The mammalian canonical transient receptor channels (TRPCs) are considered to be candidates for store-operated calcium channels (SOCCs). Many studies have addressed how TRPC3 channels are affected by depletion of intracellular calcium stores. Conflicting results have been shown for TRPC3 regarding its function, and this has been linked to its level of expression in various systems. In the present study, we have investigated how overexpression of TRPC3 interferes with the regulation of intracellular calcium stores. We demonstrate that overexpression of TRPC3 reduces the mobilization of calcium in response to stimulation of the cells with thapsigargin (TG) or the G-protein coupled receptor agonist sphingosine-1-phosphate (S1P). Our results indicate that this is the result of the expression of TRPC3 channels in the endoplasmic reticulum (ER), thus depleting ER calcium stores. OAG evoked calcium entry in cells overexpressing TRPC3, indicating that functional TRPC3 channels were also expressed in the plasma membrane. Taken together, our results show that overexpression of the putative SOCC, TRPC3, actually reduces the calcium content of intracellular stores, but does not enhance agonist-evoked or store-dependent calcium entry. Our results may, in part, explain the conflicting results obtained in previous studies on the actions of TRPC3 channels.

Genetically encoded calcium indicators (GECI) such as GCaMP3 are attracting significant attention as a good option for measuring intracellular calcium levels. Recently, a modified GCaMP3 called dCys-GCaMP3 was developed by replacing two threonine residues with cysteines. dCys-GCaMP3 proved to be a better calcium indicator, but it was not clear how and why the two cysteine residues were able to enhance the protein’s calcium sensitivity. The aim of the present study was to investigate the possible roles of these cysteine residues in dCys-GCaMP3. dCys-GCaMP3 (Thr330Cys;Thr364Cys) exhibited enhanced fluorescence intensity compared to the canonical GCaMP3 in calcium imaging experiments. However, substitution of a single residue at position 330 with cysteine (Thr330Cys) also afforded comparable sensitivity to GCaMP3. In contrast, the other single residue substitution at position 364 with cysteine (Thr364Cys) failed to enhance calcium sensitivity, showing that cysteine at position 330 is essential to improve calcium sensitivity. Thr330Cys substitution in the GCaMP3 or “Cys³³⁰–GCaMP3” showed significantly reduced background fluorescence, and the fluorescence intensity was proportional to the amount of DNA used to transfect the cells used in the study. The substitute had to be cysteine, because replacement with other amino acids such as alanine, valine, and aspartate did not improve GCaMP3‘s calcium sensitivity. Cys³³⁰–GCaMP3 outperformed a synthetic calcium-specific indicator, Fluo-3, in various calcium imaging experiments. Thus, the present study asserts that substituting the threonine at position 330 in GCaMP3 with cysteine is essential to enhance calcium sensitivity, and suggests that Cys³³⁰–GCaMP3 can be used as a potent fluorescent calcium indicator to measure intracellular calcium levels.

BACKGROUND:

Pruritus is a cardinal symptom of atopic dermatitis, and an increased cutaneous sensory network is thought to contribute to pruritus. Although the immune cell-IL-31-neuron axis has been implicated in severe pruritus during atopic skin inflammation, IL-31’s neuropoietic potential remains elusive.

OBJECTIVE:

We sought to analyze the IL-31-related transcriptome in sensory neurons and to investigate whether IL-31 promotes sensory nerve fiber outgrowth.

The pruritus and TH2associated cytokine IL31promotes growth of sensory nerves

Utilizing a novel molecular model of TRPC3, based on the voltage-gated sodium channel from Arcobacter butzleri (Na(V)AB) as template, we performed structure-guided mutagenesis experiments to identify amino acid residues involved in divalent permeation and gating. Substituted cysteine accessibility screening within the predicted selectivity filter uncovered amino acids 629-631 as the narrowest part of the permeation pathway with an estimated pore diameter of < 5.8Å. E630 was found to govern not only divalent permeability but also sensitivity of the channel to block by ruthenium red. Mutations in a hydrophobic cluster at the cytosolic termini of transmembrane segment 6, corresponding to the S6 bundle crossing structure in Na(V)AB, distorted channel gating. Removal of a large hydrophobic residue (I667A or I667E) generated channels with approximately 60% constitutive activity, suggesting I667 as part of the dynamic structure occluding the permeation path. Destabilization of the gate was associated with reduced Ca2+ permeability, altered cysteine cross-linking in the selectivity filter and promoted channel block by ruthenium red. Collectively, we present a structural model of the TRPC3 permeation pathway and localize the channel’s selectivity filter and the occluding gate. Moreover, we provide evidence for allosteric coupling between the gate and the selectivity filter in TRPC3.

A novel homology model of TRPC3 reveals allosteric coupling between gate and selectivity filter

BACKGROUND:

Substance P (SP) is linked to itch and inflammation through activation of receptors on mast cells and sensory neurons. There is increasing evidence that SP functions through Mas-related G protein-coupled receptors (Mrgprs) in addition to its conventional receptor, neurokinin-1.

OBJECTIVE:

Because Mrgprs mediate some aspects of inflammation that had been considered mediated by neurokinin-1 receptor (NK-1R), we sought to determine whether itch induced by SP can also be mediated by Mrgprs.

METHODS:

Genetic and pharmacologic approaches were used to evaluate the contribution of Mrgprs to SP-induced scratching behavior and activation of cultured dorsal root ganglion neurons from mice.

RESULTS:

SP-induced scratching behavior and activation of cultured dorsal root ganglion neurons was dependent on Mrgprs rather than NK-1R.

CONCLUSION:

We deduce that SP activates MrgprA1 on sensory neurons rather than NK-1R to induce itch.

Recent studies have shown that approximately 70% of patients with severe atopic dermatitis (AD) develop asthma. Development of AD in infancy and subsequent other atopic diseases such as asthma in childhood is referred to as atopic march. However, a causal link between the diseases of atopic march has remained largely unaddressed, possibly due to lack of a proper animal model. Recently, we developed an AD rat model showing chronically relapsing dermatitis and scratching behaviors induced by neonatal capsaicin treatment. Here, we investigated whether our model also showed asthmatic changes, with the aim of expanding our AD model into an atopic march model. First, we confirmed that capsaicin treatment (50 mg/kg within 24 h after birth) induced dermatitis and scratching behaviors until 6 weeks of age. After that, the mRNA expression of Th1 and Th2 cytokines, such as IFN-γ and TNF-α, and IL-4, IL-5, and IL-13, respectively, was quantified with quantitative real-time polymerase chain reaction in the skin and the lungs. The number of total cells and eosinophils was counted in bronchoalveolar lavage (BAL) fluid. The levels of IgE in the serum and BAL fluid were determined with enzyme-linked immunosorbent assay. Paraffin-embedded sections (4 μm) were stained with hematoxylin/eosin to analyze the morphology of the lung and the airway. Airway responsiveness was measured in terms of airway resistance and compliance using the flexiVent system. In the capsaicin-treated rats, persistent dermatitis developed, and scratching behaviors increased over several weeks. The levels of IgE in the serum and BAL fluid as well as the mRNA expression of Th2 cytokines, including IL-4, IL-5, and IL-13, in both the skin and the lungs were elevated, and the number of eosinophils in the BAL fluid was also increased in the capsaicin-treated rats compared to control rats. Morphological analysis of the airway revealed smooth muscle hypertrophy and extensive mucus plug in the capsaicin-treated rats. Functional studies demonstrated an increment of the airway resistance and a decrement of lung compliance in the capsaicin-treated rats compared to control rats. Taken together, our findings suggested that neonatal capsaicin treatment induced asthma-like airway inflammation and responses in juvenile rats.

17.06.30

Diabetic neuropathy is a severe complication of long-standing diabetes and one of the major etiologies of neuropathic pain. Diabetes is associated with an increased formation of reactive oxygen species and the electrophilic dicarbonyl compound methylglyoxal (MG). Here we show that MG stimulates heterologously expressed TRPA1 in CHO cells and natively expressed TRPA1 in MDCK cells and DRG neurons. MG evokes [Ca(2+)]i-responses in TRPA1 expressing DRG neurons but is without effect in neurons cultured from Trpa1(-/-) mice. Consistent with a direct, intracellular action, we show that methylglyoxal is significantly more potent as a TRPA1 agonist when applied to the intracellular face of excised membrane patches than to intact cells. Local intraplantar administration of MG evokes a pain response in Trpa1(+/+) but not in Trpa1(-/-) mice. Furthermore, persistently increased MG levels achieved by two weeks pharmacological inhibition of glyoxalase-1 (GLO-1), the rate-limiting enzyme responsible for detoxification of MG, evokes a progressive and marked thermal (cold and heat) and mechanical hypersensitivity in wildtype but not in Trpa1(-/-) mice. Our results thus demonstrate that TRPA1 is required both for the acute pain response evoked by topical MG and for the long-lasting pronociceptive effects associated with elevated MG in vivo. In contrast to our observations in DRG neurons, MG evokes indistinguishable [Ca(2+)]i-responses in pancreatic β-cells cultured from Trpa1(+/+) and Trpa1(-/-) mice. In vivo, the TRPA1 antagonist HC030031 impairs glucose clearance in the glucose tolerance test both in Trpa1(+/+) and Trpa1(-/-) mice, indicating a non-TRPA1 mediated effect and suggesting that results obtained with this compound should be interpreted with caution. Our results show that TRPA1 is the principal target for MG in sensory neurons but not in pancreatic β-cells and that activation of TRPA1 by MG produces a painful neuropathy with the behavioral hallmarks of diabetic neuropathy.

journal.pone.0077986