Nav channel binder containing a specific conjugation-site based on a low toxicity \u03b2-scorpion toxin

Tomoya Kubota,Francisco Bezanilla,Ana M Correa,Joao L Carvalho-De-Souza,Bobo Dang

doi:10.1038/s41598-017-16426-x

Abstract

Voltage-gated sodium (Nav) channels play a key role in generating action potentials which leads to physiological signaling in excitable cells. The availability of probes for functional studies of mammalian Nav is limited. Here, by introducing two amino acid substitutions into the beta scorpion toxin Ts1, we have chemically synthesized a novel binder [S14R, W50Pra]Ts1 for Nav with high affinity, low dissociation rate and reduced toxicity while retaining the capability of conjugating Ts1 with molecules of interests for different applications. Using the fluorescent-dye conjugate, [S14R, W50Pra(Bodipy)]Ts1, we confirmed its binding to Nav1.4 through Lanthanide-based Resonance Energy Transfer. Moreover, using the gold nanoparticle conjugate, [S14R, W50Pra(AuNP)]Ts1, we were able to optically stimulate dorsal root ganglia neurons and generate action potentials with visible light via the optocapacitive effect as previously reported. [S14R, W50Pra]Ts1 is a novel probe with great potential for wider applications in Nav-related neuroscience research.

Highlights

Voltage-gated sodium (Nav) channels are the primary initiators of action potentials in excitable cells[1]
The binding site in mammalian Nav channels has been identified to be formed by the S1-S2 linker and the S3-S4 linker of voltage sensor domain (VSD) in DII and the S5-S6 pore linker in DIII14,15
We generated Ts1-S14R conjugated with gold nanoparticles, [S14R, W50Pra(AuNP)]Ts1 and we demonstrated that this conjugate can induce action potentials in rat dorsal root ganglia (DRG) neurons by visible light stimulation via the optocapacitive effect, as shown previously for Ts1-AuNP22

Summary

Introduction

Voltage-gated sodium (Nav) channels are the primary initiators of action potentials in excitable cells[1]. Several antibodies for the Nav channel α subunits are commercially available, but most of them were generated using Nav channel cytosolic peptides as epitopes They have limited in vivo application potential for biophysical experiments or for neuroscience because the epitopes would not be accessible from the extracellular side. Using Ts1-W50Pra conjugated to a fluorescent dye Bodipy (Ts1-Bodipy), we have determined its binding location in rat skeletal muscle Nav channels (Nav1.4) using Lanthanide-based Resonance Energy Transfer (LRET), showing that this molecule is a powerful tool for biophysical research[21]. In the study reported here, we have generated a novel potent Nav binder based on Ts1-W50Pra with significantly reduced toxicity by introducing a single amino acid substitution S14R, corresponding to E15R in Css[4] toxin, [S14R, W50Pra]Ts1 (hereafter, “Ts1-S14R”). Ts1-S14R can be a powerful probe with great potential for wider applications in Nav-related research for biophysics, pharmacology and neuroscience research

Methods

Results

Discussion

Conclusion