Human TRPA1 is intrinsically cold- and chemosensitive with and without its N-terminal ankyrin repeat domain.
We have purified and reconstituted human transient receptor potential (TRP) subtype A1 (hTRPA1) into lipid bilayers and recorded single-channel currents to understand its inherent thermo- and chemosensory properties as well as the role of the ankyrin repeat domain (ARD) of the N terminus in channel behavior. We report that hTRPA1 with and without its N-terminal ARD (Δ1-688 hTRPA1) is intrinsically cold-sensitive, and thus, cold-sensing properties of hTRPA1 reside outside the N-terminal ARD. We show activation of hTRPA1 by the thiol oxidant 2-((biotinoyl)amino)ethyl methanethiosulfonate (MTSEA-biotin) and that electrophilic compounds activate hTRPA1 in the presence and absence of the N-terminal ARD. The nonelectrophilic compounds menthol and the cannabinoid Δ(9)-tetrahydrocannabiorcol (C16) directly activate hTRPA1 at different sites independent of the N-terminal ARD. The TRPA1 antagonist HC030031 inhibited cold and chemical activation of hTRPA1 and Δ1-688 hTRPA1, supporting a direct interaction with hTRPA1 outside the N-terminal ARD. These findings show that hTRPA1 is an intrinsically cold- and chemosensitive ion channel. Thus, second messengers, including Ca(2+), or accessory proteins are not needed for hTRPA1 responses to cold or chemical activators. We suggest that conformational changes outside the N-terminal ARD by cold, electrophiles, and nonelectrophiles are important in hTRPA1 channel gating and that targeting chemical interaction sites outside the N-terminal ARD provides possibilities to fine tune TRPA1-based drug therapies (e.g., for treatment of pain associated with cold hypersensitivity and cardiovascular disease).
Department of Biochemistry and Structural Biology, Center for Molecular Protein Science, Lund University, SE-221 00 Lund, Sweden;
Lavanya
L
Moparthi
Department of Biochemistry and Structural Biology, Center for Molecular Protein Science, Lund University, SE-221 00 Lund, Sweden;
Sabeen
S
Survery
Nanion Technologies GmbH, D-80636 Munich, Germany; Department of Biophysics, Jacobs University Bremen, D-27857 Bremen, Germany; and.
Mohamed
M
Kreir
Clinical Chemistry and Pharmacology, Department of Laboratory Medicine, Lund University, SE-221 85 Lund, Sweden.
Charlotte
C
Simonsen
Department of Biochemistry and Structural Biology, Center for Molecular Protein Science, Lund University, SE-221 00 Lund, Sweden;
Per
P
Kjellbom
Clinical Chemistry and Pharmacology, Department of Laboratory Medicine, Lund University, SE-221 85 Lund, Sweden edward.hogestatt@med.lu.se urban.johanson@biochemistry.lu.se.
Edward D
ED
Högestätt
Department of Biochemistry and Structural Biology, Center for Molecular Protein Science, Lund University, SE-221 00 Lund, Sweden; edward.hogestatt@med.lu.se urban.johanson@biochemistry.lu.se.
Urban
U
Johanson
Clinical Chemistry and Pharmacology, Department of Laboratory Medicine, Lund University, SE-221 85 Lund, Sweden.
Peter M
PM
Zygmunt
D017089:Ankyrin Repeat; D015220:Calcium Channels / Q000737:chemistry / Q000502:physiology*; D003080:Cold Temperature; D006801:Humans; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000502:physiology*; D018408:Patch-Clamp Techniques; D000074025:TRPA1 Cation Channel; D050051:Transient Receptor Potential Channels / Q000737:chemistry / Q000502:physiology*
D015220:Calcium Channels; D009419:Nerve Tissue Proteins; D000074025:TRPA1 Cation Channel; C473350:TRPA1 protein, human; D050051:Transient Receptor Potential Channels
TRP channels; cold sensing; irritants; pain; sensory neuron