Structural modeling and patch-clamp analysis of pain-related mutation TRPA1-N855S reveal inter-subunit salt bridges stabilizing the channel open state. (June 2015)

• Record Type: Journal Article
• Authors: Zíma, Vlastimil; Witschas, Katja; Hynkova, Anna; Zímová, Lucie; Barvík, Ivan; Vlachova, Viktorie
• Is Part Of: Neuropharmacology. Volume 93(2015)
• Page Start: 294
• Page End: 307
• Publication Date: 2015-06
• Subjects: Ankyrin receptor subtype 1 -- Transient receptor potential -- S4–S5-linker -- Mutagenesis -- Homology modeling -- Molecular dynamics; AITC allyl isothiocyanate -- CA cinnamaldehyde -- HEK293T cell human embryonic kidney-293 cell expressing the large T-antigen of SV40 (simian virus 40) -- TRP transient receptor potential -- TRPA TRP ankyrin -- MD molecular dynamics; Neuropsychopharmacology -- Periodicals; Autonomic Agents -- Periodicals; Neuropsychopharmacologie -- Périodiques; Neuropsychopharmacology; Periodicals; Electronic journals; 615.78
• DOI: 10.1016/j.neuropharm.2015.02.018
• Languages: English
• ISSNs: 0028-3908

Abstract

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