Demonstration of ion channel synthesis by isolated squid giant axon provides functional evidence for localized axonal membrane protein translation

Chhavi Mathur,Daniel Basilio,Brian A. Tong,Pablo Miranda,Miguel Holmgren,Francisco Bezanilla,Ramon Latorre,Deepa Srikumar,Kory R. Johnson

doi:10.1038/s41598-018-20684-8

Abstract

Local translation of membrane proteins in neuronal subcellular domains like soma, dendrites and axon termini is well-documented. In this study, we isolated the electrical signaling unit of an axon by dissecting giant axons from mature squids (Dosidicus gigas). Axoplasm extracted from these axons was found to contain ribosomal RNAs, ~8000 messenger RNA species, many encoding the translation machinery, membrane proteins, translocon and signal recognition particle (SRP) subunits, endomembrane-associated proteins, and unprecedented proportions of SRP RNA (~68% identical to human homolog). While these components support endoplasmic reticulum-dependent protein synthesis, functional assessment of a newly synthesized membrane protein in axolemma of an isolated axon is technically challenging. Ion channels are ideal proteins for this purpose because their functional dynamics can be directly evaluated by applying voltage clamp across the axon membrane. We delivered in vitro transcribed RNA encoding native or Drosophila voltage-activated Shaker KV channel into excised squid giant axons. We found that total K+ currents increased in both cases; with added inactivation kinetics on those axons injected with RNA encoding the Shaker channel. These results provide unambiguous evidence that isolated axons can exhibit de novo synthesis, assembly and membrane incorporation of fully functional oligomeric membrane proteins.

Highlights

IntroductionWe aimed to obtain a transcriptome using axoplasm, exclusively derived from the giant axon of the Humboldt squid (Dosidicus gigas)
The messenger RNAs (mRNAs) content of the axons was analyzed by constructing poly(A)-enriched cDNA libraries (Illumina Truseq) from D. gigas axoplasm, and subjecting them to deep sequencing (RNASeq) using Hi-Seq. 2000 RNA-seq platform (Illumina)
We have shown that the axoplasm of squid giant axon contains a transcriptome representative of a compartment with membrane protein synthesis and trafficking capabilities

Summary

Introduction

We aimed to obtain a transcriptome using axoplasm, exclusively derived from the giant axon of the Humboldt squid (Dosidicus gigas) These axons are known to be formed by fusion of axons originating from cell bodies residing in the stellate ganglion[38] that gives rise to a large electrical signal transmission unit, normally larger than 1.0 mm in diameter (Fig. 1). We monitored the incorporation of newly synthesized ion channels into axon membrane by voltage clamp, which allowed us to study directly, the function of these membrane proteins. In both cases we found an increase in potassium currents across the axolemma. Since KV channels are known to undergo post-translational processing and oligomerization in endomembranous compartments prior to membrane-targeting[45,46], our study unambiguously demonstrated that new KV channel subunits can be de novo translated, folded and hetero-tetramerized into functional ion channels that were targeted to axolemma likely using the axoplasm’s resident endomembranous machinery

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