How the TRPA1 receptor transmits painful stimuli: Inner workings revealed by electron cryomicroscopy

Abstract

A new high-resolution structure of a pain-sensing ion channel, TRPA1, provides a molecular scaffold to understand channel function. Unexpected structural features include a TRP-domain helix similar to TRPV1, a novel ligand-binding site, and an unusual C-terminal coiled coil stabilized by inositol hexakisphosphate (IP6). TRP-domain helices, which structurally act as a nexus for communication between the channel gates and its other domains, may thus be a feature conserved across the entire TRP family and, possibly, other allosterically-gated channels. Similarly, the TRPA1 antagonist-binding site could also represent a druggable location in other ion channels. Combined with known TRPA1 functional properties, the structural role for IP6 leads us to propose that polyphosphate unbinding could act as a molecular kill switch for TRPA1 inactivation. Finally, although packing of the TRPA1 membrane-proximal region hints at a mechanism for electrophile sensing, the details of how TRPA1 responds to noxious reactive electrophiles and temperature await future studies.

Keywords: TRP domain; allosteric gating; ankyrin repeats; coiled coil; electron cryomicroscopy; polyphosphates; transient receptor potential ion channels.

Figure 1

A schematic of TRPA1 maps a number of demonstrated and proposed regulatory handles. The schematic follows the color scheme from N- to C-terminus: ankyrin repeats, turquoise; linker, yellow; S1–S4, lilac; S5–S6, blue; gate residues, orange; TRP-domain helix, red; and C-terminus, green. The regions absent in the structure are shown here in grey. The antagonist A967079 is represented as a green circle and the S4–S5 pocket is marked with a green asterisk. The positions of cysteines essential for response to reactive electrophiles are marked as black circles in the membrane-proximal N-terminal region that surrounds the TRP-domain helix (red). The position of the familial episodic pain variation N855S and the paradoxical heat syndrome variation E179K are marked with a yellow star and blue triangle, respectively. IP6 is represented as a purple hexagon between the membrane-proximal ankyrin repeats (turquoise) and coiled coil (green). The N-terminus and ankyrin repeats 1–11, which are missing from the TRPA1 structure, are in grey. Curved grey lines denote the suspected flexibility of the linkage between 1–11 and the remaining ankyrin repeats. Potential interactions between the N-terminus and the membrane or membrane-associated factors are indicated by a grey arrow. Intracellular mutations reported to disturb calcium-dependent potentiation or inactivation of TRPA1 are marked with red diamonds, which cluster at the intersection of the ARD and the C-terminus.

Figure 2

CryoEM structure of TRPA1. All panels follow the color scheme outlined in Figure 1. A and B: Surface and cartoon views of the TRPA1 extracellular face, respectively. Only one subunit in (A) is colored, highlighting the domain swapping of the S1–S4 bundle (lilac) and pore-forming S5–S6 (blue). In (B) the S5 and S6 helices are grey to highlight the two pore helices, labeled in one subunit, at the pore entrance and the unexpected TRP-domain helices positioned like radiating spokes beneath the transmembrane domain. C: TRPA1 viewed from the side with one subunit as a colored cartoon to indicate the relative position of each element in the subunit. D: Key features of TRPA1 are colored on a cartoon side view, including the upper and lower gates (orange), asparagine 855 position involved in familial episodic pain syndrome (blue), tetrameric coiled coil (green), TRP-domain helix (red), and proposed β-sheet (turquoise and green).

Figure 3

Comparisons of the TRPA1 and TRPV1 channels highlight different ligand-binding sites. All panels follow the same color scheme as Figure 1 to depict TRPA1 (PDB 3j9p) on the left and TRPV1 (3j5q) as cartoons. A: Side views of TRPA1 (left) and TRPV1 (right) show the high similarity between their transmembrane domains, despite drastically different intracellular regions. DkTx is depicted as a grey mesh. B: and C: Views of the TRPA1 and TRPV1 transmembrane domains from the side and extracellular face, respectively, include densities for TRPA1 antagonist A967079 and TRPV1 agonist resiniferatoxin as green surfaces. These molecules have different binding sites in the channels. For clarity, the views in (C) are clipped to include only the regions bracketed in (B).

Figure 4

Intracellular features of TRPA1. All panels follow the same color scheme as in Figure 1.A: The transmembrane domain is hidden from the view of the extracellular channel face, revealing the presence of four IP6 molecules (purple) at interfaces of the intracellular ankyrin repeats (turquoise) and coiled coil (green). B: A side view of TRPA1 with IP6 density has the front and back subunits removed for clarity. C: Zooming into the membrane-proximal region, the positions of cysteines important for TRPA1 activation by reactive electrophiles are marked with black spheres. The approximate position of cysteine 655, not modelled in the original structure, is marked with a grey circle.