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Title of Journal: Pflugers Arch - Eur J Physiol

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Abbravation: Pflügers Archiv - European Journal of Physiology

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Springer Berlin Heidelberg

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Bimodal voltage dependence of TRPA1: mutations of a key pore helix residue reveal strong intrinsic voltage-dependent inactivation

Authors: Xia Wan, Yungang Lu, Xueqin Chen, Jian Xiong, Yuanda Zhou, Ping Li, Bingqing Xia, Min Li, Michael X. Zhu, Zhaobing Gao,

Publish Date: 2013/10/05
Volume: 466, Issue:7, Pages: 1273-1287
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Transient receptor potential A1 (TRPA1) is implicated in somatosensory processing and pathological pain sensation. Although not strictly voltage-gated, ionic currents of TRPA1 typically rectify outwardly, indicating channel activation at depolarized membrane potentials. However, some reports also showed TRPA1 inactivation at high positive potentials, implicating voltage-dependent inactivation. Here we report a conserved leucine residue, L906, in the putative pore helix, which strongly impacts the voltage dependency of TRPA1. Mutation of the leucine to cysteine (L906C) converted the channel from outward to inward rectification independent of divalent cations and irrespective to stimulation by allyl isothiocyanate. The mutant, but not the wild-type channel, displayed exclusively voltage-dependent inactivation at positive potentials. The L906C mutation also exhibited reduced sensitivity to inhibition by TRPA1 blockers, HC030031 and ruthenium red. Further mutagenesis of the leucine to all natural amino acids individually revealed that most substitutions at L906 (15/19) resulted in inward rectification, with exceptions of three amino acids that dramatically reduced channel activity and one, methionine, which mimicked the wild-type channel. Our data are plausibly explained by a bimodal gating model involving both voltage-dependent activation and inactivation of TRPA1. We propose that the key pore helix residue, L906, plays an essential role in responding to the voltage-dependent gating.Transient receptor potential A1 (TRPA1) is a nonselective cation channel highly expressed in a subpopulation of primary afferent sensory neurons of the dorsal root and trigeminal ganglia [16, 42] and implicated in somatosensory processing and pathological pain sensation, particularly inflammatory and neuropathic pain [36]. TRPA1 is activated by a plethora of natural and synthetic compounds, including both electrophilic chemicals and oxidants that covalently modify cysteine residues at the cytoplasmic N-terminus and nonelectrophilic agents that bind to the channel in noncovalent fashions [8, 47]. In addition, the channel is sensitive to intracellular Ca2+ and pH [12, 29, 44, 47, 60], as well as membrane depolarization [29]. The ability of TPRA1 to respond to multiple stimuli is consistent with its role in sensing pain stimuli as well as irritants and allergens [4, 7, 20, 25, 26, 32, 36, 42, 46].Similar to other TRP channels, e.g., TRPV1 and TRPM8 [48], TRPA1 displays voltage dependence, showing marked outward rectification, especially under weakly activated conditions, such as activation by low temperature, CO2, O2, or intracellular Ca2+ [42, 60]. The rectification becomes less pronounced as the channel is activated strongly by certain chemical ligands [19, 20] or stimulated for a long time period by electrophilic compounds [51], indicating a shift of voltage dependence to more negative potentials. In general, the commonly used TRPA1 agonists, e.g., allyl isothiocyanate (AITC) and cinnamaldehyde, elicit currents with variable degrees of outward rectification depending on the agonist concentration and stimulation duration [51]. Ironically, however, some studies also showed leveling off or inactivation at high positive potentials (e.g., at > +50 mV) for mouse and human TRPA1 expressed in CHO and HEK293 cells [1, 3, 22, 23, 24, 35, 36, 45], giving rise to an inwardly rectifying appearance in the current–voltage (I–V) relationships. It was not made clear under which conditions the inward rectification tended to occur and often both linear and inwardly rectifying I–V curves were displayed in the same study. Based on single channel measurements from cell-attached patches, the open probability of TRPA1 clearly shows inactivation at positive potentials [35]. Therefore, it appears that TRPA1 has both voltage-dependent activation and inactivation.Like other TRP channels, TRPA1 may have the same architecture as voltage-gated, Shaker-type K+ channels, for which two molecular gates exist. The inner gate is formed by bundle crossing of the four S6 transmembrane segments near the cytoplasmic side, while the outer gate involves the selectivity filter situated in the pore loop between the S5 and S6 transmembrane segments [6, 28]. Mutational analyses at the pore loops of TRPV1 [31, 39] and TRPA1 [9] revealed that residues adjacent to the selectivity filter are important for TRP channel gating, suggesting a significant contribution of the outer gate in TRP channel activation. Here, we report an unexpected finding involving L906 in the pore helix of TRPA1. When substituted by another amino acid, including cysteine and 14 others, the resultant channel displays only inward rectification, showing stronger activity at negative than at positive potentials. Such an effect was unaffected by divalent cations. Our results suggest a strong influence of pore helix in voltage-dependent gating of TRPA1.The mouse TRPA1 cDNA was a gift from Dr. Gina Story (Washington University in St. Louis). Point mutations were introduced using the QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA) and the standard PCR overlap extension technique. The mutations were verified by DNA sequencing.HEK293 cells were grown in DMEM containing 10 % (vol/vol) fetal bovine serum (FBS), 2 mM  l-glutamine at 37 °C in a humidity-controlled incubator with 5 % CO2. All cell culture reagents were purchased from Invitrogen. The conditions for transient transfection of cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in serum-free conditions were optimized. The medium was exchanged for FBS-containing DMEM 6 h after transfection. Transfection efficiency was monitored through cotransfection with an EGFP vector, coding for the enhanced green fluorescent protein. Electrophysiological recordings were performed between 24 and 36 h after transfection.Mutation of L906 at the pore helix of mouse TRPA1 to cysteine converts the channel from outward to inward rectification. a Sequence alignment of putative pore regions of mammalian TRPA1 and TRPM channels. Equivalent regions of known channel structures (KvAP, Kv1.2, KcsA, NaKbc, NaChBac, NavAb, and NavRH) are shown for comparison. Shaded areas indicate minimal spans of pore helices and selectivity filters, adapted for TRPMs from Refs. [27, 34]. Residues mutated in mouse TRPA1 in the current study (Pro904, Leu905, and Leu906) and the Asp (D918) previously shown to determine the Ca2+ selectivity of TRPA1 [51] are underlined. b Whole-cell current recording of a HEK293 cell expressing wild-type mouse TRPA1 in the normal extracellular solution containing 2 mM Ca2+ and 1 mM Mg2+. A 300-ms ramp from −100 to 100 mV was applied every 2 s, from a holding potential of 0 mV and current measured at −100 mV (filled circles) and +100 mV (open circles) during each ramp is plotted as a function of time. AITC (100 μM) was applied during the time indicated by the horizontal bar. The graph below the current traces shows changes in rectification ratio ([R = I −100/I +100] for the same cell during the same time period. The bottom plot shows current–voltage (I–V) relationships recorded from the same cell by the voltage ramp collected at basal (black line), the peak of AITC response (red line), and the end of time course (blue line). c As in b, but for a cell that expressed L906C mutant of mouse TRPA1. Note the inwardly rectifying I–V relationships and the large R valuesInward rectification of L906C is independent of divalent cations. Similar to Fig. 1b, c, but the recording was performed using a Ca2+-free (omitting Ca2+ and adding 0.5 mM EGTA) bath solution (a, b) or divalent cation-free bath (omitting Ca2+ and Mg2+ and adding 0.5 mM EGTA) and pipette (omitting Ca2+ and Mg2+ and adding 10 mM BAPTA) solutions (c, d). Representative traces are shown as for Fig. 1b, c for cells that expressed wild-type mouse TRPA1 (a, c) or its L906C mutant (b, d)



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