Intracellular cavity of sensor domain controls allosteric gating of TRPA1 channel

Abstract

Transient receptor potential ankyrin 1 (TRPA1) is a temperature-sensitive ion channel activated by various pungent and irritant compounds that can produce pain in humans. Its activation involves an allosteric mechanism whereby electrophilic agonists evoke interactions within cytosolic domains and open the channel pore through an integrated nexus formed by intracellular membrane proximal regions that are densely packed beneath the lower segment of the S1-S4 sensor domain. Studies indicate that this part of the channel may contain residues that form a water-accessible cavity that undergoes changes in solvation during channel gating. We identified conserved polar residues facing the putative lower crevice of the sensor domain that were crucial determinants of the electrophilic, voltage, and calcium sensitivity of the TRPA1 channel. This part of the sensor may also comprise a domain capable of binding to membrane phosphoinositides through which gating of the channel is regulated in a state-dependent manner.

Putative interaction site for membrane phospholipids controls activation of TRPA1 channel at physiological membrane potentials

“…5A-C). Control cells transfected with wild-type TRPA1 responded robustly to application of agonist in agreement with our previous reports (e.g., [23][24][25]). At its other side, it contacts the sensor from an adjacent subunit through stacking interactions with the side chains of F716 and H719 from S1 helix and F853 from the S4-S5 linker (Fig.…”

“…1. To stimulate the cells, we used a previously established protocol [23][24][25] in which currents were first induced by the electrophilic agonist in the absence of external Ca 2+ . (A) Schematic illustration of human TRPA1 subunit with depicted position of a poorly resolved flexible linker connecting the TRP-like domain with the C-terminal coiled coil linker region (colored region containing the calmodulin-binding domain in green, and an overlapping violet region containing predicted short a-helix).…”

“…The density map for the flexible loop T1003-Q1031 at one side extends to the vicinity of the poorly resolved intracellular S2-S3 linker of the S1-S4 sensor domain. The currents developed slowly and were further diminished by the addition of external calcium, thus resembling H719A [25]. 5B).…”

“…It is possible that mutations of other contacting residues would give similar defects in channel functioning once they are identified [26]. We have previously shown that the substitution of H719 by a small residue alanine induced serious functional defects manifested as significant changes in rectification and kinetics of the currents that were blocked but not potentiated by external calcium [25]. 5A-C).…”

“…The lower segment of the S1-S4 sensor domain with the C-terminal TRP-like helix below forms an integrated nexus that has been previously proposed to be capable of binding membrane phosphoinositides including PIP 2 [25]. Our ligand docking studies showed that the negatively charged inositol trisphosphate head group of PIP 2 may adopt several different conformations within the inner crevice formed by the S1-S4 helical bundle of the sensor to contact polar residues in the inner cavity of the sensor (H719, N722, K787, K796, R852) and the TRP-like helix (K989).…”