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

Abstract

The transient receptor potential ankyrin 1 (TRPA1) channel is a polymodal sensor of environmental irritant compounds, endogenous proalgesic agents, and cold. Upon activation, TRPA1 channels increase cellular calcium levels via direct permeation and trigger signaling pathways that hydrolyze phosphatidylinositol‐4,5‐bisphosphate (PIP2) in the inner membrane leaflet. Our objective was to determine the extent to which a putative PIP2‐interaction site (Y1006‐Q1031) is involved in TRPA1 regulation. The interactions of two specific peptides (L992‐N1008 and T1003‐P1034) with model lipid membranes were characterized by biophysical approaches to obtain information about affinity, peptide secondary structure, and peptide effect in the lipid organization. The results indicate that the two peptides interact with lipid membranes only if PIP2 is present and their affinities depend on the presence of calcium. Using whole‐cell electrophysiology, we demonstrate that mutation at F1020 produced channels with faster activation kinetics and with a rightward shifted voltage‐dependent activation curve by altering the allosteric constant that couples voltage sensing to pore opening. We assert that the presence of PIP2 is essential for the interaction of the two peptide sequences with the lipid membrane. The putative phosphoinositide‐interacting domain comprising the highly conserved F1020 contributes to the stabilization of the TRPA1 channel gate.

Human and Mouse TRPA1 Are Heat and Cold Sensors Differentially Tuned by Voltage

“…During the review process, a structural study on TRPA1 was published identifying a direct interaction between N855 and the backbone carbonyl of C1024 from a short predicted C-terminal α-helix located at the cytosol-membrane interface near S1 and S4 of the sensor domain [64]. The findings support our previous predictions [56] and may partly explain our data presented in Figure S4.…”

“…Previous studies have identified the C-terminal loop as a site for Ca 2+ /calmodulin and phosphatidylinositol-4,5-bisphophate binding [54,56]. Mutations within this region affected TRPA1 responses induced by various stimuli including cinnamaldehyde, allyl isothiocyanate, voltage, Ca 2+ and carvacrol.…”

Proximal C-Terminus Serves as a Signaling Hub for TRPA1 Channel Regulation via Its Interacting Molecules and Supramolecular Complexes

“…For the experiments described in Figure 1, the extracellular bath solution contained: 140 mM NaCl, 5 mM KCl, 2 mM MgCl 2 , 5 mM EGTA, and 10 mM FIGURE 1 | Acute PIP 2 depletion has an opposite effect on cold-dependent gating of TRPA1 to missense histidine-to-arginine mutation at position 1018. (A) Schematic diagram shows binding of Ca 2+ -CaM (calmodulin) to C terminus of TRPA1 (Macikova et al, 2019). The binding domain for CaM partly overlaps with the proposed binding pocket for PIP 2 (in red) which includes H1018.…”

“…The proximal C terminus contains a TRP-like domain that interacts with a pre-S1 helix. There are at least two sites with separate functions from which the activity of TRPA1 can be regulated by membrane phosphatidylinositol-4,5-bisphosphate (PIP 2 ) or other phosphoinositides: the first, formed by the intracellular part of VSLD and contributed to by K989 from the TRP-like domain (Samad et al, 2011;Witschas et al, 2015;Zimova et al, 2018), and the second localized between adjacent subunits (T1003-P1034), capable of directly affecting the gating of the channel through the S4-S5 linker and R975 from the TRP-like domain (Macikova et al, 2019). Mutation at the highly conserved phenylalanine F1020 located in the center of the latter interacting region produced channels with faster activation kinetics and with significantly suppressed responses at negative membrane potentials.…”

“…A large number (>150) of single-point mutations and chimeras of human TRPA1 have been functionally characterized in the literature (Meents et al, 2019), yet the published data do not enable us to fully understand the molecular details of the channel function, mostly because (1) the available structures of TRPA1 capture the channel in an intermediate or inactivated conformation (Paulsen et al, 2015;Suo et al, 2020) which does not allow to distinguish the functional states, (2) the structures lack information on a considerable (∼50%) part of the protein, (3) the impact of the interactions of TRPA1 with various important endogenous proteins and cellular signaling mechanisms has not yet been fully characterized (Zygmunt and Hogestatt, 2014;Gouin et al, 2017;Talavera et al, 2019), and (4) the extent to which TRPA1 can be regulated by its membrane environment is still only gradually being uncovered (Hirono et al, 2004;Akopian et al, 2007;Dai et al, 2007;Karashima et al, 2008;Witschas et al, 2015;Macikova et al, 2019;Startek et al, 2019). Obviously, a better understanding of all these issues is key for a precise description of the mechanisms of TRPA1 activation, and, perhaps more importantly, for rational screening of its novel modulators as potential therapeutic agents.…”