Requirement of extracellular Ca²⁺ binding to specific amino acids for heat‐evoked activation of TRPA1

Abstract

Transient receptor potential ankyrin 1 (TRPA1) is a homotetrameric non-selective cation-permeable channel that has six transmembrane domains and cytoplasmic N- and C-termini. The N-terminus is characterized by an unusually large number of ankyrin repeats. Although the 3-dimensional structure of human TRPA1 has been determined, and TRPA1 channels from insects to birds are known to be activated by heat stimulus, the mechanism for temperature-dependent TRPA1 activation is unclear. We previously reported that extracellular Ca , but not intracellular Ca , plays an important role in heat-evoked TRPA1 activation in green anole lizards (gaTRPA1). Here we focus on extracellular Ca -dependent heat sensitivity of gaTRPA1 by comparing gaTRPA1 with heat-activated TRPA1 channels from rat snake (rsTRPA1) and chicken (chTRPA1). In the absence of extracellular Ca , rsTRPA1 and chTRPA1 are activated by heat and generate small inward currents. A comparison of extracellular amino acids in TRPA1 identified three negatively charged amino acid residues (glutamate and aspartate) near the outer pore vestibule that are involved in heat-evoked TRPA1 activation in the presence of extracellular Ca . These results suggest that neutralization of acidic amino acids by extracellular Ca is important for heat-evoked activation of gaTRPA1, chTRPA1, and rsTRPA1, which could clarify mechanisms of heat-evoked channel activation.

Evolutionary tuning of TRPA1 and TRPV1 thermal and chemical sensitivity in vertebrates

“…73 Furthermore, comparative and mutagenesis analyses using TRPA1s from green anoles, rat snakes, and chickens showed that three negatively charged amino acids near the outer pore vestibule are involved in heat-evoked activation of TRPA1. 74 Presumably, calcium ions would neutralize these negatively charged amino acids to allow heat-induced opening of the channel pore. Thus, species diversity of thermoTRP channels provides informative clues for identifying critical amino acids involved in the activation and inhibitory action of these channels.…”

Heat activation is intrinsic to the pore domain of TRPV1

“…In contrast, mutagenesis or deletion studies have identified several regions that are important for heat-dependent activation throughout TRPV1 and other heat-activated TRP channels. These regions include the N-terminal ankyrin repeat domains in TRPA1 ( 9 ), a membrane-proximal N-terminal segment for TRPV1, TRPV2, and TRPV3 ( 10 , 11 ), the C terminus of TRPV1 ( 12 – 15 ), and the pore domain in TRPV1 ( 16 – 19 ), TRPV3 ( 20 ), and TRPA1 ( 21 , 22 ) channels. Indeed, it has been proposed that residues involved in temperature sensing may be scattered throughout the receptor rather than forming a defined temperature-sensing domain ( 23 ).…”

TRP channels in oxygen physiology: distinctive functional properties and roles of TRPA1 in O₂ sensing

“… 15 , 16 ) Number of studies have revealed controversy concerning temperature sensitivity of TRPA1 channels, raising a possibility that TRPA1 is more important in heat sensation than cold sensation. 17 – 19 ) TRPA1 is also robustly activated by electrophiles following covalent modification of cysteine (Cys) residues within the ankyrin repeat domain (ARD). 20 , 21 )…”