Sensory TRP Channels in Three Dimensions

Abstract

Transient receptor potential (TRP) ion channels are sophisticated signaling machines that detect a wide variety of environmental and physiological signals. Every cell in the body expresses one or more members of the extended TRP channel family, which consists of over 30 subtypes, each likely possessing distinct pharmacological, biophysical, and/or structural attributes. While the function of some TRP subtypes remains enigmatic, those involved in sensory signaling are perhaps best characterized and have served as models for understanding how these excitatory ion channels serve as polymodal signal integrators. With the recent resolution revolution in cryo-electron microscopy, these and other TRP channel subtypes are now yielding their secrets to detailed atomic analysis, which is beginning to reveal structural underpinnings of stimulus detection and gating, ion permeation, and allosteric mechanisms governing signal integration. These insights are providing a framework for designing and evaluating modality-specific pharmacological agents for treating sensory and other TRP channel-associated disorders.

Keywords: TRP channels; cryo-EM; membrane protein structure; sensory biology.

Figure 1.. Structural overview of TRPV1, TRPA1 and TRPM8

A: Structures of TRPV1, TRPA1 and TRPM8 are illustrated, together with the genetic tree of TRP channel superfamily. B: Top view of transmembrane domain of TRPV1, illustrating the domain swapped tetrameric architecture of TRP channel in general. C – E: Details of domain architectures illustrated for a single subunit for TRPV1 (C), TRPA1 (D) and TRPM8 (E).

Figure 2.. Ligand action on TRPV1

A, B: Solvent accessible pathway (A) and pore radius (B) along the ion permeation pathway calculated from apo structure shows two major restriction sites: the selectivity filter formed by G643 and M644 and the lower gate formed by I679. C: Extracellular ligand (DkTx) binding site and vanilloid binding site are illustrated. Binding of DkTx pushes VSLD backwards to couple the opening of lower gate. Binding of RTX pulls S5/S6 away from the pore to open the lower gate. D: transition between π- and α-helix rotates the lower half of S6 helix by one residue, unwinds or reform a helix turn in the joint between S6 and TRP domain. E: A phosphatidylinositol (PI) lipid resides in the vanilloid binding pocket. F: Binding of RTX to the vanilloid pocket displays resident PI. G: Double knot toxin (DkTx) binding to the extracellular surface of the channel.

Figure 3.. Structural attributes of the cold and menthol receptor TRPM8.

A, D: Structural rearrangements associated with transitioning from closed (A) to desensitized (D) states include a rigid-body tilt of the S1-S4 domain, formation of a canonical S4-S5 linker, shifts of S5, the pore helix, and S6, stabilization of the outer pore loop, and tilting of the TRP domain, such that it is parallel to the membrane bilayer. This transition is accompanied by introduction of a 3₁₀-helix in S4 and a π-helix in both the pore helix and S6. Ligand-binding sites are indicated. B: The antagonists AMTB and TC-I 2014 adopt distinct poses in the S1-S4 ligand-binding pocket. C: The agonist icilin also adopts a distinct pose in the S1-S4 ligand-binding pocket. PIP₂ binds to an interfacial cavity formed by the pre-S1 domain, the S4-S5 linker, the TRP domain, and cytosolic MHR4 domain from an adjacent subunit. E: Interactions with calcium. F: In the desensitized state, a stabilizing lipid packs between the pore helix and S6 of the neighboring subunit (modeled as CHS). Inset shows lipid density (blue mesh, 4σ contour).

Figure 4.. Cryo-EM Structures of human TRPA1

A: Closed structure of TRPA1 with irreversible fluorescent electrophile BODIPY-Iodoacetamide attached to C621 and a bound Ca2+ within the VSLD. TRPA1 was solubilized in LMNG. PDB ID: 6V9V B: Pore architecture of TRPA1 in a closed (non-conducting) conformation. The ion gate is formed by two hydrophobic residues (I957 and V961) while the selectivity filter is formed by the backbone carbonyl of G914 and the side chain carbonyl of D915. C: Ca2+ binding site at the base of the VSLD. Ca2+ is coordinated by the indicated polar and negatively charged residues’ side chains. D: Activated structure of TRPA1 with irreversible electrophile Iodoacetamide attached to C621. TRPA1 was solubilized in PMAL-C8. PDB ID: 6V9X E: Pore architecture of TRPA1 in an activated state. In an activated state, the ion gate expands and the selectivity filter undergoes upward rotation and translation, which enhances exposure of the D915 to cytoplasm. F: Model for activation of TRPA1 by electrophiles. Electrophilic addition to C621 triggers stabilization of the Activation (A-) loop in an ‘up’ conformation, which repositions K671 to the C-terminus of the TRP helix, enhancing its N-terminus dipole moment. This promotes repulsion between the symmetrically opposed TRP helices and, ultimately, opening of the ion gate. In the ‘up’ conformation, the C665 within the A-loop rotates into the ligand-binding pocket, becoming available for electrophilic modification, which supports stabilization of the A-loop and full channel activation. Panels (B) and (E) are modified and reproduced with permission from (Zhao et al., Nature, 2020)