Structural Insights into the Dynamics of the TRPA1 Activation Mechanism

Abstract

TRPA1 is a Ca2+-permeable, non-selective cation channel and one of the key pain sensors in mammals. Pain sensation mediated by TRPA1 involves modification of N-terminal cysteine residues on the channel by thiol-reactive compounds and inflammatory mediators. Recent mutagenesis and chimeric studies have suggested that N-terminal cysteine residues (C622, C642, C666) located on the flexible linker region of the channel are involved in channel activation and desensitization, but how conformational changes in the flexible linker region lead to activation and desensitization is unresolved.Capitalizing on our ability to isolate pure and functional TRPA1 channel protein, we recently provided the first insight into the channel architecture by reconstructing a TRPA1 structure to 16 A resolution in resting state using electron microscopy (EM). Fitting TRPA1 homology model into the EM density lead us to hypothesize that the critical cysteines on the flexible linker region (C622, C642, C666) are in close proximity to one another and that covalent modification of cysteines within this pocket could promote conformational changes leading to channel gating and desensitization. Furthermore, we performed a mass spectrometry analysis of the in vivo TRPA1 thiol status and discovered that, when treated with a thiol-reactive TRPA1 agonist, C622 and C666 could form a disulfide bond with each other or with two other cysteine residues.Our current structural and biophysical analyses using EM, mass spectrometry and homology modeling are elucidating ligand-induced conformational changes in the channel's flexible linker region that occur during activation and desensitization. These insights bring us a greater understanding of the structural rearrangements involved in the gating and desensitization mechanisms of the TRPA1 ion channel.