Figure 1.
Cysteine scanning on TRPV1 and TRPV3 channels.
(A) Schematic of MTSET labeling on a cysteine residue by covalent bond formation. (B–D) Chemical- and temperature- dose-response curves of cysteine-less TRPV1 after application of MTSET or buffer control (B), of cysteine-less TRPV3 after application of MTSET or buffer control (C), of cysteine-less and wild-type TRPV1 (D), and of cysteine-less and wild-type TRPV3 (E). For chemical dose-responses data are averages of five measurements (mean ± s.d.) and for temperature-responses averages of 16 wells (mean ±2× s.e.). (F) Reaction schemes of GSH with MTSET and GSH with OPA. (G) Fluorescence signal of obtained from competing reactions in (F) as a function of MTSET concentration at 4°C and 40°C. n>20 with from three independent experiments. Error bars are mean ± s.d.
Figure 2.
Temperature-dependent labeling in TRPV1.
(A) Temperature as a function of time during FLIPR temperature-activation assay. (B–D) Representative examples of fluorescence responses upon temperature stimulation of TRPV1 N652C (B), A657C (C) and Y53C (D) after incubation of MTSET at 20°C (blue) and 42°C (red). For both temperatures, buffer as a negative control is colored gray; n>8 wells. Error bars are 2× s.e. (E) Average basal fluorescence change of TRPV1 Y653C after incubation of MTSET at 20°C and 42°C. The basal fluorescence is averaged fluorescence level between 0 to 20 sec. For each experiment, the increase of basal fluorescence was obtained by subtracting buffer control from MTSET incubation. Five independent experiments were performed for 20°C and three for 40°C with n>5 wells per experiment. Two-tailed t-test, *p = 0.036. Error bars are mean ± s.e.
Figure 3.
Temperature-dependent labeling in TRPV3.
(A) Temperature as a function of time during FLIPR temperature-activation assay. (B) Representative examples of fluorescence responses upon temperature stimulation of TRPV3 I652C. n>8 wells. Error bars are mean ±2x s.e. (C) Average basal fluorescence change of TRPV3 I652C after incubation of MTSET at 20°C and 40°C. For each experiment, the basal fluorescence is an average of fluorescence between 0 and 20 sec. Fluorescence change is the difference of MTSET incubation and buffer incubation basal fluorescence. Numbers of independent experiments are shown in the bar graph and n>8 wells per experiment. Error bars are mean ± s.e. Two-tailed t-test, ***p<0.0001. (D) Representative examples of fluorescence responses upon temperature stimulation of L655, n>8 wells, Error bars are mean ±2× s.e. (E) Average basal fluorescence change of TRPV3 L655C after incubation of MTSET at 20°C and 40°C. Number of experiments is shown in the bar graph and n>8 wells per experiment. Error bars are mean ± s.e. Two-tailed t-test, **p = 0.0062. (F) The basal fluorescence change of TRPV3 L655C after incubation of MTSET at 20°C and 40°C as a function of MTSET concentration. The incubation time was 10 min. n>5 wells. Error bars are mean ± s.d. Straight lines are exponential fits to the data.
Figure 4.
Electrophysiological characterization of temperature-dependent MTSET accessibility of TRPV1 Y653C.
(A) Above: Temperature profile for a whole-cell voltage-step protocol. Middle: Voltage-step protocols were applied before (I) and after (II) MTSET application at 20°C or 40°C. Bottom: Example of current traces of Y653C before and after 2 mM MTSET at 20°C. (B) Average change of plateau current (+100 mV) upon application of MTSET at 20°C or 40°C. Data are averages from five patches. Two-tailed t-test, **p = 0.00034. (C) Current-voltage (IV) curves from whole-cell measurement. Error bars are mean ± s.e.
Figure 5.
Location of temperature-dependent accessible residues.
(A) Sequence alignment of pore domains of rat TRPV1 and mouse TRPV3. Predicted structural domains are indicated above. Residues with temperature-independent accessibility are highlighted in cyan and residues with temperature-dependent accessibility are in pink. (B) Homology models of pore domains. Same color coding was used as the above.