Single-residue molecular switch for high-temperature dependence of vanilloid receptor TRPV3

Beiying Liu; Feng Qin

doi:10.1073/pnas.1615304114

Edited by Ramon Latorre, Centro Interdisciplinario de Neurociencias de Valparaíso, Facultad de Ciencias, Universidad de Valparaíso, Valparaiso, Chile, and approved January 4, 2017 (received for review September 13, 2016)

Significance

Thermal TRP channels are principal molecular entities of transducing thermal and noxious stimuli. The mechanisms by which they detect temperature remain elusive, however. Our and others’ recent studies show that thermal channels, such as the vanilloid receptor transient receptor potential 3 (TRPV3), are strongly use-dependent. Here, by exploring this distinct feature using fast temperature jumps, we have identified a molecular basis for temperature-dependent gating of ion channels.

Abstract

Thermal transient receptor potential (TRP) channels, a group of ion channels from the transient receptor potential family, play important functions in pain and thermal sensation. These channels are directly activated by temperature and possess strong temperature dependence. Furthermore, their temperature sensitivity can be highly dynamic and use-dependent. For example, the vanilloid receptor transient receptor potential 3 (TRPV3), which has been implicated as a warmth detector, becomes responsive to warm temperatures only after intensive stimulation. Upon initial activation, the channel exhibits a high-temperature threshold in the noxious temperature range above 50 °C. This use dependence of heat sensitivity thus provides a mechanism for sensitization of thermal channels. However, how the channels acquire the use dependence remains unknown. Here, by comparative studies of chimeric channels between use-dependent and use-independent homologs, we have determined the molecular basis that underlies the use dependence of temperature sensitivity of TRPV3. Remarkably, the restoration of a single residue that is apparently missing in the use-dependent homologs could largely eliminate the use dependence of heat sensitivity of TRPV3. The location of the region suggests a mechanism of temperature-dependent gating of thermal TRP channels involving an intracellular region assembled around the TRP domain.

Temperature detection is a vital ability of all homeothermic organisms. In mammals, thermal transduction is mediated by designated peripheral sensory neurons known as thermoreceptors or nociceptors, including mainly the Aδ and C fibers. Prominent candidate molecules underlying temperature transduction in these neurons are members of the transient receptor potential (TRP) channels family that have been discovered in recent years (1). They include, for example, the vanilloid receptors TRPV1–4, the menthol receptor TRPM8, and the anykrin repeat receptor TRPA1. These temperature-dependent TRP channels are expressed in places known to mediate thermal reception, such as peripheral sensory neurons or skin keratinocytes. Their genetic ablation causes deficiency in thermal sensation and heat-induced hyperalgesia.

Different thermal TRP channels are generally known to possess distinct temperature activation thresholds that coincide with temperatures for eliciting distinct sensations of being noxious cold, cold, warm, and noxious hot. In this paradigm, the vanilloid receptor TRPV1 is responsible for detection of noxious heat above 42 °C (2). Another heat-gated vanilloid receptor, TRPV2, also responds to noxious heat but above 51 °C (3), whereas two other TRPV subfamily members, TRPV3 and TRPV4, have apparent thresholds in innocuous temperature ranges (4 ⇓⇓–7), and thus have been implicated in warmth detection. On the other hand, the TRPM8 receptor is involved in detection of cool temperatures below 21 °C (8, 9), whereas TRPA1 may mediate noxious cold sensitivity (10). Together, their responsiveness spans the entire range of physiologically relevant temperatures.

Recent studies, however, have demonstrated that some thermal channels can have more complex thermal sensitivity than implicated by their known physiological functions. In particular, their activation profiles, including temperature thresholds, may change drastically as a result of prior stimulation. The activation of TRPV3 was first noticed to undergo sensitization over repeated stimulation by chemical agonists or heat. Mechanisms such as pore blockade or Ca²⁺-dependent intracellular regulations have been proposed to account for the sensitization effect (11, 12). However, other findings show that the gating of the channel itself is susceptible to hysteresis, which causes activations to be strongly use-dependent (5, 13). With fast temperature jumps, we have resolved that the channel, albeit generally classified as a warmth receptor, requires high noxious temperatures for initial activation (>50 °C). Only after repetitive stimulation does the channel become responsive to warm temperatures. Similar use dependence has also been found for the vanilloid receptor TRPV2 (14), which initially displayed a noxious activation threshold >50 °C but became warmth-sensitive following heat stimulation. Concomitant to changes in the activation threshold, the slope sensitivity of temperature dependence of the channels is also dynamic. Strong temperature dependence is a hallmark feature of thermal channels. However, heat stimulation can significantly reduce the temperature dependence of the use-dependent thermal channels, causing them to have only moderate temperature dependence.

The use dependence occurring with vanilloid receptors can have functional consequences of sensitizing nociceptive channels to become responsive to otherwise innocuous temperatures, and thus has implications in thermal hyperalgesia (14). Although they are believed to incur from changes on the level of intrinsic gating, the underlying molecular and structural mechanisms are yet to be elucidated. Here, we present a molecular study of the use dependence of thermal channels, focusing on the vanilloid receptor TRPV3 to elucidate its molecular basis and to draw novel insights on mechanisms underlying temperature gating of the channel.

Results

Use Dependence of TRPV3.

We first examined the wild-type temperature response of TRPV3 and its use dependence evoked by fast temperature jumps. Because the initial activation requires a high temperature >50 °C, we have limited temperature pulses to 100 ms so that the channel could be activated in a repetitive manner. Fig. 1A illustrates a train of such heat responses of TRPV3 at 53 °C. The initial current was small, but subsequent stimulations caused progressive increases in responses, which, on average, reached an extent of >10-fold after 12–14 repetitions (Fig. 1B).

Fig. 1.

Use dependence of wild-type TRPV3. (A) Heat responses evoked by a series of identical temperature jumps (53 °C). Each temperature pulse was 100 ms. (Inset) Enlarged view of a single pulse response. (B) Statistical plot of fold increase of peak response over repetitive stimulation. Data were normalized to the first pulse response (n = 7). (C) Responses to a family of temperature jumps ranging from 32–59 °C for the first round of activation (Left) and for repeated activation in the same patch (Right). (D) Temperature (T)-dependent responsiveness curves. Data from 10 independent experiments are superimposed. (E) van’t Hoff plots of current responses for determination of energetics. Linear fittings correspond to ΔH = 86 ± 6 kcal/mol (n = 10) for the first run and ΔH = 32 ± 1 kcal/mol (n = 10) for the repeated run. The upper portion of the plot for the second run has a shallower slope, corresponding to ΔH = 18 ± 1 kcal/mol (n = 10). The holding potential was −60 mV. I/Imax, normalized current to the maximum at 59 °C.

To see how the sensitization induced by repetitive stimulation affects the temperature sensitivity of TRPV3, we activated the channel by repeated applications of a family of temperature pulses ranging from 30–60 °C (Fig. 1C). Initially, the channel began to be activated above 50 °C; however, during the repeated run, significant activity occurred at warm temperatures. Fig. 1D compares the current–temperature relationships of the responses between the two runs. Concomitant to the decrease of the activation threshold, the slope sensitivity of the temperature dependence was also drastically reduced. However, the maximum currents between the two runs were similar. Thus, the stimulation mainly altered the steepness of temperature dependence, effectively broadening the responsiveness curve so that the channel became responsive at lower temperatures.

To quantify the change of temperature dependence, we analyzed the van’t Hoff plot of the temperature responsiveness curve [Fig. 1E; applicability of the analysis is discussed by Liu and Qin (14)] The responses of the first run exhibited a linear relationship up to ∼57 °C (black line), with a slope corresponding to an enthalpy change ΔH ∼ 86 kcal/mol. The second run displayed a bimodal linear dependence, with a slightly steeper slope at lower temperatures up to ∼45 °C, followed by a shallower slope at higher temperatures. Such bimodal temperature dependence could be expected if the gating of the channel became saturating at high temperatures. Thus, we have chosen to fit the lower portion of the curve for the temperature dependence of heat activation, which gave ΔH ∼ 32 kcal/mol. Stimulation thus considerably reduced the energetics of gating, indicating that the temperature dependence of TRPV3 is strongly use-dependent.

Membrane Proximal N Terminus Determines Use Dependence.

If the temperature dependence of TRPV3 is use-dependent, a perturbation in the structure for temperature sensing would be expected to alter the use dependence. A region that has been identified for temperature sensing by vanilloid receptors is the membrane proximal domain (MPD) on the N terminus, located between the ankyrin repeats and the first transmembrane segment (15). Thus, we investigated whether the same region mediates the use dependence of TRPV3. Because the heat sensitivity of TRPV1 is stable (14), we transferred the MPD of TRPV1 into TRPV3 and evaluated the use dependence of heat sensitivity of the chimeric channel [TRPV3/V1(366–441), where amino acid numbering refers to mouse TRPV3].

Fig. 2A shows heat responses of the chimera evoked by repetitive temperature jumps at 53 °C. In contrast to wild-type responses, the initial activity of the chimera was large. Furthermore, over a course of >10 repetitions, the peak current stayed comparable (Fig. 2B). There was a small increase during the first two or three pulses, but the change was minor, which could have happened due to thermal relaxation of the patch after high-temperature exposures. Fig. 2C shows the activation of the chimera with a family of temperature jumps. Here, significant activation was seen at warm temperatures during the first run of stimulation. The time course and temperature dependence of the responses resembled the time course and temperature dependence of the wild-type during repeated activations. Fig. 2D compares the resulting current–temperature relationships between two runs. They were nearly superimposed, indicating that the chimera was stable over repetitive stimulation.

Fig. 2.

N-terminal MPD mediates use dependence. (A) Responses of the resulting chimera to repetitive temperature pulses (53 °C). (B) Average plot of fold increase of peak response with respect to repetition of stimulation (n = 11). (C) Responses to a family of temperature jumps in two consecutive runs. (D) Comparison of temperature-dependent responsiveness curves between initial and repeated activations (n = 10). (E) Comparison of energetics (Left, n = 10) and maximum current responses (Right, n = 10). The relative change of the response at 59 °C was plotted. The holding potential was −60 mV.

The slope sensitivity of the chimera was estimated with ΔH ∼ 38 kcal/mol for both the initial and repeated activations (Fig. 2E). Thus, the same energetic was retained during repeated activations. These energetics were considerably less than the wild-type energetics upon initial activations, but were similar to the energetics during repeated activations, supporting that the chimeric channel indeed resembled the sensitized wild-type channel. The maximum currents were again similar between runs (Fig. 2E). Thus, the overall heat activation profile of the chimera stayed largely unchanged over repetitive stimulation, supporting that the MPD delineates the use dependence of heat sensitivity of TRPV3.

Delineation of Molecular Basis of Use Dependence.

We next sought to determine whether the whole membrane-proximal N terminus is required or if a minimal subregion exists to mediate the use dependence of TRPV3. The N-terminal domain we have exchanged is still large, consisting of nearly 80 residues, many of which are not conserved between vanilloid homologs (Fig. 3A). To narrow down possible effective subregions, we further constructed a series of chimeras between TRPV3 and TRPV1 by exchanging smaller fragments within the domain. Summarized in Fig. 3B are some of the subregions we have successfully exchanged.

Fig. 3.

Identification of molecular regions underlying use dependence of heat sensitivity. (A) Sequence alignment of the N-terminal MPD between TRPV3 and TRPV1. Identical residues are highlighted in black. (B) Schematic diagrams showing subregions within the MPD that were exchanged between TRPV3 and TRPV1. Residue numbering is according to mouse TRPV3. (C and D) Representative chimeric responses of TRPV3 evoked by repetitive temperature pulses (53 °C). Chimeras containing exchange of region 410–414 or greater resulted in stable activity. Means of 10–11 independent recordings, normalized to sensitized responses, are shown for each average plot. (E and F) Representative chimeric responses evoked by a family of temperature jumps applied consecutively (30–59 °C). Average plots were each from 10 to 11 experiments. (G) Energetics of representative chimeras. (H) Relative change of the maximum current response. The holding potential was −60 mV.

We first evaluated the heat activation of the chimeras by repetitive identical temperature pulses (Fig. 3C). When fragments on the N-terminal end of the MPD were exchanged (e.g., residues 365–398), the resulting chimeras still showed use-dependent sensitization (Fig. 3C, Top), which, on average, resulted in greater than sevenfold increases in responses (Fig. 3D, black trace). Next, we extended the exchanged region incrementally toward the C-terminal end of the MPD. When the region between residues 365–414 was exchanged, the chimera exhibited stable responses over repetitive stimulations (Fig. 3 C, Middle and D, red). The results thus suggest that the C-terminal end, instead of the N-terminal end, of the MPD is involved in the use dependence of TRPV3. In confirmation, the exchange of fragment 410–414 only recapitulated parent chimeric responses involving the exchange between fragments 365 and 414 (Fig. 3 C, Bottom and D).

Fig. 3 E and F assessed the stability of temperature dependences of chimeras using a family of temperature jumps. The large changes with the wild-type channel between two runs of activations persisted in the N-terminal chimera at residues 365–398, but were diminished when the more C-terminal portion of MPD was exchanged (e.g., residues 365–414, residues 410–414). These C-terminal chimeras displayed fast, low-threshold activations with similar temperature dependencies upon both initial and repeated stimulations.

The van’t Hoff analyses of temperature responsiveness curves confirmed similar energetics between runs for chimeras involving exchanges encompassing the subregion 410–414 (Fig. 3G). These energetics were also similar to the wild-type energetics during repeated activations. On the other hand, the residue 365–398 chimera had a large enthalpy upon initial activation (90 ± 6 kcal/mol, n = 11), comparable to the wild-type channel. The maximum responses for all chimeras were largely the same between consecutive runs (Fig. 3H). Together, our initial chimeric screening delineated a small, discrete subregion around residues 410–414 for the use dependence of TRPV3.

Loop Region Around Residues 412–414 Is Critical.

With position 414 as the C-terminal end, we continued to vary the N-terminal starting position to narrow down the molecular region encoding the use dependence of TRPV3. A minimal region was found to locate around residues 412–414, which corresponds to the loop (residues 404–407) linking two short helices underneath the TRP helix in TRPV1 (Fig. 4A).

Fig. 4.

Loop region 412–414 (404–407 in TRPV1) controls use dependence in TRPV3. (A) Structural diagram for illustration of the loop and its adjacent elements (Protein Data Bank ID code 3J5P). MPD is shown in red, and ankyrin repeats are shown in orange. (B) Replacement of the loop in TRPV3 abolished use-dependent sensitization evoked by repetitive stimulation. The fold increase relative to the initial response was shown for the average plot (n = 11). (C) Time-resolved activation by a family of temperature jumps from 31 to 57 °C [initial activation (Left) and repeated run (Right)]. (D) Comparison of temperature-dependent response curves between runs (n = 10). (E) Enthalpy changes for the initial and repeated activations (Left, n = 10) and relative change of the maximum response (Right, 57 °C). The holding potential was −60 mV.

Fig. 4B shows that heat responses of the chimera [TRPV3/V1(412–414)] evoked by repetitive temperature pulses at 53 °C were stable. The responses to a family of temperature jumps also resembled the responses of the parent chimeras (Fig. 4C), yielding overlapping temperature responsiveness curves between consecutive runs (Fig. 4D). The initial activation of the chimera was estimated with ΔH ∼ 47 kcal/mol, whereas the repeated activation involved 45 kcal/mol (Fig. 4E). The maximum activity (at 57 °C) was similar between two runs (Fig. 4E). These results indicate that the replacement of the small-loop region between residues 412 and 414 suffices to attain stable heat responses, suggesting that the loop is a major molecular determinant of the use dependence of the heat sensitivity of TRPV3.

A Single-Residue Molecular Switch for Use Dependence.

Sequence alignments revealed one notable difference in the loop region (residues 412–414) between different vanilloid receptors: The homologs with use dependence are one residue shorter than homologs with stable heat sensitivity, such as TRPV1 (Fig. 3A). To elucidate the roles of individual residues in the loop, we first examined whether the missing residue (S404 in rat TRPV1) is pertinent to the use dependence of TRPV3. As shown in Fig. 5, the insertion of a serine residue between positions 411 and 412 in TRPV3 resulted in responses with markedly improved stability. The plot of the current–temperature relationship (Fig. 5D) suggests that the second run of activation involved a slightly reduced temperature dependence compared with the first run (ΔH ∼ 53 kcal/mol for the first run and ΔH ∼ 34 kcal/mol for the second run; Fig. 5G). These estimates indicate that the mutant is still use-dependent, but to a considerably less degree than the wild-type channel. Thus, the deletion of the serine residue at position 412 in the loop played a major role in inducing the strong use dependence of the wild-type TRPV3.

Fig. 5.

Identification of critical residues in the loop region. (A) Insertion of a serine residue at position 412 (412S) resulted in a relatively stable heat response over repetitive stimulation. (B) Average plot for fold change of current at each repetition relative to the initial response (n = 10). (C) Responses of the mutant (412S) evoked by a family of temperature jumps (31–57 °C). (D) Temperature-dependent response curves of the 412S mutant (black for the first run and red for the second run). (E and F) Temperature-dependent response curves for mutants containing replacement of residues at other locations on the loop (black for the first run and red for the second run). (G) Summary plot of ΔH of mutant channels. The holding potential was −60 mV.

Other markedly different residues in the loop region between TRPV3 and TRPV1 include N412 and D414, which are E405 and P407, respectively, in TRPV1. To probe the importance of these residues, we substituted them accordingly in wild-type TRPV3, one at a time, for their counterresidues in TRPV1. However, both mutants (N412E and D414P) retained wild-type responsiveness (Fig. 5 E–G). Thus, the mutations at these positions were relatively ineffective in altering the use dependence of the wild-type channel.

Loop Conformation Controls Heat Sensitivity.

To draw insights into how the loop mediates heat sensitivity and its use dependence in TRPV3, we examined insertions of other residues at position 412 on the loop. The question of particular interest is whether the serine residue as identified above is the best substituent for stable heat sensitivity.

Fig. 6 summarizes the results for various substitutions at position 412, with side chains spanning different sizes and polarity, including G, A, V, T, and N. Among these side chains, the valine insertion was found to be the most effective in elimination of use dependence of TRPV3 (Fig. 6 A and B), yielding virtually overlapping temperature responsiveness curves between two consecutive runs. The second most effective insertion was threonine (Fig. 6C), for which the responsiveness curves were only slightly shifted between runs. On the other hand, the glycine and alanine insertions were largely ineffective (Fig. 6 D and E), albeit alanine slightly reduced the ΔH of initial activation (Fig. 6G), whereas the insertion of an asparagine residue, which is polar and also larger in size, showed an intermediate effect (Fig. 6 E and H).

Fig. 6.

Influence of side-chain structure on use dependence. (A and B) Activation time courses and temperature responsiveness curves for the most effective insertion of valine at position 412. Averages from 10 to 12 independent recordings were plotted (the same below). (C–F) Temperature responsiveness curves for insertions with other residues (Thr, Gly, Ala, and Asn) at position 412. (G) Summary plot of ΔH (n = 10–12). The holding potential was −60 mV.

Overall, the data support that heat activation of TRPV3 strongly depends on the side-chain properties of residues in the loop, particularly at position 412. The ineffectiveness of the glycine insertion implies that the loop flexibility is not a critical determinant, whereas the effectiveness of valine rules out the significance of polarity but favors a role for the size of the side chain. The order of the relative effectiveness of insertions between G, A, and S is also consistent with an effect of the side-chain size. The optimal size appears to be achieved with valine or threonine with branched chains. The results thus support that the conformation of the loop, which is dependent on side-chain sizes, is important for heat sensitivity and its use dependence of TRPV3.

Discussion

By application of rapid temperature jumps, we have resolved a more complex temperature activation profile of the vanilloid receptor TRPV3 than commonly thought as a warmth detector (4 ⇓–6). The channel possesses a high noxious activation threshold >50 °C, similar to TRPV2 (3), upon initial activation. It is only after intense stimulation that it becomes responsive to warm temperatures. TRPV3 thus can function as either a nociceptor or a warmth receptor. The change of the activation threshold of the channel does not occur by a simple shift of the gating curve, as usually occurs for sensitization of a channel due to messenger-based regulations. Instead, it results from the change of the steepness of the gating curve. The use-dependent sensitization thus occurs at the expense of a decreased discriminative power over small temperature gradients.

The sharp steepness of temperature dependence of thermal channels is generally associated with the large energetic involved in temperature sensing by the channels. Thus, a straightforward interpretation of the large change in the slope sensitivity of TRPV3 is that the process of temperature sensing by the channel becomes altered during activation. For more complex gating mechanisms such as an allosteric paradigm (16), where the stimulus sensor domain is allosterically coupled to the gate, the slope of the gating curve may depend on both stimulus sensor properties and strength of allosteric coupling. However, as illustrated by simulations in Supporting Information, a change in the coupling strength in this case predicted opposite effects on activation threshold and slope sensitivity. By fitting explicitly with an allosteric scheme (Supporting Information), we also found that such a model was difficult to reconcile with the responsiveness curves of TRPV3 both before and after sensitization if temperature sensor properties were unchanged. These analyses suggest that a change in allosteric coupling alone could not account for the type of simultaneous reductions of the activation threshold and slope sensitivity of TRPV3, and thus still support changes in intrinsic temperature dependence of the channel. Consistently, the loop region underlying the use dependence of TRPV3 is a part of the N-terminal MPD. Among the various molecular regions that have been found to affect activations of thermal channels (15, 17 ⇓⇓⇓–21), the MPD has been implicated for temperature sensing by vanilloid receptors (15). Thus, the location of the loop also supports altered temperature sensing underlying the use dependence of TRPV3.

The new structures of TRPV1 reveal several intriguing features in the identified loop region that may be pertinent to the use dependence of TRPV3 (22; Supporting Information). The critical serine at position 404 is in proximity to the S2–S3 linker, with distances to V508 (closed: 4.48 Å, open: 3.93 Å) and D509 (closed: 4.73 Å, open: 4.4 Å) in the range for van der Waals interactions. Two other residues on the loop, T406 and P407, are H-bonded to ankyrin repeats via residue S342 in the closed state and residue G344 in the open state, respectively. Thus, the loop interfaces with the S2–S3 linker on the top and the ankyrin repeats on the bottom. This arrangement of the loop and its dynamics suggest a possible “click-and-hold” model for activation of TRPV3. Because the serine residue is missing in TRPV3, the loop may become dislocated from the S2–S3 linker. Then, the recovery of S404 in TRPV3 mutants presumably plays a role in restoring the interactions with the S2–S3 linker. The change of the loop position can incur further changes in local structures, such as the MPD and adjacent ankyrin repeats, with a functional impact on heat sensitivity and stability. On the other hand, the comparison of the closed and open structures of TRPV1 indicates that the distances between S404 and the S2–S3 linker become shorter in the open state than in the closed state, suggesting a trend that the loop moves toward the S2–S3 linker during channel opening. In the wild-type TRPV3, such a movement upon initial activation may bring the loop sufficiently close to the S2–S3 linker to forge new interactions between them, leading the channel to adopt a structure subsequently similar to the serine insertion mutant. The appearance of this new structure would underlie the hysteresis of the wild-type TRPV3. According to our energetic analyses, the opening from the new structure, which corresponds to the activation during repeated stimulation, involved ΔH ∼ 30 kcal/mol, compared with ΔH ∼ 90 kcal/mol for the initial activation. This change places an estimate of an energetic change of ΔH ∼ 60 kcal/mol between the initial and new structures. In summary, the structural and functional data both support that the positioning of the loop has influences on structures of TRPV3, especially the elements for temperature sensing, and predict its dynamic interactions with the S2–S3 linker as a mechanism for the use dependence of the channel.

Materials and Methods

Temperature Jumps.

Temperature jumps were produced by laser irradiation (23). Constant temperature steps were generated using the current of an electrode for feedback control. Temperature was calibrated offline from the electrode current based on temperature coefficient (Q₁₀) of electrolyte conductivity.

Other Materials and Methods.

Details for the method described above and for cell culture, expression, electrophysiology, and data analysis are provided in Supporting Information.

SI Use Dependence of Wild-Type TRPV3 at a Positive Membrane Potential

Thermal TRP channels are responsive to depolarization of membranes. In particular, it has been suggested that temperature activates these channels through a voltage-dependent gating mechanism (24, but also 25, 26). Thus, we have examined whether the use dependence as observed at a negative membrane potential also occurs at a depolarizing voltage. We first examined the wild-type TRPV3 channel. Fig. S1 shows heat responses of the channel at +60 mV. The temperature stimulation protocols remained the same as at −60 mV. When stimulated repetitively by temperature pulses at 53 °C, the channel exhibited similarly sensitized responses. When activated by a family of temperature jumps, the second round of activation also showed considerably reduced threshold and slope sensitivity. Thus, the heat activation of the wild-type TRPV3 is similarly use-dependent at a positive voltage. The energetic analyses also support large comparable changes in the energetics of activations during the initial and repeated stimulations (Fig. S1E).

Fig. S1.

Heat responses of wild-type TRPV3 at +60 mV. (A) Heat-evoked currents by repetitive temperature pulses at 53 °C. Each temperature pulse was 100 ms, and the interpulse duration was 5–10 s. (B) Average plot of relative changes in peak current with respect to repetition of stimulation (n = 5). (C) Heat responses to a family of temperature jumps [first run (Left) and second run (Right)]. (D) Comparison of current–temperature relationships between two consecutive runs. (E) Quantitative analyses of temperature dependence. The slope of the linear fit on the lower portion of the curve provides ΔH = 86 ± 1.2 kcal/mol for initial activation and ΔH = 25 ± 1.4 kcal/mol for repeated activation (n = 5). Recordings were from transiently transfected HEK293 cells. The holding potential is +60 mV. I/Imax, normalized current to the maximum at 57 °C; T, temperature.

SI Use Dependence of TRPV3 Mutants at a Positive Membrane Potential

Conversely, we examined whether the mutant channels lacking use dependence continued to have stable responses at a depolarizing potential. Fig. S2 summarizes the heat responses of the chimera TRPV3/V1(412–414) at +60 mV. The currents evoked by temperature pulses at 53 °C remained stable over repetitive activations. The temperature responsiveness curves resulting from activations by a family of temperature jumps were nearly overlapping between two consecutive runs. The activation profiles also resembled the activation profiles of the sensitized wild-type channel, with the threshold of activation shifted to warm temperatures. The identified loop region thus remained effective in controlling the use dependence of activation at a positive membrane potential.

Fig. S2.

Heat responses of chimera TRPV3/V1(412–414) at +60 mV. (A) Heat-evoked currents by repetitive temperature pulses at 53 °C. (B) Relative changes of responses with respect to repetition of stimulation, averaged from n = 5 independent recordings. (C) Heat responses to repeated applications of a family of temperature jumps [first run (Left) and second run (Right)]. (D and E) Comparison of temperature dependence of heat responses between two consecutive runs of activations. The lower linear portions of the plots in E have slopes corresponding to ΔH = 27 ± 0.4 kcal/mol for the first run of activation and ΔH= 24 ± 0.2 kcal/mol for the second run of activation (n = 5). Recordings were from transiently transfected HEK293 cells. The holding potential is +60 mV.

Figs. S3 and S4 illustrate heat activation profiles of single-residue insertion mutants TRPV3-412S and TRPV3-412V, respectively. In both cases, the insertion of a single residue between positions 411 and 412 was still largely able to suppress the use-dependent changes incurring from repeated stimulations. Also consistent with the observation at a negative voltage, the valine insertion appeared more effective than the serine insertion in diminishing use dependence (e.g., the serine mutant continued to show a minor change in temperature dependence corresponding to about 4 kcal/mol between two successive rounds of activations, whereas the valine mutant exhibited a more negligible change). These observations were parallel to those observations at a negative voltage, and thus support the generality of the findings.

Fig. S3.

Heat responses of mutant TRPV3-412S at +60 mV. (A) Heat-evoked currents by repetitive stimulation with a temperature pulse of 53 °C. (B) Relative changes of responses with respect to repetition of stimulation (n = 5). (C) Heat responses to repeated applications of a family of temperature jumps [first run (Left) and second run (Right)]. (D and E) Comparison of temperature dependence of heat responses between two runs. The lower linear portions of the plots in E have slopes corresponding to ΔH = 27 ± 0.6 kcal/mol for the first run of activation and ΔH= 23 ± 0.2 kcal/mol for the second run of activation (n = 5). Recordings were from transiently transfected HEK293 cells. The holding potential is +60 mV.

Fig. S4.

Heat responses of mutant TRPV3-412V at +60 mV. (A) Heat-evoked currents by repetitive stimulation at 53 °C. (B) Relative changes of currents over the course of repetition of stimulation (n = 5). (C) Heat responses to repeated applications of a family of temperature jumps [initial stimulation (Left) and repeated stimulation (Right)]. (D and E) Comparison of temperature dependence of heat responses between two consecutive runs of activations. The lower linear portions of the plots in E have slopes corresponding to ΔH = 25 ± 0.7 kcal/mol for the first run of activation and ΔH = 25 ± 1 kcal/mol for the second run of activation (n = 5). Recordings were from transiently transfected HEK293 cells. The holding potential is +60 mV.

SI Voltage Activation of TRPV3 Is Stable

In another experiment to address whether voltage affects the use dependence of heat sensitivity of TRPV3, we directly measured voltage responses of the channel and examined their stability. First, similar to heat sensitivity, we tested voltage sensitivity in response to repetitive identical voltage pulses. Fig. S5 A and B illustrates responses of TRPV3 to a series of depolarization pulses (+140 mV per 100 ms). A large current was seen during initial activation, and the response was stable during subsequent activations. Then, we tested voltage sensitivity by repeated applications of a family of voltage steps. Fig. S5 C–E shows responses to voltage steps from −140 mV to +160 mV in increments of 20 mV. The responses also appeared similar between the first and second runs. Fig. S6 F–K summarizes the voltage responsiveness of several mutant channels [TRPV3/V1(412–414), TRPV3-412S, and TRPV3-412V]. In all cases, the resulting current–voltage relationships were superimposing, whereas the maximal current at +160 mV stayed comparable. Thus, our data support that the voltage sensitivity of TRPV3 is not use-dependent, suggesting that the heat activation differs mechanistically from the voltage activation.

Fig. S5.

Voltage sensitivity of wild-type mutant TRPV3 channels. (A and B) Responses of wild-type TRPV3 evoked by voltage pulses (+140 mV) were stable over repetitive stimulation. Each voltage pulse was 100 ms long. The average value represents the mean of n = 5 independent recordings. (C–E) Responses of wild-type TRPV3 evoked by voltage steps from −140 mV to +160 mV (with an increment of 20 mV) were also stable between repetitions. Current–voltage relationships were overlapping (D), whereas the maximum current at +160 mV stayed comparable (E). Data points were averages of n = 6 independent patches. Parallel plots for chimera and mutant TRPV3 channels [TRPV3/V1(412–414) (F and G), TRPV3-412S (H and I), and TRPV3-412V (J and K)]. Analyses were based on n = 5 independent experiments. Recordings were all from transiently transfected HEK293 cells at the ambient temperature. Vm, membrane potential.

Fig. S6.

Control experiments showing effectiveness of polylysine (PLL) for screening membrane PIP₂. (A) Addition of 0.01% PLL in pipette abolished responses of TRPM8 evoked by menthol (100 μM). (B) Average plot of inhibition of TRPM8 by PLL (n = 6). Currents were normalized to their initial responses after breaking in. Recordings were all from transiently transfected HEK293 cells at the ambient temperature. The holding potential is −60 mV.

SI Phosphatidylinositol 4,5-Bisphosphate Does Not Mediate Use Dependence of TRPV3

The hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate (PIP₂) has been reported to have a sensitization effect on TRPV3 (27). Thus, we examined whether the use dependence of TRPV3 is PIP₂-dependent. In the first experiment, we exploited polylysine to screen membrane PIP₂. The effectiveness of the assay was demonstrated on TRPM8 (Fig. S6), where the dialysis of polylysine into cells through the patch electrode resulted in profound inhibition of the current of TRPM8. Then, we examined the heat sensitivity of TRPV3 in the presence of polylysine. Fig. S7 A and B illustrates heat responses at 53 °C. The repetitive stimulation still caused progressively increased responses. Fig. S7 C–E shows responses evoked by repeated applications of a family of temperature jumps. Again, the repetition of stimulation profoundly altered the heat activation profile, leading to changes in both the activation threshold and slope sensitivity, as observed in the absence of polylysine.

Fig. S7.

Use dependence of TRPV3 remains after screening of membrane PIP₂ by polylysine. (A and B) Heat responses evoked by temperature pulses at 53 °C (100 ms) were still sensitized by repetitive stimulation in the presence of polylysine (n = 5). (C) Activations by a family of temperature pulses continued to have distinct profiles between repetitions. Experiments used fewer and more moderate temperature pulses to help maintain patch stability. (D and E) Comparison of temperature dependence of heat responses between initial and repeated activations. Linear slopes of the plots in E resulted in estimates of ΔH = 86 ± 5 kcal/mol for initial activation and 28 ± 1 kcal/mol for repeated activation (n = 8). Recordings were from transiently transfected HEK293 cells. The holding potential is −60 mV. Polylysine (0.01%) was applied through a patch pipette.

In the other experiment, we used receptor-mediated hydrolysis to deplete membrane PIP₂. We coexpressed the rat M1 receptor with either TRPM8 or TRPV3. The receptor was activated by a maximum concentration of carbachol (CCH; 30 μM). The effectiveness of the assay was again confirmed using TRPM8 as a control (Fig. S8 A and B). The persistent application of CCH caused continuous inhibition of the TRPM8 current. With the same assay, however, TRPV3 still displayed a high activation threshold (>50 °C) upon the first run of activation (Fig. S8C). Only after repeated stimulation was the activation threshold, along with the slope sensitivity, decreased. The differences resembled the differences under control conditions (Fig. S9 D and E), albeit quantitative analyses suggested a slightly reduced ΔH for the initial activation (67 kcal/mol). Together, our experiments argue that the use dependence of heat sensitivity of TRPV3 was not due to depletion of membrane PIP₂.

Fig. S8.

Use dependence of TRPV3 remained after receptor-mediated depletion of membrane PIP₂. (A and B) Control experiments showing effectiveness of rat M1-mediated depletion of membrane PIP₂. Persistent stimulation of rat M1 by carbachol (CCH; 30 μM) in cells coexpressing rat M1 and TRPM8 resulted in sustained inhibition of menthol (100 μM) responses. Data shown were the average of n = 6 independent experiments. (C–E) Heat responses of TRPV3 following PIP₂ depletion. TRPV3 was coexpressed with rat M1 receptors and was activated, whereas rat M1 was stimulated by CCH. Responses remained sensitized during repeated activations, with changes in time course, activation threshold, and slope sensitivity resembling those changes under control conditions. Linear fitting of slopes in E resulted in ΔH = 67 ± 5 kcal/mol for initial activation and 24 ± 0.2 kcal/mol for repeated activation (n = 6). Recordings were from transiently transfected HEK293 cells. The holding potential is −60 mV.

Fig. S9.

Simulation of allosteric models. (A) Simulated dose–response curves from an allosteric model showing that the increase of the allosteric coupling factor has opposite effects on the midpoint and slope sensitivity of the curve; that is, it decreases the midpoint while increasing the slope. Simulation was based on a MWC (Monod–Wyman–Changeux) model, Po = 1/(1 + L⁻¹ [(1 + [Ca²⁺]/K_d)/(1 + c * [Ca²⁺]/K_d)]⁴), where L is intrinsic opening and c is allosteric coupling, with parameters adapted from the parameters of BK (big potassium) channels (K_d = 32 μM, L = 7.9 × 10⁻³, and c = 2.4 or 24). (B) Heat responsiveness curves of wild-type TRPV3 fitted by an allosteric model. The black line shows the fit for initial responses upon the first round of activation. The red line represents the best fit for responses upon the second round of activation while holding temperature sensor properties (J₀ and ΔH as below) the same as for the fit in the first round of activation. The model response was calculated by I = I₀ * exp(ΔH_i/RT)/[1 + L⁻¹(1 + J)/(1 + cJ)], where I₀ * exp(ΔH_i/RT) describes the maximum current and its temperature dependence (ΔH_i was fixed to 4 kcal/mol), and J = J₀exp(ΔH/RT) describes temperature sensor equilibrium (first fit: L = 1.15 × 10⁻³, c = 4,303, ΔH = 90 kcal/mol, J₀ = 2.85 × 10⁻⁵⁹, and I₀ = 1; second fit: L = 1.1e-3, c = 1.4 × 10⁶, ΔH = 90 kcal/mol, and I₀ = 0.35).

Fig. S10.

Cryo-EM structures of TRPV1 in the identified loop region (colored in red). Closed [Left, PDB (Protein Data Bank) ID code 5irx] and open (Right, PDB ID code 5irz) cryo-EM structures are shown. Residue S404 is the critical serine residue missing in TRPV3. The residue is in proximity to the S2–S3 linker (colored in orange), with distances to V508 (closed: 4.48 A; open: 3.93A) and D509 (closed: 4.73A; open: 4.4A) in the range for van der Waals interactions. The lower side of the loop interfaces with the last two ankyrin repeats (colored in yellow), where residue T406 is H-bonded to S342 in the closed structure and P407 is H-bonded G344 in the open structure (H-bonds shown in green).

SI Materials and Methods

Cell Culture and Expression.

HEK293 cells were grown in DMEM containing 10% (vol/vol) FBS (HyClone Laboratories, Inc.) and were incubated at 37 °C in a humidified incubator gassed with CO₂. Transfection was made at a space confluence of ∼80% by calcium phosphate precipitation. Monomeric red fluorescent protein (mRFP) was cotransfected for laser beam positioning. Experiments took place usually 10–28 h after transfection. The mouse TRPV3 clone was provided by Ardem Patapoutian, The Scripps Research Institute, La Jolla, CA; and Mike Zhu, University of Texas Health Science Center at Houston, TX.

Electrophysiology.

Patch-clamp recordings were made in whole cells and excised patch configurations. Currents were amplified using an Axopatch 200B amplifier (Axon Instruments); low-pass-filtered at 5–10 kHz through the built-in, eight-pole Bessel filter; and sampled at 10–20 kHz with a multifunctional data acquisition card (National Instruments). Data acquisition was controlled by a homemade program capable of synchronous input/output and simultaneous control of the laser and patch-clamp amplifier. Patch pipettes were fabricated from borosilicate glass capillary (Sutter Instrument) and fire-polished to a resistance of <5 MΩ when filled with 150 mM CsCl solution. Pipette series resistance and capacitance were compensated using the built-in circuitry of the amplifier (50–70%), and the liquid junction potential between the pipette and bath solutions was zeroed before seal formation. Currents were evoked from a holding potential of −60 mV.

Bath solutions for whole-cell recording consisted of 150 mM NaCl, 5 mM EGTA, and 10 mM Hepes (pH 7.4) (adjusted with NaOH). Electrodes were filled with 140 mM CsCl, 10 mM Hepes, and 1 mM EGTA (pH 7.4) (adjusted with CsOH). Symmetrical solutions of 150 NaCl were used for excised patch recording. The pH of the Hepes-buffered solutions changed by ≤0.4 unit over 22–55 °C. All chemicals were purchased from Sigma.

Fast Temperature Jump.

Temperature jumps were produced by laser irradiation using a single-emitter, high-power, infrared laser diode as previously described (23). In brief, the diode was mounted on a cooling block and operates at room temperature. Laser emissions from the laser diode were launched into a multimode fiber with a 100-μm core diameter and 0.2 N.A. The other end of the fiber was positioned close to cells, as the perfusion pipette normally was. The laser diode was driven by a pulsed quasi-CW (continuous wave) current power supply (Lumina Power). Pulsing of the controller was controlled from the computer through the data acquisition card using a custom program. A green laser line (532 nm) was coupled to the fiber to aid alignment. The beam spot on the coverslip was identified by illumination of mRFP-expressing cells using the green laser.

Constant temperature steps were generated by irradiating the tip of an open pipette and using the current of the electrode as the readout for feedback control. The laser was first powered on for a brief duration (<0.75 ms) to reach the target temperature, and was subsequently modulated to maintain a constant pipette current. The modulation pulses were stored, and subsequently played back to apply temperature jumps to whole cells or membrane patches. Between consecutive temperature pulses, laser power was adjusted manually and the adjustment generally took less than a minute. Temperature was calibrated offline from the pipette current based on temperature dependence of electrolyte conductivity.

Data Analysis.

The enthalpy change for channel opening was estimated by linear fitting of the van’t Hoff plot of the temperature-responsiveness curve (applicability of the approach is discussed in ref. 14). The leak current in recordings held at negative potentials was extrapolated from the response at the holding temperature (22 °C) preceding temperature jumps using temperature coefficient Q₁₀ = 1.2 and was subtracted from measurement. The temperature dependence of the unitary conductance was not subtracted. However, because it is relatively small (4 kcal/mol for Q₁₀ = 1.2), the reported ΔH is predominated by the ΔH of gating.

Acknowledgments

The authors would like to thank Wen Han for assisting with illustration of protein structures. This work was supported by NIH Grant R01 GM104521.

Footnotes

↵¹To whom correspondence should be addressed. Email: qin{at}buffalo.edu.

Author contributions: B.L. and F.Q. designed research; B.L. and F.Q. performed research; B.L. and F.Q. analyzed data; and F.Q. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1615304114/-/DCSupplemental.

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1. Xiao R, et al.
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1. Chung MK,
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1. Liu B,
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4. Qin F
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1. Liu B,
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1. Yao J,
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1. Diaz-Franulic I,
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5. Latorre R
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1. Grandl J, et al.
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OpenUrl Abstract/FREE Full Text
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1. Yao J,
2. Liu B,
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(2010) Pore turret of thermal TRP channels is not essential for temperature sensing. Proc Natl Acad Sci USA 107(32):E125, author reply E126–E127.
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1. Brauchi S,
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5. Latorre R
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OpenUrl Abstract/FREE Full Text
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1. Cordero-Morales JF,
2. Gracheva EO,
3. Julius D
(2011) Cytoplasmic ankyrin repeats of transient receptor potential A1 (TRPA1) dictate sensitivity to thermal and chemical stimuli. Proc Natl Acad Sci USA 108(46):E1184–E1191.
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1. Gao Y,
2. Cao E,
3. Julius D,
4. Cheng Y
(2016) TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action. Nature 534(7607):347–351.
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1. Yao J,
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3. Qin F
(2009) Rapid temperature jump by infrared diode laser irradiation for patch-clamp studies. Biophys J 96(9):3611–3619.
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1. Voets T, et al.
(2004) The principle of temperature-dependent gating in cold- and heat-sensitive TRP channels. Nature 430(7001):748–754.
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↵
1. Brauchi S,
2. Orio P,
3. Latorre R
(2004) Clues to understanding cold sensation: Thermodynamics and electrophysiological analysis of the cold receptor TRPM8. Proc Natl Acad Sci USA 101(43):15494–15499.
.
OpenUrl Abstract/FREE Full Text
↵
1. Matta JA,
2. Ahern GP
(2007) Voltage is a partial activator of rat thermosensitive TRP channels. J Physiol 585(Pt 2):469–482.
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OpenUrl CrossRef PubMed
↵
1. Doerner JF,
2. Hatt H,
3. Ramsey IS
(2011) Voltage- and temperature-dependent activation of TRPV3 channels is potentiated by receptor-mediated PI(4,5)P2 hydrolysis. J Gen Physiol 137(3):271–288.
.
OpenUrl Abstract/FREE Full Text

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Biophysics and Computational Biology

Proceedings of the National Academy of Sciences: 114 (7)

Table of Contents

Submit

[1] ↵
Clapham DE
(2003) TRP channels as cellular sensors. Nature 426(6966):517–524.
.
OpenUrl CrossRef PubMed

[2] Clapham DE

[3] ↵
Caterina MJ, et al.
(1997) The capsaicin receptor: A heat-activated ion channel in the pain pathway. Nature 389(6653):816–824.
.
OpenUrl CrossRef PubMed

[4] Caterina MJ, et al.

[5] ↵
Caterina MJ,
Rosen TA,
Tominaga M,
Brake AJ,
Julius D
(1999) A capsaicin-receptor homologue with a high threshold for noxious heat. Nature 398(6726):436–441.
.
OpenUrl CrossRef PubMed

[6] Caterina MJ,

[7] Rosen TA,

[8] Tominaga M,

[9] Brake AJ,

[10] Julius D

[11] ↵
Peier AM, et al.
(2002) A heat-sensitive TRP channel expressed in keratinocytes. Science 296(5575):2046–2049.
.
OpenUrl Abstract/FREE Full Text

[12] Peier AM, et al.

[13] ↵
Xu H, et al.
(2002) TRPV3 is a calcium-permeable temperature-sensitive cation channel. Nature 418(6894):181–186.
.
OpenUrl CrossRef PubMed

[14] Xu H, et al.

[15] ↵
Smith GD, et al.
(2002) TRPV3 is a temperature-sensitive vanilloid receptor-like protein. Nature 418(6894):186–190.
.
OpenUrl CrossRef PubMed

[16] Smith GD, et al.

[17] ↵
Güler AD, et al.
(2002) Heat-evoked activation of the ion channel, TRPV4. J Neurosci 22(15):6408–6414.
.
OpenUrl Abstract/FREE Full Text

[18] Güler AD, et al.

[19] ↵
McKemy DD,
Neuhausser WM,
Julius D
(2002) Identification of a cold receptor reveals a general role for TRP channels in thermosensation. Nature 416(6876):52–58.
.
OpenUrl CrossRef PubMed

[20] McKemy DD,

[21] Neuhausser WM,

[22] Julius D

[23] ↵
Peier AM, et al.
(2002) A TRP channel that senses cold stimuli and menthol. Cell 108(5):705–715.
.
OpenUrl CrossRef PubMed

[24] Peier AM, et al.

[25] ↵
Story GM, et al.
(2003) ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 112(6):819–829.
.
OpenUrl CrossRef PubMed

[26] Story GM, et al.

[27] ↵
Xiao R, et al.
(2008) Calcium plays a central role in the sensitization of TRPV3 channel to repetitive stimulations. J Biol Chem 283(10):6162–6174.
.
OpenUrl Abstract/FREE Full Text

[28] Xiao R, et al.

[29] ↵
Chung MK,
Güler AD,
Caterina MJ
(2005) Biphasic currents evoked by chemical or thermal activation of the heat-gated ion channel, TRPV3. J Biol Chem 280(16):15928–15941.
.
OpenUrl Abstract/FREE Full Text

[30] Chung MK,

[31] Güler AD,

[32] Caterina MJ

[33] ↵
Liu B,
Yao J,
Zhu MX,
Qin F
(2011) Hysteresis of gating underlines sensitization of TRPV3 channels. J Gen Physiol 138(5):509–520.
.
OpenUrl Abstract/FREE Full Text

[34] Liu B,

[35] Yao J,

[36] Zhu MX,

[37] Qin F

[38] ↵
Liu B,
Qin F
(2016) Use dependence of heat sensitivity of vanilloid receptor TRPV2. Biophys J 110(7):1523–1537.
.
OpenUrl CrossRef PubMed

[39] Liu B,

[40] Qin F

[41] ↵
Yao J,
Liu B,
Qin F
(2011) Modular thermal sensors in temperature-gated transient receptor potential (TRP) channels. Proc Natl Acad Sci USA 108(27):11109–11114.
.
OpenUrl Abstract/FREE Full Text

[42] Yao J,

[43] Liu B,

[44] Qin F

[45] ↵
Diaz-Franulic I,
Poblete H,
Miño-Galaz G,
González C,
Latorre R
(2016) Allosterism and structure in thermally activated transient receptor potential channels. Annu Rev Biophys 45:371–398.
.
OpenUrl

[46] Diaz-Franulic I,

[47] Poblete H,

[48] Miño-Galaz G,

[49] González C,

[50] Latorre R

[51] ↵
Grandl J, et al.
(2008) Pore region of TRPV3 ion channel is specifically required for heat activation. Nat Neurosci 11(9):1007–1013.
.
OpenUrl CrossRef PubMed

[52] Grandl J, et al.

[53] ↵
Yang F,
Cui Y,
Wang K,
Zheng J
(2010) Thermosensitive TRP channel pore turret is part of the temperature activation pathway. Proc Natl Acad Sci USA 107(15):7083–7088.
.
OpenUrl Abstract/FREE Full Text

[54] Yang F,

[55] Cui Y,

[56] Wang K,

[57] Zheng J

[58] ↵
Yao J,
Liu B,
Qin F
(2010) Pore turret of thermal TRP channels is not essential for temperature sensing. Proc Natl Acad Sci USA 107(32):E125, author reply E126–E127.
.
OpenUrl

[59] Yao J,

[60] Liu B,

[61] Qin F

[62] ↵
Brauchi S,
Orta G,
Salazar M,
Rosenmann E,
Latorre R
(2006) A hot-sensing cold receptor: C-terminal domain determines thermosensation in transient receptor potential channels. J Neurosci 26(18):4835–4840.
.
OpenUrl Abstract/FREE Full Text

[63] Brauchi S,

[64] Orta G,

[65] Salazar M,

[66] Rosenmann E,

[67] Latorre R

[68] ↵
Cordero-Morales JF,
Gracheva EO,
Julius D
(2011) Cytoplasmic ankyrin repeats of transient receptor potential A1 (TRPA1) dictate sensitivity to thermal and chemical stimuli. Proc Natl Acad Sci USA 108(46):E1184–E1191.
.
OpenUrl Abstract/FREE Full Text

[69] Cordero-Morales JF,

[70] Gracheva EO,

[71] Julius D

[72] ↵
Gao Y,
Cao E,
Julius D,
Cheng Y
(2016) TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action. Nature 534(7607):347–351.
.
OpenUrl CrossRef PubMed

[73] Gao Y,

[74] Cao E,

[75] Julius D,

[76] Cheng Y

[77] ↵
Yao J,
Liu B,
Qin F
(2009) Rapid temperature jump by infrared diode laser irradiation for patch-clamp studies. Biophys J 96(9):3611–3619.
.
OpenUrl CrossRef PubMed

[78] Yao J,

[79] Liu B,

[80] Qin F

[81] ↵
Voets T, et al.
(2004) The principle of temperature-dependent gating in cold- and heat-sensitive TRP channels. Nature 430(7001):748–754.
.
OpenUrl CrossRef PubMed

[82] Voets T, et al.

[83] ↵
Brauchi S,
Orio P,
Latorre R
(2004) Clues to understanding cold sensation: Thermodynamics and electrophysiological analysis of the cold receptor TRPM8. Proc Natl Acad Sci USA 101(43):15494–15499.
.
OpenUrl Abstract/FREE Full Text

[84] Brauchi S,

[85] Orio P,

[86] Latorre R

[87] ↵
Matta JA,
Ahern GP
(2007) Voltage is a partial activator of rat thermosensitive TRP channels. J Physiol 585(Pt 2):469–482.
.
OpenUrl CrossRef PubMed

[88] Matta JA,

[89] Ahern GP

[90] ↵
Doerner JF,
Hatt H,
Ramsey IS
(2011) Voltage- and temperature-dependent activation of TRPV3 channels is potentiated by receptor-mediated PI(4,5)P2 hydrolysis. J Gen Physiol 137(3):271–288.
.
OpenUrl Abstract/FREE Full Text

[91] Doerner JF,

[92] Hatt H,

[93] Ramsey IS

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Single-residue molecular switch for high-temperature dependence of vanilloid receptor TRPV3

Significance

Abstract

Results

Use Dependence of TRPV3.

Membrane Proximal N Terminus Determines Use Dependence.

Delineation of Molecular Basis of Use Dependence.

Loop Region Around Residues 412–414 Is Critical.

A Single-Residue Molecular Switch for Use Dependence.

Loop Conformation Controls Heat Sensitivity.

Discussion

Materials and Methods

Temperature Jumps.

Other Materials and Methods.

SI Use Dependence of Wild-Type TRPV3 at a Positive Membrane Potential

SI Use Dependence of TRPV3 Mutants at a Positive Membrane Potential

SI Voltage Activation of TRPV3 Is Stable

SI Phosphatidylinositol 4,5-Bisphosphate Does Not Mediate Use Dependence of TRPV3

SI Materials and Methods

Cell Culture and Expression.

Electrophysiology.

Fast Temperature Jump.

Data Analysis.

Acknowledgments

Footnotes

References

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Significance

Abstract

Results

Use Dependence of TRPV3.

Membrane Proximal N Terminus Determines Use Dependence.

Delineation of Molecular Basis of Use Dependence.

Loop Region Around Residues 412–414 Is Critical.

A Single-Residue Molecular Switch for Use Dependence.

Loop Conformation Controls Heat Sensitivity.

Discussion

Materials and Methods

Temperature Jumps.

Other Materials and Methods.

SI Use Dependence of Wild-Type TRPV3 at a Positive Membrane Potential

SI Use Dependence of TRPV3 Mutants at a Positive Membrane Potential

SI Voltage Activation of TRPV3 Is Stable

SI Phosphatidylinositol 4,5-Bisphosphate Does Not Mediate Use Dependence of TRPV3

SI Materials and Methods

Cell Culture and Expression.

Electrophysiology.

Fast Temperature Jump.

Data Analysis.

Acknowledgments

Footnotes

References

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