Human TRPA1 is an inherently mechanosensitive bilayer-gated ion channel

Abstract

The Transient Receptor Potential Ankyrin 1 (TRPA1) channel is an intrinsic chemo-and thermo-sensitive ion channel with distinct sensory signaling properties. Although a role of TRPA1 in mammalian mechanosensory transduction in vivo seems likely, it remains to be shown that TRPA1 has the inherent capability to respond to mechanical stimuli. Here we have used the patch-clamp technique to study the response of human purified TRPA1 (hTRPA1), reconstituted into artificial lipid bilayers, to changes in bilayer pressure. We report that hTRPA1 responded with increased single-channel open probability (Po) within the applied pressure interval of 7.5 to 60 mmHg with a half maximum Po (P50) value of 38.0 ± 2.3 mmHg. The Po value reached a maximum close to 1 (0.87 ± 0.02) at 60 mmHg. Within the same pressure interval, hTRPA1 without its N-terminal ankyrin repeat domain (Δ1-688 hTRPA1) responded fully opened (0.99 ± 0.01) at 60 mmHg and with a P50 value of 39.0 ± 1.1 mmHg. The pressure-evoked responses of hTRPA1 and Δ1-688 hTRPA1 at 45 mmHg were inhibited by the TRPA1 antagonist HC030031, and the activity of purified hTRPA1 at 45 mmHg was abolished by the thiol reducing agent tris(2-carboxyethyl)phosphine (TCEP). In conclusion, hTRPA1 is an inherent mechanosensitive ion channel gated by force-from-lipids. The hTRPA1 mechanosensitivity is dependent on the redox environment, and it is suggested that oxidative stress shifts hTRPA1 into a protein conformation sensitive to mechanical stimuli.

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

“…A-967079 inhibits hTRPA1 with an IC50 value of 67 nM and is about 100 times more potent than HC030031 (15,16), which was used in this study at a concentration that abolished purified hTRPA1 and Δ1-688 hTRPA1 single-channel activity evoked by chemical ligands, temperature and mechanical stimuli (17)(18)(19)(20). As also reported previously (17)(18)(19)(20), no basal TRPA1 activity was observed at 20-22°C (not shown). Likewise, no channel activity was observed in membranes without the purified hTRPA1 proteins exposed to 10 and 100 µM calcium (n=3, not shown).…”

“…To get further insight into the functional consequences of calcium-induced structural changes of hTRPA1 with and without its N-ARD, we reconstituted the purified hTRPA1 proteins into artificial lipid bilayers for studies of electrical activity as in previous studies of hTRPA1 intrinsic chemo-thermo-and mechanosensitivity (17)(18)(19)(20). At calcium concentrations that evoked half and near maximal activation of heterologously expressed TRPA1 in isolated insideout cell membrane patches (6)(7)(8), we recorded intense activity of both hTRPA1 and Δ1-688 hTRPA1.…”

Lipid Raft Destabilization Impairs Mouse TRPA1 Responses to Cold and Bacterial Lipopolysaccharides

“…In this sense, as done previously in structure-function analyses (see for review [13,28,29,53]), future studies may test the role of lipid-channel interactions exploiting the fact that, depending on the species, TRPA1 channels can be activated by heat [13,16,[54][55][56][57][58], by cold [14,17,18,57] or insensitive to cold [13,59]. Of particular interest is the new technical possibility of studying TRPA1 in artificial membranes [16,17,25], which in principle may allow determining the influence of individual lipid components on the membrane expression pattern and the functional properties of the channel.…”

“…This seems to be particularly important for sensory signaling, as the modification of the cholesterol content at the plasma membrane alters the function of several sensory Transient Receptor Potential (TRP) cation channels [7][8][9][10][11][12]. One of these proteins, the ankyrin-rich TRPA1, is arguably the most versatile polymodal sensor, being activated by thermal [13][14][15][16][17][18][19][20][21][22] and mechanical [15,[23][24][25][26][27] stimuli, and by an impressive variety of chemicals [28][29][30].…”

A nonlinear and time-dependent leak current in the presence of calcium fluoride patch-clamp seal enhancer

“…Indeed the compensation may affect the shapes of the observed current, but given the size of the current, membrane capacitance, and series resistance, both the time dependence (time constant) and the size of the effect (current size) due to membrane capacitance and series resistance are in a completely different scale (and in fact opposite direction) compared to the nonlinear time-dependent current that was observed. A comparison of the capacitance and series resistance effect is shown in Lei et al, 2020 [1]. Furthermore, the compensations were turned on for the measurements in Figure 2.…”