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N-terminal tetrapeptide T/SPLH motifs contribute to multimodal activation of human TRPA1 channel.

ABSTRACT: Human transient receptor potential ankyrin channel 1 (TRPA1) is a polymodal sensor implicated in pain, inflammation and itching. An important locus for TRPA1 regulation is the cytoplasmic N-terminal domain, through which various exogenous electrophilic compounds such as allyl-isothiocyanate from mustard oil or cinnamaldehyde from cinnamon activate primary afferent nociceptors. This major region is comprised of a tandem set of 17 ankyrin repeats (AR1-AR17), five of them contain a strictly conserved T/SPLH tetrapeptide motif, a hallmark of an important and evolutionarily conserved contribution to conformational stability. Here, we characterize the functional consequences of putatively stabilizing and destabilizing mutations in these important structural units and identify AR2, AR6, and AR11-13 to be distinctly involved in the allosteric activation of TRPA1 by chemical irritants, cytoplasmic calcium, and membrane voltage. Considering the potential involvement of the T/SP motifs as putative phosphorylation sites, we also show that proline-directed Ser/Thr kinase CDK5 modulates the activity of TRPA1, and that T673 outside the AR-domain is its only possible target. Our data suggest that the most strictly conserved N-terminal ARs define the energetics of the TRPA1 channel gate and contribute to chemical-, calcium- and voltage-dependence.

SUBMITTER: Hynkova A

PROVIDER: S-EPMC4922051 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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N-terminal tetrapeptide T/SPLH motifs contribute to multimodal activation of human TRPA1 channel.

Hynkova Anna A Marsakova Lenka L Vaskova Jana J Vlachova Viktorie V

Scientific reports 20160627

Human transient receptor potential ankyrin channel 1 (TRPA1) is a polymodal sensor implicated in pain, inflammation and itching. An important locus for TRPA1 regulation is the cytoplasmic N-terminal domain, through which various exogenous electrophilic compounds such as allyl-isothiocyanate from mustard oil or cinnamaldehyde from cinnamon activate primary afferent nociceptors. This major region is comprised of a tandem set of 17 ankyrin repeats (AR1-AR17), five of them contain a strictly conserv ...[more]

PMID: 27345869

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Project description:The mechanism of pain in dentine hypersensitivity is poorly understood but proposed to result from the activation of dental sensory neurons in response to dentinal fluid movements. Odontoblasts have been suggested to contribute to thermal and mechanosensation in the tooth via expression of transient receptor potential (TRP) channels. However, a mechanism by which odontoblasts could modulate neuronal activity has not been demonstrated. In this study, we investigated functional TRP channel expression in human odontoblast-like cells and measured ATP release in response to TRP channel activation. Human immortalized dental pulp cells were driven toward an odontoblast phenotype by culture in conditioned media. Functional expression of TRP channels was determined with reverse transcription polymerase chain reaction and ratiometric calcium imaging with Fura-2. ATP release was measured using a luciferin-luciferase assay. Expression of mRNA for TRPA1, TRPV1, and TRPV4 but not TRPM8 was detected in odontoblasts by reverse transcription polymerase chain reaction. Expression of TRPV4 protein was detected by Western blotting and immunocytochemistry. The TRPA1 agonists allyl isothiocyanate and cinnamaldehyde and the TRPV4 agonist GSK1016790A caused a concentration-dependent increase in intracellular Ca(2+) concentration that was inhibited by the selective antagonists HC030031, AP18, and HC067047, respectively. In contrast, exposure to the TRPV1 agonist capsaicin or the TRPM8 agonist icilin had no effect on intracellular Ca(2+) concentration. Treatment with allyl isothiocyanate, cinnamaldehyde, or GSK1016790A caused an increase in ATP concentration in culture medium that was abolished by preincubation with TRP channel antagonists. These data demonstrate that activation of TRPA1 and TRPV4 channels in human odontoblast-like cells can stimulate ATP release. We were unable to confirm the presence of thermosensitive TRPV1 and TRPM8 that has previously been reported in odontoblasts.

Dataset Information

N-terminal tetrapeptide T/SPLH motifs contribute to multimodal activation of human TRPA1 channel.

Publications

N-terminal tetrapeptide T/SPLH motifs contribute to multimodal activation of human TRPA1 channel.

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