activation potential, indicating changes to this region of the molecule are tolerated. A BTX derivative having a conformationally flexible ester, BTX-yne, uniquely alters NaV inactivation. With the exception of BTX-yne, BTX and all synthetic ester derivatives completely eliminate both fast and slow inactivation, distinct mechanisms that regulate NaV availability. In contrast, BTX-yne selectively eliminates fast inactivation while allowing channels to undergo slow inactivation. Electrophysiology experiments with rNaV1.4 and a prokaryotic sodium channel, NaVAe, were performed to fully characterize the behavior of this novel pharmacological tool compound. A novel sodium channel mutant, rNaV1.4 A438K, that is incapable of fast inactivation was characterized. The behavior of unmodified A438K resembles that of wild-type channels modified by BTX-yne, demonstrating that slow inactivation can be studied without the influence of fast inactivation with small molecules and mutant NaVs. The experiments in this thesis lay the groundwork for future work to understand the molecular elements underlying the dynamic conformational rearrangements that underlie channel gating.
Description
| Type of resource | text |
|---|---|
| Form | electronic resource; remote; computer; online resource |
| Extent | 1 online resource. |
| Place | California |
| Place | [Stanford, California] |
| Publisher | [Stanford University] |
| Copyright date | 2018; ©2018 |
| Publication date | 2018; 2018 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | MacKenzie, Timothy Michael Gale |
|---|---|
| Degree supervisor | Du Bois, Justin |
| Thesis advisor | Du Bois, Justin |
| Thesis advisor | Boxer, Steven G. (Steven George), 1947- |
| Thesis advisor | Wender, Paul A |
| Degree committee member | Boxer, Steven G. (Steven George), 1947- |
| Degree committee member | Wender, Paul A |
| Associated with | Stanford University, Department of Chemistry. |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Timothy Michael Gale MacKenzie. |
|---|---|
| Note | Submitted to the Department of Chemistry. |
| Thesis | Thesis Ph.D. Stanford University 2018. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2018 by Timothy Michael Gale MacKenzie
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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