N-terminal tetrapeptide T/SPLH motifs contribute to multimodal activation of human TRPA1 channel

Human transient receptor potential ankyrin channel 1 (TRPA1) is a polymodal sensor implicated in pain, inflammation and itching. An important locus for TRPA1 regulation is the cytoplasmic N-terminal domain, through which various exogenous electrophilic compounds such as allyl-isothiocyanate from mustard oil or cinnamaldehyde from cinnamon activate primary afferent nociceptors. This major region is comprised of a tandem set of 17 ankyrin repeats (AR1-AR17), five of them contain a strictly conserved T/SPLH tetrapeptide motif, a hallmark of an important and evolutionarily conserved contribution to conformational stability. Here, we characterize the functional consequences of putatively stabilizing and destabilizing mutations in these important structural units and identify AR2, AR6, and AR11-13 to be distinctly involved in the allosteric activation of TRPA1 by chemical irritants, cytoplasmic calcium, and membrane voltage. Considering the potential involvement of the T/SP motifs as putative phosphorylation sites, we also show that proline-directed Ser/Thr kinase CDK5 modulates the activity of TRPA1, and that T673 outside the AR-domain is its only possible target. Our data suggest that the most strictly conserved N-terminal ARs define the energetics of the TRPA1 channel gate and contribute to chemical-, calcium- and voltage-dependence.

proteins represented as a sequence logo generated by a WebLogo server (Crooks et al., 2004).
The alignment was obtained from 80 species sequences and is numbered according to human TRPA1. The height of the particular amino acid at each position indicates its probability of occurrence at that position. Above, highly conserved consensus amino acid sequence of the ankyrin repeat proteins. Note the high inter-species conservation of T/SPLH motifs of the ankyrin repeats 2, 6, 11, 12 and 13, and also note a highly conserved and substantial difference from this motif in ankyrin repeat 10 ( 377 NFLH).  Table S2. The modeling indicates that mutations in the conserved T/SPLH motifs in ankyrin repeat domain impact the energetics of channel opening and interfere with the allosteric mechanism of coupling of putative voltage-sensing domain and gate opening.

Cell viability assay
The cells were incubated with 7 μM propidium iodide (Sigma-Aldrich) in Opti-MEM media supplemented with 5% fetal bovine serum for 30 min at 37 o C, 5% CO 2 . Then cells were washed and analysed in control bath solution (see Methods). Fluorescence images of the cells were recorded with a Cell^R imaging system based on an Olympus IX81 inverted microscope. The excitation light was generated with a Polychrome V polychromator (Till Photonics, Grafelfing, Germany), and the fluorescence emission was detected with a Hamamatsu Orca-ER camera (Hamamatsu Photonics, Hamamatsu City, Japan). The excitation wavelength was 480 ± 7.5 nm (GFP) or 540 ± 7.5 nm (propidium iodide), and emission was collected using 515 nm (GFP) or 575 nm (propidium iodide) long pass filters .
Data were collected using CellˆR software (Olympus), and the analysis was done using the program ImageJ (National Institutes of Health, Bethesda, MD).

Biotinylation of cell surface proteins
The HEK293T cells were transfected with 1.5 μg of cDNA plasmid encoding wild-type or mutant C-terminally GFP-tagged human TRPA1 (in the pCMV6-AC-GFP vector; OriGene) with Lipofectamine 2000 and cultured in a 6-well plate. At 48 h post-transfection, cells were washed with ice-cold phosphate buffered saline (PBS) buffer three times and incubated with 0.5 mg/ml EZ-link Sulfo-NHS-LC-biotin (Thermo Scientific) in PBS for 30 min at 4 °C.
After quenching the reaction (50 mM glycine in PBS), the cells were homogenized and crude plasma membrane fraction was prepared as described by Chaudhury et al. (2011). The membrane fraction was incubated with streptavidin-agarose beads (Thermo Scientific) at room temperature with constant rotation for 2 hours, followed by four washes. The Sensitivity chemiluminiscent substrate (Thermo Scientific). Immunoblots were digitized and quantified with ImageJ 1.41v software (National Institutes of Health).

Molecular modelling and stability change predictions
Structural hypotheses were tested by mapping the residues onto the homology models of the N-terminal ankyrin repeat domain region made using the software Yasara (version 15.9.6) (Krieger and Vriend, 2014) in conjunction with the Swiss-Model and I-Tasser protein modeling servers (Arnold et al., 2006;Kopp and Schwede, 2006;Zhang, 2008;Bordoli et al., 2009;Kiefer et al., 2009;Roy et al., 2010;Yang et al., 2015). Allosteric gating model predictions for specific mutants G-V curves for voltage-induced gating were normalized to G max estimated from the Boltzmann fit. The allosteric model for channel activation was used, assuming that two independent equilibria interact allosterically (Horrigan and Aldrich, 2002;Brauchi et al., 2004;Brauchi and Orio, 2011). The first equilibrium is between resting and activated state of  . G-V curves were fitted by using the allosteric model described by Equation 1, where J 0 is the equilibrium constant for voltage-sensor activation at 0 mV, z gating valence, L the equilibrium constant for gate opening, D the allosteric coupling factor.