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. 2010 Apr 13;107(15):7083-8.
doi: 10.1073/pnas.1000357107. Epub 2010 Mar 29.

Thermosensitive TRP Channel Pore Turret Is Part of the Temperature Activation Pathway

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Free PMC article

Thermosensitive TRP Channel Pore Turret Is Part of the Temperature Activation Pathway

Fan Yang et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Temperature sensing is crucial for homeotherms, including human beings, to maintain a stable body core temperature and respond to the ambient environment. A group of exquisitely temperature-sensitive transient receptor potential channels, termed thermoTRPs, serve as cellular temperature sensors. How thermoTRPs convert thermal energy (heat) into protein conformational changes leading to channel opening remains unknown. Here we demonstrate that the pathway for temperature-dependent activation is distinct from those for ligand- and voltage-dependent activation and involves the pore turret. We found that mutant channels with an artificial pore turret sequence lose temperature sensitivity but maintain normal ligand responses. Using site-directed fluorescence recordings we observed that temperature change induces a significant rearrangement of TRPV1 pore turret that is coupled to channel opening. This movement is specifically associated to temperature-dependent activation and is not observed during ligand- and voltage-dependent channel activation. These observations suggest that the turret is part of the temperature-sensing apparatus in thermoTRP channels, and its conformational change may give rise to the large entropy that defines high temperature sensitivity.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Thermodynamic characterization of the temperature-dependent gating of thermoTRPs. (A and B) Inside-out patch recordings of temperature-driven activation of TRPV1 (A) or TRPM8 (B) expressed in HEK293 cells. Currents in response to 10 μM TRPV1 agonist capsaicin (CAP) seen in (A) confirm that they are mediated by TRPV1 channels. (C and D) Van't Hoff plots for the heat-elicited TRPV1 current (C) and cold-elicited TRPM8 current (D). The symbols in (C) represent current responses to four temperature jumps shown in A. Lines represent fits of the Van't Hoff equation, from which ΔH and ΔS are estimated. (E) Measured ΔH values (filled bars, left axis) and ΔS values (open bars, right axis) of thermoTRPs and CLC-0 channels. (F) Box-and-whisker plot of half-activation temperature T0.5. The whisker top, box top, line inside the box, box bottom, and whisker bottom represent the maximum, 75th percentile, median, 25th percentile, and minimum value of each pool of T0.5 measurements, respectively. Square dots indicate the average T0.5 value. The data in E and F represent measurements from 3–14 patches.
Fig. 2.
Fig. 2.
Determination of a distinct temperature-sensitive activation pathway. (A and B) Changes in ΔH and ΔS of the temperature-driven activation measured under various conditions for TRPV1 (A) and TRPM8 (B). Filled bars represent ΔH values (left axis), and open bars correspond to ΔS values (right axis). **Significant difference at the level of P < 0.01. n = 4–13 patches. (C and D) Representative inside-out patch recordings demonstrating additive effects of capsaicin (C) or voltage (D) on heat-induced TRPV1 activation. Leak currents are not subtracted.
Fig. 3.
Fig. 3.
Replacement of the pore turret sequence eliminates temperature-dependent gating. (A) A structural model of the channel pore showing the replaced turret sequence (in red) and the two cysteines, C617 and C622 (in yellow) used for fluorescence labeling. The structure is based on a model of the human TRPV1 (50). (B) Mutant channels containing the G4PG4SG4S sequence are insensitive to temperatures up to 45 °C but can be activated by capsaicin. A representative trace of 10 inside-out patches is shown; 2 other patches showed spontaneous channel activities at room temperature. (C) Representative single-channel traces recorded from inside-out patches at +80 mV. (D) Dose–response relationship for the WT and G4PG4SG4S mutant channels. Curves represent fits of a Hill equation. The EC50 and Hill slope values are as follows: WT, 0.98 μM and 1.9 (n = 3); mutant, 0.35 μM and 1.3 (n = 5). (E) The relationship between open probability and temperature for the WT channels (n = 12) and mutant channels (n = 4).
Fig. 4.
Fig. 4.
Fluorescence recording of the temperature-induced structural changes in the TRPV1 pore turret. (A) Top view of the modeled TRPV1 pore structure, with the distance between Cβ of C622 of neighboring subunits labeled. (B) Spectral images of a cell expressing WT TRPV1 labeled with FM and TMRM at low and high temperatures. The corresponding spectra on the right are normalized by their FM peaks. (C) Simultaneous whole-cell current and fluorescence recordings from WT TRPV1 channels when the temperature is changed. (D) FRET efficiency measured from the outer pore positions as well as from two positions at the S1-–S2 linker (L461C and N467C) and one position at the S3-S4 linker (K536C). “Low T” and “high T” indicate temperature below 25 °C and above 40 °C, respectively. Measurements with capsaicin (10 μM) and voltage were performed at room temperature. Cys-less indicates a mutant channel missing both C617 and C622. *P < 0.05. n = 3–5. (E) A schematic model of the proposed gating conformational changes in TRPV1 induced by heat, capsaicin, and voltage. Green stars represent fluorophores attached to the pore turret.

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