Design of saxitoxin-based tools for studying voltage-gated sodium channel physiology
Abstract/Contents
- Abstract
- Electrical signaling, a key method of cellular communication, relies on voltage gradients across cell membranes. Voltage-gated sodium channels (NaVs) modulate cell permeability to sodium ions in response to changes in membrane potential and are therefore crucial to action potential generation and propagation. In humans, the nine NaV isoforms (NaV1.1--1.9) are differentially expressed across the nervous system, heart, and muscle tissues, and NaV dysfunction has been implicated in myriad diseases, including epilepsy, cardiac arrhythmia, and chronic pain. The role of aberrant channel expression, trafficking, subcellular distribution, and function in pathogenesis continues to be an active area of research, in part due to the limitations of the molecular tools available for studying these channel properties. Herein, we present efforts towards developing both NaV subtype-selective probes and reversible NaV imaging probes to expand the toolkit for interrogating NaV physiology. The naturally-occurring, bis-guanidinium toxin saxitoxin (STX) is a potent NaV inhibitor (IC50 = 3 nM vs. rat NaV1.4 (rNaV1.4)) and therefore an ideal template for developing such tools. A practical approach towards developing a subtype-selective probe is to design a toxin derivative that is selective for an NaV bearing one or more point mutations: a modified toxin-mutant channel pair, in which the modified toxin is ≥100 times less potent against wild-type (WT) NaVs, can be used to functionally 'knock out' single channel subtypes, enabling investigations into the function of individual isoforms. Towards this end, seven novel STX derivatives, all featuring C10- or C10/C11-modification, were prepared through de novo synthesis. The majority of these toxin analogs bear a cyclopropane unit spanning C10 and C11 and are highly potent against WT NaVs (IC50 = 3--143 nM vs. rNaV1.4). Further electrophysiological characterization of these analogs has revealed multiple point mutations that confer enhanced potency, providing a path towards developing the desired modified toxin-mutant channel pair. Towards the design of NaV imaging probes, STX-fluorophore conjugates, including an STX-fluorescein derivative, as well as STX derivatives bearing bioorthogonal groups (i.e., biotin, tetrazine) were prepared. All of these probes exhibit poor potency, low brightness, and/or a propensity for nonspecific binding to the plasma membrane, precluding their use for reversibly labeling plasma membrane-bound WT NaVs in model systems and/or primary neurons. However, characterization of these probes by electrophysiology and multiple microscopy methods has elucidated promising strategies for generating viable STX-based imaging probes. Preparation of NaV subtype-selective tools for modulating channel function and NaV imaging probes for visualizing dynamic channel properties will enable studies that have the potential to advance our understanding of the link between NaV dysfunction and disease, a critical step towards treating NaV-based channelopathies.
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 | 2022; ©2022 |
| Publication date | 2022; 2022 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Hajare, Holly Shivashri |
|---|---|
| Degree supervisor | Du Bois, Justin |
| Thesis advisor | Du Bois, Justin |
| Thesis advisor | Bertozzi, Carolyn R, 1966- |
| Thesis advisor | Wender, Paul A |
| Degree committee member | Bertozzi, Carolyn R, 1966- |
| Degree committee member | Wender, Paul A |
| Associated with | Stanford University, Department of Chemistry |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Holly Shivashri Hajare. |
|---|---|
| Note | Submitted to the Department of Chemistry. |
| Thesis | Thesis Ph.D. Stanford University 2022. |
| Location | https://purl.stanford.edu/tm267ym1589 |
Access conditions
- Copyright
- © 2022 by Holly Shivashri Hajare
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
Also listed in
Loading usage metrics...