Background: TRPA1 channel is modulated by Ca 2ϩ , but the molecular mechanisms are unclear. Results: Mutations in the distal C-terminal acidic domain altered Ca 2ϩ dependence of TRPA1. Conclusion: The C-terminal acidic cluster is involved in the Ca 2ϩ -induced potentiation and inactivation of TRPA1. Significance: Identification of the Ca 2ϩ -dependent domain is important for understanding the role of TRPA1 in chemical nociception.
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.
Gain-of-function (GOF) mutations in ion channels are rare events, which lead to increased agonist sensitivity or altered gating properties, and may render the channel constitutively active. Uncovering and following characterization of such mutants contribute substantially to the understanding of the molecular basis of ion channel functioning. Here we give an overview of some GOF mutants in polymodal ion channels specifically involved in transduction of painful stimuli – TRPV1 and TRPA1, which are scrutinized by scientists due to their important role in development of some pathological pain states. Remarkably, a substitution of single amino acid in the S4-S5 region of TRPA1 (N855S) has been recently associated with familial episodic pain syndrome. This mutation increases chemical sensitivity of TRPA1, but leaves the voltage sensitivity unchanged. On the other hand, mutations in the analogous region of TRPV1 (R557K and G563S) severely affect all aspects of channel activation and lead to spontaneous activity. Comparison of the effects induced by mutations in homologous positions in different TRP receptors (or more generally in other distantly related ion channels) may elucidate the gating mechanisms conserved during evolution.
strips placed below. The membrane had 0.4 mm pores that allowed the cytokines released from the CD4þT cells to reach the SM but prevented direct contact between cells. We found a significant increase in Vmax when the SM were in contact with the T cells (0.3250.02 l/s) compared to the co-culture without contact (0.2550.01 l/s) or compared to the SM alone (0.2450.01 l/s; p=0.01). Thus, contact between SM and CD4þT cells is necessary for modulation of SM contractile properties and this may be mediated, at least in part, by increases in (þ)insert myosin heavy chain and MLCK expression. Supported by: CIHR, NIH-RO1HL103405.
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