TRPA1 Is Functionally Expressed Primarily by IB4-Binding, Non-Peptidergic Mouse and Rat Sensory Neurons

Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.
PLoS ONE (Impact Factor: 3.23). 10/2012; 7(10):e47988. DOI: 10.1371/journal.pone.0047988
Source: PubMed


Subpopulations of somatosensory neurons are characterized by functional properties and expression of receptor proteins and surface markers. CGRP expression and IB4-binding are commonly used to define peptidergic and non-peptidergic subpopulations. TRPA1 is a polymodal, plasma membrane ion channel that contributes to mechanical and cold hypersensitivity during tissue injury, making it a key target for pain therapeutics. Some studies have shown that TRPA1 is predominantly expressed by peptidergic sensory neurons, but others indicate that TRPA1 is expressed extensively within non-peptidergic, IB4-binding neurons. We used FURA-2 calcium imaging to define the functional distribution of TRPA1 among peptidergic and non-peptidergic adult mouse (C57BL/6J) DRG neurons. Approximately 80% of all small-diameter (<27 µm) neurons from lumbar 1-6 DRGs that responded to TRPA1 agonists allyl isothiocyanate (AITC; 79%) or cinnamaldehyde (84%) were IB4-positive. Retrograde labeling via plantar hind paw injection of WGA-Alexafluor594 showed similarly that most (81%) cutaneous neurons responding to TRPA1 agonists were IB4-positive. Additionally, we cultured DRG neurons from a novel CGRP-GFP mouse where GFP expression is driven by the CGRPα promoter, enabling identification of CGRP-expressing live neurons. Interestingly, 78% of TRPA1-responsive neurons were CGRP-negative. Co-labeling with IB4 revealed that the majority (66%) of TRPA1 agonist responders were IB4-positive but CGRP-negative. Among TRPA1-null DRGs, few small neurons (2-4%) responded to either TRPA1 agonist, indicating that both cinnamaldehyde and AITC specifically target TRPA1. Additionally, few large neurons (≥27 µm diameter) responded to AITC (6%) or cinnamaldehyde (4%), confirming that most large-diameter somata lack functional TRPA1. Comparison of mouse and rat DRGs showed that the majority of TRPA1-responsive neurons in both species were IB4-positive. Together, these data demonstrate that TRPA1 is functionally expressed primarily in the IB4-positive, CGRP-negative subpopulation of small lumbar DRG neurons from rodents. Thus, IB4 binding is a better indicator than neuropeptides for TRPA1 expression.

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Available from: Cheryl Stucky, Mar 11, 2014
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    • "These results suggest that Tmem100 is primarily expressed in peptidergic DRG neurons (Figure 1E), many of which are TRPV1 and TRPA1 double positive (Figure 1F) (Bautista et al., 2006; Story et al., 2003). A recent study has shown that TRPA1 is functionally expressed in IB4 + nonpeptidergic neurons (Barabas et al., 2012). The culture conditions of dissociated DRG neurons can influence the expression of TRPA1. "
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    ABSTRACT: TRPA1 and TRPV1 are crucial pain mediators, but how their interaction contributes to persistent pain is unknown. Here, we identify Tmem100 as a potentiating modulator of TRPA1-V1 complexes. Tmem100 is coexpressed and forms a complex with TRPA1 and TRPV1 in DRG neurons. Tmem100-deficient mice show a reduction in inflammatory mechanical hyperalgesia and TRPA1- but not TRPV1-mediated pain. Single-channel recording in a heterologous system reveals that Tmem100 selectively potentiates TRPA1 activity in a TRPV1-dependent manner. Mechanistically, Tmem100 weakens the association of TRPA1 and TRPV1, thereby releasing the inhibition of TRPA1 by TRPV1. A Tmem100 mutant, Tmem100-3Q, exerts the opposite effect; i.e., it enhances the association of TRPA1 and TRPV1 and strongly inhibits TRPA1. Strikingly, a cell-permeable peptide (CPP) containing the C-terminal sequence of Tmem100-3Q mimics its effect and inhibits persistent pain. Our study unveils a context-dependent modulation of the TRPA1-V1 complex, and Tmem100-3Q CPP is a promising pain therapy. Copyright © 2015 Elsevier Inc. All rights reserved.
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    • "Previous experiments showed maximal responses to 1 mmol/L menthol in patch clamp experiments, and observed calcium transients in response to both 10 lmol/L and 100 lmol/L menthol [44]. In addition, the EC50 for cinnamaldehyde acting upon DRG neurons or transfected CHO cells is approximately 70 lmol/L [3] [4]. Thus, a submaximal concentration of either 100 lmol/L menthol [35] or 70 lmol/L cinnamaldehyde [3] was applied for 3 minutes. "
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    • "Several studies on rat and mouse DRG neurons showed a preferential ASIC expression in IB4-negative neurons (Dirajlal et al., 2003; Liu et al., 2004; Poirot et al., 2006). In mouse, IB4-negative DRG neurons express almost exclusively neuropeptides, while this negative correlation is much weaker in rat (Barabas et al., 2012; Price and Flores, 2007). PcTx1-sensitive currents were found only in 5% of rat DRG neurons in one study (Deval et al., 2008), and in ~ 30% of DRG neurons in our previous study (Poirot et al., 2006). "
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    ABSTRACT: Inflammatory mediators induce neuropeptide release from nociceptive nerve endings and cell bodies, causing increased local blood flow and vascular leakage resulting in edema. Neuropeptide release from sensory neurons depends on an increase in intracellular Ca(2+) concentration. In this study we investigated the role of two types of pH sensors in acid-induced Ca(2+) entry and neuropeptide release from dorsal root ganglion (DRG) neurons. The transient receptor potential vanilloid 1 channel (TRPV1) and acid-sensing ion channels (ASICs) are both H(+)-activated ion channels present in these neurons, and are therefore potential pH sensors for this process. We demonstrate with in situ hybridization and immunocytochemistry that TRPV1 and several ASIC subunits are co-expressed with neuropeptides in DRG neurons. Activation of ASICs and of TRPV1 led to an increase in intracellular Ca(2+) concentration. While TRPV1 has a high Ca(2+) permeability and allows direct Ca(2+) entry when activated, we show here that ASICs of DRG neurons mediate Ca(2+) entry mostly by depolarization-induced activation of voltage-gated Ca(2+) channels and only to a small extent via the pore of Ca(2+)-permeable ASICs. Extracellular acidification led to release of the neuropeptide calcitonin gene-related peptide from DRG neurons. The pH dependence and the pharmacological profile indicated that TRPV1, but not ASICs, induced neuropeptide secretion. In conclusion, this study shows that although both TRPV1 and ASICs mediate Ca(2+) influx, TRPV1 is the principal sensor for acid-induced neuropeptide secretion from sensory neurons.
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