Molecular tools to modulate voltage-gated sodium channels
Abstract/Contents
- Abstract
- Voltage-gated sodium channels (NaVs) are responsible for sodium influx upon cellular depolarization and, as such, are fundamental to action potential initiation and propagation. There are nine voltage-sensing channel subtypes, NaV1.1--1.9, which are differentially distributed throughout the body. NaV dysfunction—whether in terms of channel activity, expression, or localization—is implicated in a host of diseases, including epilepsy, multiple sclerosis, and neuropathic pain. Efforts to fully elucidate the mechanisms of NaV-dependent pathogenesis are limited by a lack of rapid, efficient, and selective methods for the manipulation of NaV subpopulations (Chapter 1). Accordingly, we have developed tools for subtype-selective (Chapter 2) and location-specific (Chapters 3--4) channel inhibition. Toward the goal of subtype-selective channel inhibition, we designed a mutant NaV-electrophile pair for the covalent labelling of individual NaV subtypes. Building off work from Drs. William Parsons and Darren Finkelstein, we determined that a reactive saxitoxin (STX) derived probe, saxitoxin ethyl maleimide, selectively and irreversibly inhibits engineered NaVs expressing appropriately positioned cysteine point mutations. Labelling occurs rapidly and efficiently at low probe concentrations—i.e., up to 40% irreversible inhibition after 6-minute application of 500 nM toxin. Modification of STX ethyl maleimide with a biotin moiety or 'clickable' tetrazine tag should enable live cell imaging of target NaV subtypes. The efficacy of NaV labelling by such trifunctional probes has been demonstrated by patch clamp electrophysiology; efforts continue to translate these results to channel visualization with confocal microscopy and immunoblotting. Toward the goal of location-specific channel inhibition, one-photon (Chapter 3) and two-photon (Chapter 4) photocleavable groups were appended to saxitoxin. Derivatization of the photocage with carboxylate groups destabilized STX binding to the NaV. As such, inert photocaged STX compounds (STX PCs) could be applied to cells prior to selective, light-induced, focal release. Several rounds of optimization yielded STX PCs with large differences in potency pre- vs. post-uncaging—5.2 µM vs. 90 nM against rat hippocampal neurons for the optimal one-photon STX PC; 2.1 µM vs. 100 nM for the analogous two-photon STX PC—following exposure to only 5 ms of 130 mW UV light. These large differences in potency enable precise control of action potential firing in hippocampal neurons, with tandem laser--STX PC application acting as a molecular switch for electrical activity. Collaborations using these STX PCs to examine the role of NaVs in epilepsy are ongoing. The precise control of NaV function, whether through the modulation of a single channel subtype or a localized subpopulation, will help disentangle the complex biochemistry underlying electrogenesis and ectopic excitability, thereby aiding efforts to develop better treatments for electrical signaling disorders.
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 | 2021; ©2021 |
| Publication date | 2021; 2021 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Elleman, Anna Virginia |
|---|---|
| Degree supervisor | Du Bois, Justin |
| Thesis advisor | Du Bois, Justin |
| Thesis advisor | Khosla, Chaitan, 1964- |
| Thesis advisor | Wender, Paul A |
| Degree committee member | Khosla, Chaitan, 1964- |
| Degree committee member | Wender, Paul A |
| Associated with | Stanford University, Department of Chemistry |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Anna Virginia Elleman. |
|---|---|
| Note | Submitted to the Department of Chemistry. |
| Thesis | Thesis Ph.D. Stanford University 2021. |
| Location | https://purl.stanford.edu/db498ws0292 |
Access conditions
- Copyright
- © 2021 by Anna Virginia Elleman
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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