TRPA1 is a polyunsaturated fatty acid sensor in mammals

Abstract

Fatty acids can act as important signaling molecules regulating diverse physiological processes. Our understanding, however, of fatty acid signaling mechanisms and receptor targets remains incomplete. Here we show that Transient Receptor Potential Ankyrin 1 (TRPA1), a cation channel expressed in sensory neurons and gut tissues, functions as a sensor of polyunsaturated fatty acids (PUFAs) in vitro and in vivo. PUFAs, containing at least 18 carbon atoms and three unsaturated bonds, activate TRPA1 to excite primary sensory neurons and enteroendocrine cells. Moreover, behavioral aversion to PUFAs is absent in TRPA1-null mice. Further, sustained or repeated agonism with PUFAs leads to TRPA1 desensitization. PUFAs activate TRPA1 non-covalently and independently of known ligand binding domains located in the N-terminus and 5(th) transmembrane region. PUFA sensitivity is restricted to mammalian (rodent and human) TRPA1 channels, as the drosophila and zebrafish TRPA1 orthologs do not respond to DHA. We propose that PUFA-sensing by mammalian TRPA1 may regulate pain and gastrointestinal functions.

Figure 1. TRPA1 is critical for sensory nerve detection of PUFAs.

(A) Fluo-4 fluorescence images of DHA-responsive neurons (top panel) and a non-responsive neuron (bottom panel). Scale bar is 40 µm. (B&C) Representative Ca²⁺ transients evoked by DHA (100 µM), AITC (1 mM), KCl (140 mM) in DRG neurons (B, DHA responsive; C, DHA-unresponsive). (D) Ca²⁺ transients evoked by DHA (100 µM) or capsaicin (CAP, 100 nM) in DRG neurons from TRPA1-null mice. (E) The percentage of WT or TRPA1-null neurons responsive to DHA (WT, n = 25 of 143; TRPA1-null, n = 1 of 60), LA (WT, n = 2 of 58), AITC (WT, n = 39 of 201) or CAP (TRPA1-null, n = 16 of 60).

Figure 2. TRPA1 mediates taste aversion to PUFAs.

(A) Percentage consumption of gelatin with or without DHA (0.5 or 5 mM) in a two-taste preference test by wild-type (n = 10) or TRPA1-null mice (n = 7). *p<0.05 T-test. (B) Percentage consumption of DHA (5 mM) or LA (5 mM, n = 3) in a two-taste preference test. (C) Percentage of DHA (5 mM) or LA (5 mM) consumed in a single taste test compared to lipid-free gelatin consumed the previous day (wild-type only, n = 4).

Figure 3. DHA activates rat TRPA1 in transfected HEK293 cells.

(A) Representative I–V relationship for DHA (100 µM) and AITC (1 mM) in a voltage-clamped TRPA1-expressing HEK293 cell. (B&C) N-acetyl-L-cysteine (NAC, 15 mM) prevents the activation of TRPA1 current by AITC (1 mM) but not DHA (100 µM). (D) Normalized EPA-evoked currents (% of 1 mM AITC) in control buffer (n = 4), NAC (15 mM, n = 4), or Ascorbic Acid (15 mM, n = 4). (E) DHA activates TRPA1 currents in a dose- and voltage-dependent manner (n = 3–5; holding potential −60 mV or +120 mV). The smooth lines show the best-fits to a Hill equation yielding an E_max of 49±6%, EC₅₀ of 41±5 µM and Hill Coefficient of 3.8±1.3 for −60 mV, and E_max of 100%, EC₅₀ of 13.2±1.8 µM and Hill Coefficient of 1.0±0.1 for +120 mV. (F) Normalized increase in intracellular [Ca²⁺] evoked by AITC in TRPA1-expressing HEK293 cells with or without pre-treatment with DHA (100 µM, 10 min, n = 40–50). DHA was washed out for 2 min prior to AITC challenge. (G) Representative currents evoked by repeated application of DHA (100 µM) and AITC (500 µM) in a voltage-clamped TRPA1-expressing HEK293 cell.

Figure 4. Activation of TRPA1 depends on fatty acid chain length and unsaturation.

(A) Structures of fatty acids. (B) Normalized currents (% of 1 mM AITC) evoked by fatty acids (100 µM) at −60 or +100 mV in HEK293 cells transfected with rat TRPA1 (n = 4–8).

Figure 5. DHA selectively activates mammalian TRPA1 channels.

(A) Normalized responses to 100 µM DHA at −60 and +100 mV in HEK293 cells transfected with either drosophila, zebrafish A, zebrafish B, mouse, rat or human TRPA1 (n = 3–6).

Figure 6. PUFAs activate TRPA1 independently of the N-terminus or transmembrane domain 5.

A. Mean current responses in rTRPA1-expressing HEK293 cells (+100 mV; n = 3–4) for control, extracellular DHA (20 µM or 100 µM) and DHA-CoA (100 µM), or intracellular application of DHA-CoA (20 µM) and DHA (20 µM). The background current is not subtracted. (B–E) I–V relationship for responses to DHA (100 µM) and AITC (1 mM) in HEK293 cells expressing chimeric drosophila-mouse TRPA1 channels.

Figure 7. DHA activates TRPA1 and secretion in enteroendocrine cells.

(A) Representative current trace showing response of voltage-clamped STC-1 cell to successive applications of AITC (500 µM). (B) Concentration-response curve for AITC evoked currents in STC-1 cells (−60 mV, n = 3–4 for each point). The smooth lines show the best-fit to a Hill equation yielding an EC₅₀ of 29±3 µM and Hill Coefficient of 1.2±0.1 (C) DHA (100 µM) activates an inward current in an STC-1 cell that is blocked reversibly by HC030031 (20 µM). (D) I–V relationship in a voltage-clamped STC-1 cell demonstrating response to DHA (100 µM) and block by the TRPA1 antagonist, HC030031 (50 µM). (E) I–V relationship for DHA (100 µM) with and without HC030031 (50 µM) in a voltage-clamped RIN14B cell. (F) CCK secretion from STC-1 cells stimulated by 100 µM DHA, DHA plus 50 µM HC030031, 500 µM LA, 100 µM AITC and 140 mM KCl (n = 4–12) T-test **p<0.01.