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

Lavanya Moparthi,Satish Babu Moparthi,Jérôme Wenger,Peter M Zygmunt

doi:10.1016/j.ceca.2020.102228

Abstract

Extracellular influx of calcium or release of calcium from intracellular stores have been shown to activate mammalian TRPA1 as well as to sensitize and desensitize TRPA1 electrophilic activation. Calcium binding sites on both intracellular N- and C-termini have been proposed. Here, we demonstrate based on Förster resonance energy transfer (FRET) and bilayer patch-clamp studies, a direct calmodulin-independent action of calcium on the purified human TRPA1 (hTRPA1), causing structural changes and activation without immediate subsequent desensitization of hTRPA1 with and without its N-terminal ankyrin repeat domain (N-ARD). Thus, calcium alone activates hTRPA1 by a direct interaction with binding sites outside the N-ARD.

Highlights

The mammalian pain receptor transient receptor potential ankyrin 1 (TRPA1) was initially identified as a noxious cold sensor and chemoreceptor of electrophilic irritants as well as non-electrophilic cannabinoids [1,2,3]
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 [5,6,7]. 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 [8,9,10,11,13,14,15,16]
Study, we have addressed the possibility of a direct interaction between calcium and purified human TRPA1 (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 [5,7,15]

Summary

Introduction

The mammalian pain receptor TRPA1 was initially identified as a noxious cold sensor and chemoreceptor of electrophilic irritants as well as non-electrophilic cannabinoids [1,2,3]. TRPA1 was shown to be activated by an increase in the intracellular free calcium concentration [2]. Whereas electrophiles are believed to promote TRPA1 channel opening primarily by binding to cysteine residues within the cytoplasmic N-terminus, the mechanism by which calcium modulates TRPA1 activity is not obvious [4,5,6,7]. Studies have shown a direct activation of TRPA1 by calcium in cell-membrane inside-out patches containing heterologously expressed TRPA1 [8,9,10]. Some studies have focused on the modulatory role of calcium on electrophilic TRPA1 activation, and with regard to calmodulin dependence [11,12,13,14,15]. Further studies are needed to better understand the dynamics behind calcium modulation and activation of TRPA1

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