Precise spatiotemporal control of voltage-gated sodium channels by photocaged saxitoxin

Anna V Elleman,Christopher D Makinson,Allison L Haynes,J Du Bois,John R Huguenard,Gabrielle Devienne

doi:10.1038/s41467-021-24392-2

Anna V Elleman, Christopher D Makinson + Show 4 more

Open Access

https://doi.org/10.1038/s41467-021-24392-2

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Abstract

Here we report the pharmacologic blockade of voltage-gated sodium ion channels (NaVs) by a synthetic saxitoxin derivative affixed to a photocleavable protecting group. We demonstrate that a functionalized saxitoxin (STX-eac) enables exquisite spatiotemporal control of NaVs to interrupt action potentials in dissociated neurons and nerve fiber bundles. The photo-uncaged inhibitor (STX-ea) is a nanomolar potent, reversible binder of NaVs. We use STX-eac to reveal differential susceptibility of myelinated and unmyelinated axons in the corpus callosum to NaV-dependent alterations in action potential propagation, with unmyelinated axons preferentially showing reduced action potential fidelity under conditions of partial NaV block. These results validate STX-eac as a high precision tool for robust photocontrol of neuronal excitability and action potential generation.

Highlights

We report the pharmacologic blockade of voltage-gated sodium ion channels (NaVs) by a synthetic saxitoxin derivative affixed to a photocleavable protecting group
In order to achieve spatiotemporal control of NaVs, a photocaging group was appended to a synthetic derivative of saxitoxin (STX), a naturally occurring bis-guanidinium toxin that inhibits the action of six of nine NaV subtypes (e.g., IC50 = 1.2 nM vs. rat NaV 1.2)[33]
Our findings demonstrate that focally uncaging 5 enables studies of action potential initiation and propagation, and that this tool compound could be useful for modulating compound action potentials (CAPs) in unmyelinated axons

Summary

Results

In order to evaluate the uncaging efficiencies of photocaged STXs, voltage-clamped NaV1.2 CHO cells bathed in reagent were pulsed with 355 nm light (5 × 5 ms) while monitoring changes in channel block (Fig. 2b and Supplementary Fig. 2). N2 was more sensitive to uncaged 5, with N2 peak sink amplitude reduced at all three concentrations of this compound–25% at 100 nM, 36% at 250 nM, and 58% at 500 nM (Fig. 5d and Supplementary Table 1) These results indicate that transmission in unmyelinated callosal axons is more susceptible to partial sodium channel block than in myelinated fibers. If the differential effect of focally uncaged 5 on N1 vs N2 might be due to an underlying sensitivity in the NaVs responsible for action potential initiation or propagation in myelinated vs unmyelinated fibers To address this possibility, we tested the sensitivity of N1 and N2 to global, steady-state (i.e., bath) application of the active parent compound, STX-ea 1. In contrast with targeted sodium channel inhibition by photouncaging 5, CAP velocity is significantly slowed for both N1 and N2 with bath application of 1: from 1.36 ± 0.16 m/s to 1.00 ± 0.08 m/s for N1, and from 0.53 ± 0.01 m/s to 0.43 ± 0.03 m/s for N2 (Supplementary Table 2)

Discussion

Methods

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