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.

The propofol binding sites of prokaryotic voltage-gated sodium channels

“…To directly identify the propofol binding site in prokaryotic Navs, we employed an unbiased photoaffinity labeling (PAL) approach that has been extensively used to identify anesthetic binding sites of numerous proteins (Hall, 2010; Chiara, 2013; Weiser, 2013; Jayakar, 2014; Woll, 2016, 2017, 2018; Bensel, 2017). Here, we used AziP m ( m- Azipropofol), an alkyl-diazirinyl derivative of propofol, to label propofol binding sites in NaChBac and NavMs (Hall, 2010).…”

“…Using photoaffinity labeling, previous work from our laboratories have provided strong evidence to suggest that the S4-S5 linker and neighboring regions of Kv and TRPA1 channels are critical structural determinants of their modulation by volatile (sevoflurane) and intravenous (propofol) anesthetics (Bu et al, 2017; Woll et al, 2017). Based on the new results reported here, we can reach a similar conclusion for prokaryotic Navs.…”

Recent progress on the molecular pharmacology of propofol

“…TRPA-1, in particular, is known to be modulated by propofol in a biphasic manner: activated at low, clinically relevant concentrations and inhibited at higher concentrations. The mechanism or mechanisms by which propofol produces these actions are suggested by recent mutagenesis 82 and photolabeling results with aziP m 83 . For example, the canonical “hinge” region of these channels, connecting sensor to pore domains and typically located in and around the cytoplasmic face of the transmembrane region, forms a binding site whereby, when occupied, the open state is stabilized, enhancing current flow ( Figure 2 ).…”

Location and Character of Volatile General Anesthetics Binding Sites in the Transmembrane Domain of TRPV1

“…Further work by photolabeling has yielded supporting evidence for this site, highlighting the residues the V954 and E969 from the S6 transmembrane helix as crucial in the activation of TRPA1 by propofol. 20 Activation and sensitization of TRPV1 induced by local anesthetics is thought to involve a domain that is similar but not identical to the vanilloid-binding domain, the area of the protein that interacts with its agonist. 17 Volatile general anesthetics (VGAs) span a group of chemicals that are able to reversibly inhibit the central nervous system activity, rendering patients unresponsive to stimuli in contrast to local anesthetics.…”

“…The side-pocket labelled site II located on the opposite side of the vanilloid binding pocket is flanked by the S4-S5 linker of one subunit, and the S6 P-loop of another subunit, similar to a propofol binding site reported for TRPA1. 20 Chloroform makes contacts with Y584, F580, M581, L664, T670, and I672…”