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

Abstract

Our understanding of the general principles of the polymodal regulation of transient receptor potential (TRP) ion channels has grown impressively in recent years as a result of intense efforts in protein structure determination by cryo-electron microscopy. In particular, the high-resolution structures of various TRP channels captured in different conformations, a number of them determined in a membrane mimetic environment, have yielded valuable insights into their architecture, gating properties and the sites of their interactions with annular and regulatory lipids. The correct repertoire of these channels is, however, organized by supramolecular complexes that involve the localization of signaling proteins to sites of action, ensuring the specificity and speed of signal transduction events. As such, TRP ankyrin 1 (TRPA1), a major player involved in various pain conditions, localizes into cholesterol-rich sensory membrane microdomains, physically interacts with calmodulin, associates with the scaffolding A-kinase anchoring protein (AKAP) and forms functional complexes with the related TRPV1 channel. This perspective will contextualize the recent biochemical and functional studies with emerging structural data with the aim of enabling a more thorough interpretation of the results, which may ultimately help to understand the roles of TRPA1 under various physiological and pathophysiological pain conditions. We demonstrate that an alteration to the putative lipid-binding site containing a residue polymorphism associated with human asthma affects the cold sensitivity of TRPA1. Moreover, we present evidence that TRPA1 can interact with AKAP to prime the channel for opening. The structural bases underlying these interactions remain unclear and are definitely worth the attention of future studies.

Calcium activates purified human TRPA1 with and without its N-terminal ankyrin repeat domain in the absence of calmodulin

“…The effect of calcium on TRPA1 is mechanistically intriguing, and encompasses channel activation by calcium alone as well as calcium channel sensitization/desensitization of ligand activation (5). These effects of calcium may occur by a direct interaction with TRPA1 or as a result of association with a calcium binding partner such as calmodulin (6)(7)(8)(9)(11)(12)(13)(14). The site of action may be on the cytoplasmic N-and C-termini (5).…”

“…The site of action may be on the cytoplasmic N-and C-termini (5). In this, study, we have addressed the possibility of a direct interaction between calcium and purified hTRPA1 without any interplay with other calcium-sensitive proteins including calmodulin, TRPV1 and A-kinase anchoring protein (AKAP) that can associate with TRPA1 and influence its function (5,13).…”

“…However, compared to activation of purified hTRPA1 by most ligands and temperature (17)(18)(19), using the same experimental set-up and conditions, calcium seemed to evoke more variability in open probability and conductance levels of both hTRPA1 proteins. Thus, in a physiological context, there must be a need for intracellular co-factors such as calmodulin to tightly control and fine-tune the otherwise wild activity of TRPA1 triggered by calcium (9,13). Importantly, in a mass spectrometry study on hTRPA1 and Δ1-688 hTRPA1 (22), we did not find any calcium binding partners including calmodulin associated with hTRPA1 and Δ1-688 hTRPA1, which could have been reminiscent of the expression and purification of the hTRPA1 proteins although performed in Pichia pastoris.…”

“…However, an effect solely by calcium has been questioned as membrane bound calcium co-mediators such as calmodulin may still be present in isolated membrane patches (9). Some studies have focused on the modulatory role of calcium on electrophilic TRPA1 activation, and also with regard to calmodulin dependence (9)(10)(11)(12)(13). Furthermore, structures within the intracellular N-and C-termini have been suggested to contain calcium binding sites coupled to the gating of TRPA1 (7-9, 11, 13).…”

“…Furthermore, structures within the intracellular N-and C-termini have been suggested to contain calcium binding sites coupled to the gating of TRPA1 (7-9, 11, 13). Interestingly, a calcium binding amino acid cluster has been identified in the C-terminus, and recently in the cytoplasmic end of transmembrane domain 2 and 3 of TRPA1 (11,13,14).…”