Characterization of a ligand binding site in the human transient receptor potential ankyrin 1 pore

Abstract

The pharmacology and regulation of Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel activity is intricate due to the physiological function as an integrator of multiple chemical, mechanical, and temperature stimuli as well as differences in species pharmacology. In this study, we describe and compare the current inhibition efficacy of human TRPA1 on three different TRPA1 antagonists. We used a homology model of TRPA1 based on Kv1.2 to select pore vestibule residues available for interaction with ligands entering the vestibule. Site-directed mutation constructs were expressed in Xenopus oocytes and their functionality and pharmacology assessed to support and improve our homology model. Based on the functional pharmacology results we propose an antagonist-binding site in the vestibule of the TRPA1 ion channel. We use the results to describe the proposed intravestibular ligand-binding site in TRPA1 in detail. Based on the single site substitutions, we designed a human TRPA1 receptor by substituting several residues in the vestibule and adjacent regions from the rat receptor to address and explain observed species pharmacology differences. In parallel, the lack of effect on HC-030031 inhibition by the vestibule substitutions suggests that this molecule interacts with TRPA1 via a binding site not situated in the vestibule.

Figure 1

Sequence alignment of Kv1.2 (2R9R) with human and rat TRPA1. (Shading is according to similarity; amino acids with similar properties have identical tone.)

Figure 2

Docking poses of AZ868 and A-967079 in the pore region of TRPA1 according to the homology model prediction. Structures of AZ868 and A-967079 are shown in the panels below the docking poses.

Figure 3

(Left) Illustrating the selectivity filter, S5–S6 linker, and S6 fold according to the homology model. The single and double substitutions studied are shown at predicted positions on the backbone fold (solid strand). (Right) Illustrating the complete construct with the 12 rat residues substituted in the human receptor replacing human residues with the corresponding rat residues at predicted positions according to the homology model.

Figure 4

(A) One application cycle of the assay illustrating type of application: buffer type or sample. Flow rate and length of application as indicated (horizontal bars). (B) Examples of corresponding current traces from an application cycle and a test application using an antagonist. First application scale showing buffer changes as in panel A. (Vertical scale bar) Current amplitude. (Horizontal bar) Timescale for all traces. (C) Example current traces from control and test application obtained using the application cycle shown in panels A and B. The current traces are recorded from, and comparing, TRPA1 WT; rat residues substituted in to the human TRPA1 receptor, ratified; and the TRPA1 F944A substitution in control and with 1 μM AZ868 applied as indicated above the traces. The scale bars show current amplitude and timecourse for all three receptor constructs.

Figure 5

Box plot of the mean inhibition percentage of the maximal current amplitude for AZ868 (1 μM), A-967079 (0.2 μM), and HC-030031 (10 μM) as indicated above each panel. The TRPA1 construct type is listed on the y axis and the level of inhibition scale is shown on the x axis. Wild-type inhibition percentage is written above the vertical line and the inhibition percentage on the individual substitutions is shown as boxes drawn from the WT level. n = 2–16 (see Table 2 for tabulated list of inhibition and significance).