Differentiation dependent expression of TRPA1 and TRPM8 channels in IMR-32 human neuroblastoma cells.

TRPA1 and TRPM8 are transient receptor potential (TRP) channels involved in sensory perception. TRPA1 is a non-selective calcium permeable channel activated by irritants and proalgesic agents. TRPM8 reacts to chemical cooling agents such as menthol. The human neuroblastoma cell line IMR-32 undergoes a remarkable differentiation in response to treatment with 5-bromo-2-deoxyuridine. The cells acquire a neuronal morphology with increased expression of N-type voltage gated calcium channels and neurotransmitters. Here we show using RT-PCR, that mRNA for TRPA1 and TRPM8 are strongly upregulated in differentiating IMR-32 cells. Using whole cell patch clamp recordings, we demonstrate that activators of these channels, wasabi, allyl-isothiocyanate (AITC) and menthol activate membrane currents in differentiated cells. Calcium imaging experiments demonstrated that AITC mediated elevation of intracellular calcium levels were attenuated by ruthenium red, spermine, and HC-030031 as well as by siRNA directed against the channel. This indicates that the detected mRNA level correlate with the presence of functional channels of both types in the membrane of differentiated cells. Although the differentiated IMR-32 cells responded to cooling many of the cells showing this response did not respond to TRPA1/TRPM8 channel activators (60% and 90% for AITC and menthol respectively). Conversely many of the cells responding to these activators did not respond to cooling (30%). This suggests that these channels have also other functions than cold perception in these cells. Furthermore, our results suggest that IMR-32 cells have sensory characteristics and can be used to study native TRPA1 and TRPM8 channel function as well as developmental expression.

Louhivuori LM ，Bart G ，Larsson KP ，Louhivuori V ，Näsman J ，Nordström T ，Koivisto AP ，Akerman KE ... -  《-》

Menthol derivative WS-12 selectively activates transient receptor potential melastatin-8 (TRPM8) ion channels.

Ma S ，G G ，Ak VE ，Jf D ，H H ... -  《Pakistan Journal of Pharmaceutical Sciences》

Enhanced activation of the transient receptor potential channel TRPA1 by ajoene, an allicin derivative.

TRPA1 is a calcium-permeable, nonselective cation channel expressed in the dorsal root ganglion and trigeminal ganglia nociceptive neurons. It is activated by the pungent compounds in mustard oil (AITC, allyl isothiocyanate), cinnamon (cinnamaldehyde), garlic (allicin), and is believed to mediate the inflammatory actions of environmental irritants and proalgesic agents. Thiosulfinate (allicin) and isothiocyanate (AITC) compounds contain reactive electrophilic chemical groups that react with cysteine residues within the TRPA1 channel N terminus, leading to channel activation. Ajoene also contains reactive electrophilic chemical groups likely to target TRPA1 channel. Here, we have used voltage-clamp recordings to show that TRPA1-responses are enhanced by ajoene application in a Xenopus oocyte expression system. Though ajoene alone did not activate TRPA1, subsequent application of ajoene enhanced the AITC-, allicin- and depolarization-induced responses of TRPA1. Moreover, when increasing concentrations of ajoene were applied along with constant concentrations of allicin or AITC, stronger responses were elicited. These findings suggest that ajoene is a novel TRPA1 channel enhancer, operating in a channel-opening-dependent manner.

Yassaka RT ，Inagaki H ，Fujino T ，Nakatani K ，Kubo T ... -  《-》

NPPB structure-specifically activates TRPA1 channels.

TRPA1 channels have been found to play an important role in mammalian pain sensation, especially when the pain is caused by chemicals on site of inflammation. A large number of structurally diverse chemicals are found to activate TRPA1 channels, implicating a potential chemosensor in neuronal nociception. Identification of the channel activation by cysteine modification through covalent chemical reaction provides arguments for the diversity of the agonist structures. However, it is largely unknown how nonreactive compounds activate TRPA1 channels. Here, we report that NPPB, a classic Cl(-) channel blocker, potently activated human TRPA1 channels overexpressed in mammalian HEK-293 cells. This effect was confirmed in Ca(2+) imaging assay, patch clamp whole cell and single channel recordings. The NPPB response was quick, fully reversible and replicable, contrary to the effect of covalent modification by AITC. The mutagenesis studies revealed a refreshed look at several mutations known to be critical for the actions of AITC and menthol. The blocking profile of NPPB on these mutants showed that the NPPB activation was similar to that of FTS and different from AITC and menthol. The results indicated a possible close interaction between S5 and N-terminal domains of the channel. We also tested a group of NPPB analogs on TRPA1 channel activities. The results demonstrated that NPPB activation was tightly associated with chemical structure. None of the single chemical group was sufficient to activate the channel, indicating that NPPB activated TRPA1 through a structure-specific mechanism.

Liu K ，Samuel M ，Ho M ，Harrison RK ，Paslay JW ... -  《-》

Activation of TRPA1 channels by the fatty acid amide hydrolase inhibitor 3'-carbamoylbiphenyl-3-yl cyclohexylcarbamate (URB597).

Niforatos W ，Zhang XF ，Lake MR ，Walter KA ，Neelands T ，Holzman TF ，Scott VE ，Faltynek CR ，Moreland RB ，Chen J ... -  《MOLECULAR PHARMACOLOGY》