Bimodal voltage dependence of TRPA1 mutations of

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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Abstract

Transient receptor potential A1 TRPA1 is implicated in somatosensory processing and pathological pain sensation Although not strictly voltagegated 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 voltagedependent 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 wildtype channel displayed exclusively voltagedependent 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 wildtype channel Our data are plausibly explained by a bimodal gating model involving both voltagedependent activation and inactivation of TRPA1 We propose that the key pore helix residue L906 plays an essential role in responding to the voltagedependent gatingTransient 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 Nterminus 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 46Similar to other TRP channels eg 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 eg 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 eg 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 cellattached patches the open probability of TRPA1 clearly shows inactivation at positive potentials 35 Therefore it appears that TRPA1 has both voltagedependent activation and inactivationLike other TRP channels TRPA1 may have the same architecture as voltagegated Shakertype 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 voltagedependent gating of TRPA1The mouse TRPA1 cDNA was a gift from Dr Gina Story Washington University in St Louis Point mutations were introduced using the QuikChange II sitedirected mutagenesis kit Stratagene La Jolla CA and the standard PCR overlap extension technique The mutations were verified by DNA sequencingHEK293 cells were grown in DMEM containing 10  vol/vol fetal bovine serum FBS 2 mM  lglutamine at 37 °C in a humiditycontrolled 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 serumfree conditions were optimized The medium was exchanged for FBScontaining 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 transfectionMutation 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 Kv12 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 Wholecell current recording of a HEK293 cell expressing wildtype mouse TRPA1 in the normal extracellular solution containing 2 mM Ca2+ and 1 mM Mg2+ A 300ms 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 05 mM EGTA bath solution a b or divalent cationfree bath omitting Ca2+ and Mg2+ and adding 05 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 wildtype mouse TRPA1 a c or its L906C mutant b d

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