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

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.

Toward Axonal System Biology: Genome Wide Views of Local mRNA Translation

“…Complexity estimation of a giant axon cDNA library indicated hundreds of mRNAs . This figure has now been extended to around 8000 mRNAs by RNA‐seq, showing, in addition, that membrane proteins (a Drosophila K v channel) can be synthesized and inserted in the axonal membrane, as observed earlier by van Minnen . This makes it clear that having methods to obtain mature axoplasm in large quantities is highly useful.…”

“…This progress was possible thanks to advances both in neuronal cell culture techniques (allowing for the separation of cell bodies from axons) and in mRNAs identification techniques (first microarrays and later more sensitive and comprehensive RNA‐Seq methods). There are currently almost 20 publications where axonal transcriptome of different species, diverse neuronal types, and physiological conditions were studied (Table ), most of them obtained from in vitro cell culture systems …”

The local translation of KNa in dendritic projections of auditory neurons and the roles of KNa in the transition from hidden to overt hearing loss

“…Local protein translation has been identified to be essential for axonal maintenance [28], dendritic functions [29–31], and synaptic plasticity [32]. Indeed, localized axonal K + channel translation has been reported [33]. We examined the expression of K Na 1.1 and K Na 1.2 in different compartments of SGNs.…”

“…Found predominantly as a developmental occurrence in the dynamic process at growth cones, β-actin and other structural protein mRNA were localized and translated in response to netrin I gradient in growth cones [56, 57]. Axoplasm extracts from giant axons have also been found to contain ribosomal RNA and transcripts that encode for protein-synthesis machinery proteins, indicating that local protein synthesis is a basic property of axons [33, 58, 59]. Indeed, axonal transcriptome from motoneurons and sensory neurons all contain membrane protein mRNA, including K + channels [1, 60].…”

Influences: The Cell Physiology Laboratory in Montemar, Chile

“…Just recently, we used the giant axon as a preparation to demonstrate the synthesis of functional membrane proteins in the absence of a soma. This was only possible because of the large size of the Humboldt squid axon, which allowed the injection of foreign RNA and subsequent voltage-clamping plus the characterization of RNA species in the axoplasm by extruding it from a single axon (16). This has opened up the possibility of studying the details of protein synthesis in an axon preparation with high resolution and using a variety of techniques, with little interference from other cells.…”