Pinacidil, a Katp channel opener, identified as a novel agonist for TRPA1

Abstract

The transient receptor potential Ankyrin 1 (TRPA1) cation channel is activated by various pungent and irritant compounds, and it also mediates the perception of noxious cold. Identification of different agonists for this channel is important for understanding its activation mechanism. Therefore, a screen for novel TRPA1 agonists was performed using an agonist-induced calcium influx assay. Out of 90 compounds screened, pinacidil was identified as a novel agonist for this channel. Pinacidil is a known opener of the K atp channel, for which it has an EC 50 value of 1-3 μmol/L. In comparison, the EC 50 value of pinacidil for TRPA1 is relatively high (260 μmol/L). Recombinant HEK-TRPA1 cells did not respond to P1075, another K atp channel opener, suggesting that the effect of pinacidil on TRPA1 was highly specific. Further studies revealed that the agonist activity of pinacidil could be blocked by the TRP channel inhibitors, ruthenium red and HC-030031. Using glutathione (GSH) and site-specific mutagenesis, we demonstrated that pinacidil could activate TRPA1 by covalent modification of the critical amino acids C619, C639 and C663 in the N-terminus of TRPA1. TRPA1, pinacidil, covalent modification Citation:Ma L H, Deng Y, Zhang B, et al. Pinacidil, a K atp channel opener, identified as a novel agonist for TRPA1.

Cardamonin, a Novel Antagonist of hTRPA1 Cation Channel, Reveals Therapeutic Mechanism of Pathological Pain

“…Recombinant HEK293/TRPA1, HEK293/TRPV1, and HEK293/TRPV4 cell lines were generated by HD Bioscience Co. Ltd (Shanghai, China) following standard procedures as reported previously [44]. HEK293/TRPA1 and HEK293/TRPV4 cells were cultured in complete DMEM medium with 100 μg/mL hygromycin B, 50 U/mL penicillin, and 100 μg/mL streptomycin.…”

Identification of Natural Compound Carnosol as a Novel TRPA1 Receptor Agonist

“…HEK 293 cell lines stably expressing human TRPA1, TRPM8 and TRPV3 channels, respectively, were established by HD Bioscience Co. Ltd (SH, China) according to the cell line generation procedure in published articles of our laboratory [51,52]. All HEK293 cell lines used in this study were routinely maintained in DMEM containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere of 5% CO 2 at 37 °C.…”

The cardiac TRPA1 channel drives calcium-mediated mechano-arrhythmogenesis

“…We thus sought to determine the source of the overall increase in mechano-arrhythmogenicity with pinacidil. While pinacidil is thought to be a specific activator of KATP channels, it has also been shown to increase the activity of transient receptor potential kinase ankyrin 1 (TRPA1) channels in HEK293 cells 24 . TRPA1 channels are inherently mechanosensitive 5 (contributing to mechanically-activated currents in neurons 6 and vertebrate hair cells 7 ), have been shown to be highly expressed and functional in mouse ventricular myocytes 25 and to drive changes in rhythm in response to mechanical stimulation in the Drosophila heart 8 , and are important in a range of cardiovascular functions and pathologies 12 .…”

“…Interestingly, while pinacidil is generally thought of as a specific activator of K ATP channels, it has been shown to also increase the activity of transient receptor potential kinase ankyrin 1 (TRPA1) channels in HEK293 cells, resulting in a depolarising trans-membrane current and Ca 2+ influx. 29 Importantly in the context of mechano-arrhythmogenesis, TRPA1 channels: (i) are inherently mechano-sensitive 5 and contribute to mechanically-evoked electrical responses (measured as trans-membrane currents and deduced from AP initiation) in sensory neurons, 6–8 astrocytes, 9 and vertebrate hair cells; 10 (ii) have been found to be functionally expressed in murine ventricular CM 30 (although not all studies have found this) 31 and to drive changes in Drosophila heart rhythm in response to mechanical stimulation; 11 and (iii) are important for cardiovascular function and disease progression. 13 We hypothesised that, if TRPA1 channels are functionally expressed in rabbit left ventricular CM, then they could contribute to the increase in mechano-arrhythmogenesis seen with pinacidil treatment.…”

“…As a result, their activation in ventricular myocytes will always contribute to repolarisation, so they cannot account for the stretch-induced excitation seen in this and other studies (in fact, they would be expected to oppose depolarisation). Alternatively, while pinacidil is generally thought of as a specific activator of KATP channels, it has also been shown to increase the activity of transient receptor potential kinase ankyrin 1 (TRPA1) channels in HEK293 cells, 29 resulting in a depolarising trans-membrane influx. Importantly, TRPA1 channels: (i) are inherently mechano-sensitive 5 and contribute to mechanically-evoked action potentials 6 and currents in sensory neurons, 7,8 astrocytes, 9 and vertebrate hair cells; 10 (ii) have been shown to be functionally expressed in ventricular myocytes of mice 30 (although others have found conflicting results 31 ) and to drive changes in rhythm in response to mechanical stimulation in the Drosophila heart; 11 and (iii) are important in a range of cardiovascular functions and pathologies.…”