TNFα induces co-trafficking of TRPV1/TRPA1 in VAMP1-containing vesicles to the plasmalemma via Munc18-1/syntaxin1/SNAP-25 mediated fusion.

Jianghui Meng,Jiafu Wang,Martin Steinhoff,James Oliver Dolly

doi:10.1038/srep21226

Abstract

Transient receptor potential (TRP) A1 and V1 channels relay sensory signals, yet little is known about their transport to the plasmalemma during inflammation. Herein, TRPA1 and TRPV1 were found on vesicles containing calcitonin gene-related peptide (CGRP), accumulated at sites of exo- and endo-cytosis, and co-localised on fibres and cell bodies of cultured sensory neurons expressing both. A proinflammatory cytokine, TNFα, elevated their surface content, and both resided in close proximity, indicating co-trafficking. Syntaxin 1–interacting protein, Munc18–1, proved necessary for the response to TNFα, and for TRPV1-triggered CGRP release. TNFα-induced surface trafficking of TRPV1 and TRPA1 required a synaptic vesicle membrane protein VAMP1 (but not 2/3), which is essential for CGRP exocytosis from large dense-core vesicles. Inactivation of two proteins on the presynaptic plasma membrane, syntaxin-1 or SNAP-25, by botulinum neurotoxin (BoNT)/C1 or /A inhibited the TNFα-elevated delivery. Accordingly, enhancement by TNFα of Ca2+ influx through the upregulated surface-expressed TRPV1 and TRPA1 channels was abolished by BoNT/A. Thus, in addition, the neurotoxins’ known inhibition of the release of pain transmitters, their therapeutic potential is augmented by lowering the exocytotic delivery of transducing channels and the resultant hyper-sensitisation in inflammation.

Highlights

Considerable research has focused on the vanilloid (V1) and ankyrin (A1) varieties of transient receptor potential (TRP) cation channels in sensory neurons, because of their pivotal roles in the transduction of pain signals[1]
TRPV1 was visualized on the cell bodies and co-localized on neurites with large dense-core vesicles (LDCVs) markers, as well as synaptic vesicle protein 2 (SV2) (Fig. 1a); Pearson’s correlation coefficients were 0.93 ± 0.05 for TRPV1&secretogranin II (SgII), 0.91 ± 0.02 for TRPV1&calcitonin gene-related peptide (CGRP), 0.89 ± 0.03 for TRPV1& substance P (SP), 0.9 ± 0.04 for TRPV1&SV2 calculated from 20 images from 3 independent cultures
To ascertain whether LDCVs containing TRPV1 and TRPA1 can fuse with the plasmalemma in live trigeminal ganglion neurons (TGNs), the sensory neurons were labelled with a tagged marker of exo-/endo-cytosis, an antibody (Syt-ecto) against a luminal domain of the synaptic vesicle protein, synaptotagmin I42,43

Summary

Introduction

Considerable research has focused on the vanilloid (V1) and ankyrin (A1) varieties of transient receptor potential (TRP) cation channels in sensory neurons, because of their pivotal roles in the transduction of pain signals[1]. Activation of peripheral TRPA1 plays a critical role in the development of TNFα -induced mechanical hyperalgesia, and in sustaining the mechanical hyperalgesia observed after intra-articular injection of complete Freund’s adjuvant to induce arthritis-like symptoms in mice[35] These data suggest that negating TRPV1 and TRPA1 may reduce neuropathic and arthritic pain. Expressing TRPV1-V5 tagged fusion facilitated a proximity ligation assay (PLA), which demonstrated interaction and co-trafficking of this channel with TRPA1 upon stimulation with TNFα The latter seems to involve, at least, a portion of LDCVs that release CGRP from cultured neurons. Specific knockdown of Munc[], a syntaxin 1-interacting protein, provided the first evidence for its involvement These new findings should aid the designing of novel therapeutics for normalizing the increased surface appearance of transducing channels and associated neuronal hyper-excitability upon inflammatory/painful stimulation

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Conclusion