Sites Contributing to TRPA1 Activation by the Anesthetic Propofol Identified by Photoaffinity Labeling

Abstract

In addition to inducing anesthesia, propofol activates a key component of the pain pathway, the transient receptor potential ankyrin 1 ion channel (TRPA1). Recent mutagenesis studies suggested a potential activation site within the transmembrane domain, near the A-967079 cavity. However, mutagenesis cannot distinguish between protein-based and ligand-based mechanisms, nor can this site explain the complex modulation by propofol. Thus more direct approaches are required to reveal potentially druggable binding sites. Here we apply photoaffinity labeling using a propofol derivative, meta-azipropofol, for direct identification of binding sites in mouse TRPA1. We confirm that meta-azipropofol activates TRPA1 like the parent anesthetic, and identify two photolabeled residues (V954 and E969) in the S6 helix. In combination with docking to closed and open state models of TRPA1, photoaffinity labeling suggested that the A-967079 cavity is a positive modulatory site for propofol. Further, the photoaffinity labeling of E969 indicated pore block as a likely mechanism for propofol inhibition at high concentrations. The direct identification of drug-binding sites clarifies the molecular mechanisms of important TRPA1 agonists, and will facilitate drug design efforts to modulate TRPA1.

Figure 1

mTRPA1-FLAG channel is photoaffinity-labeled by AziPm. (A) Chemical structures of propofol and AziPm. (B) Protection by propofol (100 μM) from photoaffinity-labeling of mTRPA1-FLAG in Sf21 cell membranes with 1 μM [³H]AziPm. [³H]AziPm labeling was measured as dpm per intensity (OD of the Coomassie blue-stained bands multiplied by the band area in mm²). Results were analyzed by Mann-Whitney t-test (^∗p < 0.05; mean ± SE; n = 4). (C) Mass spectra of photoaffinity labeled residues V954 (top) and E969 (bottom). (D) Sequence alignment of the S6 helices for mouse TRPA1 (mTRPA1), human TRPA1 (hTRPA1), and Drosophila TRPA1 (dTRPA1). Residues photolabeled by AziPm in mTRPA1, Val954 and Glu969, are indicated in green and with stars. To see this figure in color, go online.

Figure 2

Location of photolabeled residues in closed- and open-state models of hTRPA1. The photolabeled residues V954 and E969 in mTRPA1-FLAG align with V951 and E966 in hTRPA1, respectively (see Fig. 2). (A) Side view of a cartoon representation of the cryo-EM structure of hTRPA1, with the approximate location of the membrane indicated with black bars, each subunit colored a different shade, and the Cα atoms of photolabeled residues shown as red spheres. (B and C) Docking experiments of propofol and AziPm in closed state (model 1) (B) and open state (model 2) (C). All residues facing cavities were made flexible while backbone structure remained rigid during docking experiments. The yellow dotted Connolly surface representations are of five propofol and five AziPm in the highest scored poses predicted by AutoDockVina (25). Red spheres indicate the Cα atoms of V951 (corresponding to V954 photolabeled in mTRPA1), and blue spheres indicate the Cα atoms of residues discussed in the main text. (D and E) Cytosolic surface view of the transmembrane domain pore of model 1 (D) and model 2 (E). Lower pore gate residues I957 and V961 are shown as stick representations. E966, corresponding to E969 photolabeled in mTRPA1, is shown as a yellow stick representation. To see this figure in color, go online.