Structural basis of temperature sensation by the TRP channel TRPV3

Columbia University Irving Medical Center press release

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In mammals and other animals, temperature perception is mediated by primary afferent neurons, however, the identity of the molecular sensors of temperature remained enigmatic until it was found that a member of the transient receptor potential (TRP) superfamily of ion channels, TRPV1, acts as a thermoreceptor¹. This discovery resulted in the rapid identification of ten other members of the TRP channel superfamily, collectively described as the thermo-TRPs. Thermo-TRPs exhibit unusually high temperature coefficient (Q₁₀) values compared to non-temperature sensitive ion channels^2-4, allowing them to open and close in response to changes in temperature within a physiologically relevant range.  Thermo-TRPs are polymodal by nature and respond synergistically to distinct chemical stimuli, membrane potential, and temperature⁵.

There are four thermo-TRPs in the vanilloid subfamily of TRP channels: TRPV1 and TRPV2 are activated by noxious heat (>43°C), while TRPV3 and TRPV4 can respond to warm temperatures (< 33°C)^6-9. The non-vanilloid TRP channels TRPM8^10,11 and TRPC5¹² open at moderately cold (< 24°C) temperatures, while TRPA1^13,14 is activated by noxious cold (< 17°C). Furthermore, TRPM2-TRPM5^15-18 also contribute to temperature sensation with variable temperature thresholds for activation. The genetic ablation of individual thermo-TRPs did not substantially perturb the thermo-phenotype of mice^19-23, suggesting that thermo-TRPs have partially overlapping temperature thresholds in vivo. TRPV1, TRPA1 and TRPM3 triple knockout mice, however, lacked the pain response to noxious heat²⁴, emphasizing the importance of functionally redundant thermo-TRPs in temperature and pain perception. As a result of their physiological roles, the thermo-TRPs are promising analgesic drug targets.

TRPV3 is predominantly expressed in skin keratinocytes and mediates warm and pain sensation^6,9. Temperature-dependent activation of TRPV3 is use-dependent and hysteretic; its initial activation requires a higher temperature threshold (>50°C), while any subsequent activation can occur at a relatively lower temperature (~33°C)²⁵. Previously solved structures of TRPV3 have provided an insight into its architecture and ligand-dependent gating^26,27. However, how thermo-TRPs, including TRPV3, respond to changes in temperature at the molecular level remained largely unknown^28-30. To answer this question, we captured TRPV3 in different conformational states using heat. Our structures inform the molecular basis of temperature sensation by TRPV3 and thermo-TRPs in general.  We have structurally identified two conformational steps of TRPV3’s heat activation²⁹. During the first step, sensitization, the lipid occupancy in binding sites 1 and 2 is reduced, and the S1-S4 and pore domains move closer together. As S6 moves closer to S4, it undergoes an a-to-p helical transition and bends over the S4-S5 linker. While the ion channel pore remains hydrophobically sealed, the a-to-p transition results in the ~100° rotation of the C-terminal half of S6, which exposes different residues to the channel pore. Concurrently, S6 becomes two helical turns longer while the TRP helix becomes two helical turns shorter.

The second step of TRPV3’s activation, pore opening, is accompanied by removal of the lipids from the binding sites 1 and 2 and further movement of the S1-S4 and pore domains closer to each other. Additionally, the S6 helices splay away from the pore axis, the TRP helices tilt towards the S4-S5 linker, and the linker domain undergoes conformational changes, which are stabilized by the unlatching of the C-terminus from the inter-subunit interfaces. Grossly, the conformational rearrangements accompanying pore opening are more dramatic, when compared to those that occur during sensitization, and include shortening of the ion channel and rotation of the intracellular skirt domain.

To isolate the channel’s transition from the sensitized to open state, we used TRPV3_Y564A, which appears sensitized even at 4 °C. This mutant exhibits a high open probability in the 22-42 °C temperature range and has a low Q₁₀ value. This suggests that the transition from the sensitized to open state is weakly temperature-dependent. Correspondingly, we hypothesize that the strong temperature dependence and high Q₁₀ of TRPV3_WT originate from the closed-to-sensitized state transition. Our hypothetical two-step temperature-induced activation mechanism of TRPV3 is reminiscent of the activation mechanisms of TRPM8 and TRPV1, which exhibit two contrasting Q₁₀ values as well as entropy and enthalpy changes during transitional and stabilized channel gating^31-35. Because the closed-to-sensitized state transition is accompanied by conformational changes that take place only in the transmembrane domain, its components – the S1-S4 domain, the pore domain and the TRP helix – may contribute to a TRPV3 temperature sensor. However, gating of the ion channel, which culminates in the ion conductance through the TRPV3 pore, is a multistep process that requires both sensitization and pore opening. For this reason, a change in any of the activation steps may alter the apparent response of the channel²⁹. Correspondingly, the linker domain and the C-terminus, which regulate the sensitized-to-open state transition, are not necessarily responsible for temperature sensing but contribute to heat activation and appear important for temperature-dependent gating, in addition to the transmembrane domain^5,34,36-44.

What are the physical bases of the strong temperature dependence of the closed-to-sensitized state transition? The laws of thermodynamics dictate that this transition must involve an unusually large change in enthalpy between the closed and sensitized states^29,30,45,46. Since the transition between these two states involves relatively small conformational changes, the presumed large changes in enthalpy can be accounted for by significant changes in heat capacity^39,40,45,47. In proteins, the heat capacity changes appear to result mainly from the hydration of residues; exposure of hydrophobic residues to the aqueous environment is associated with a positive change in heat capacity, while that of polar or charged residues is associated with a negative change. During the TRPV3 sensitization step, the change in heat capacity could, therefore, arise from different exposure of the transmembrane domain elements to the membrane versus solvent in the closed and sensitized states. Indeed, the 100° axial rotation of the bottom of S6, its elongation and shortening of the TRP helix during sensitization provide ~8 residues per subunit of the TRPV3 tetramer that change their hydrophobic environment. Analogous conformational changes in the homologous region of TRPV1 were proposed to contribute to TRPV1 activation via the altered wetting of the pore and of the four peripheral cavities located between each S6 and S4-S5 linker⁴⁸.

TRPV3-like conformational rearrangements were also observed during TRPV6 gating, which is not temperature-dependent⁴⁹. On the other hand, TRPV6 does not show such strong gating-associated changes in annular lipid occupancy as does TRPV3, highlighting a possibility of the important role of lipids in TRPV3 thermosensitivity. Since lipid bilayers undergo temperature-dependent phase transitions, melting of annular lipids surrounding the transmembrane domain of TRPV3 with increasing temperature could result in an energy sufficient for its thermal activation²⁹. Correspondingly, the large change in enthalpy between the closed and sensitized states of TRPV3 may originate, in part, from different interactions of the surrounding lipids with the altered S1-S4-pore domain interfaces. Cholesterol molecules, which strongly modulate fluidity of lipid membranes, may represent some of these annular lipids. In fact, the shape of the cholesterol molecule resembles the shape of putative lipids in TRPV3 structure, very different from a typical two-tail appearance of phospholipids. Further supporting the important role of lipids in TRPV3 thermosensitivity, the Y564A mutation in the putative lipid binding site 2 renders TRPV3_Y564A channels weakly temperature-sensitive. Additional research is required to better understand the role of lipids, hydrophobic interactions and residue solvation in the temperature-dependent gating of thermo-TRPs and our structures provide a foundation for such studies.

This work was done in collaboration with Dr. Zakharian group from University of Illinois College of Medicine Peoria.

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