Fluorescent Saxitoxins for Live Cell Imaging of Single Voltage-Gated Sodium Ion Channels beyond the Optical Diffraction Limit
Abstract
A desire to better understand the role of voltage-gated sodium channels (NaVs) in signal conduction and their dysregulation in specific disease states motivates the development of high precision tools for their study. Nature has evolved a collection of small molecule agents, including the shellfish poison (+)-saxitoxin, that bind to the extracellular pore of select NaV isoforms. As described in this report, de novo chemical synthesis has enabled the preparation of fluorescently labeled derivatives of (+)-saxitoxin, STX-Cy5, and STX-DCDHF, which display reversible binding to NaVs in live cells. Electrophysiology and confocal fluorescence microscopy studies confirm that these STX-based dyes function as potent and selective NaV labels. The utility of these probes is underscored in single-molecule and super-resolution imaging experiments, which reveal NaV distributions well beyond the optical diffraction limit in subcellular features such as neuritic spines and filopodia.
Additional Information
Ⓒ 2012 Elsevier Ltd. Received: February 24, 2012. Revised: May 24, 2012. Accepted: May 25, 2012. Published: July 26, 2012. A.E.O., H.L., S.I., B.M.A., W.E.M., and J.D. designed research; A.E.O., H.L., S.I., and W.H.P. performed research; A.E.O., H.L., and S.I. analyzed data; and A.E.O., H.L., W.E.M., and J.D. wrote the paper. The authors thank Professor R.J. Twieg and J.C. Williams for the gift of DCDHF-NHS. We are grateful to Professor Merritt Maduke for allowing use of her electrophysiology equipment and for many helpful discussions. This work was supported in part by R01-GM086196 (to W.E.M.) and R01-NS45684 (to J.D.) from the National Institute of General Medical Sciences and the National Institute of Neurological Disorders and Stroke, respectively, and by a grant from the Tobacco-Related Disease Research Program (to J.D.). W.H.P. is the recipient of a Stanford Interdisciplinary Graduate Fellowship. J.D. is a cofounder of SiteOne Therapeutics, Inc.Attached Files
Accepted Version - nihms493307.pdf
Supplemental Material - 1-s2.0-S1074552112002025-mmc1.pdf
Supplemental Material - 1-s2.0-S1074552112002025-mmc2.mp4
Files
Additional details
- PMCID
- PMC3731772
- Eprint ID
- 81079
- DOI
- 10.1016/j.chembiol.2012.05.021
- Resolver ID
- CaltechAUTHORS:20170901-132021426
- R01-GM086196
- NIH
- R01-NS45684
- NIH
- California Tobacco-Related Disease Research Program
- Stanford University
- Created
-
2017-09-05Created from EPrint's datestamp field
- Updated
-
2022-04-25Created from EPrint's last_modified field