Structural modeling and patch-clamp analysis of pain-related mutation TRPA1-N855S reveal inter-subunit salt bridges stabilizing the channel open state.

摘要: The ankyrin transient receptor potential channel TRPA1 is a polymodal sensor for noxious stimuli, and hence promising target treating chronic pain. This tetrameric six-transmembrane segment (S1-S6) can be activated by various pungent chemicals, such as allyl isothiocyanate or cinnamaldehyde, but also intracellular Ca(2+) depolarizing voltages. Within the S4-S5 linker of human TRPA1, gain-of-function mutation, N855S, was recently found to underlie familial episodic pain syndrome, manifested bouts severe upper body pain, triggered physical stress, fasting, cold. To clarify structural basis this channelopathy, we derive model combining homology modeling, molecular dynamics simulations, point mutagenesis electrophysiology. In vicinity N855, reveals inter-subunit salt bridges between E854 K868. Using heterologous expression recombinant wild-type mutant channels in HEK293T cells, indeed that charge-reversal mutants E854R K868E exhibited dramatically reduced responses chemical voltage whereas charge-swapping mutation E854R/K868E substantially rescued their functionalities. Moreover, analysis highly conserved charged residues within region revealed phenotype R852E with an increased basal activity, loss Ca(2+)-induced potentiation accelerated Ca(2+)-dependent inactivation. Based on comparison atomic-level structure related TRPV1, propose adjacent regions are crucial stabilizing conformations associated chemically voltage-induced gating ion channel.