Liu B and Qin F (2015), The Xenopus tropicalis orthologue of TRPV3 is h...

XB-ART-51450

J Gen Physiol 2015 Nov 01;1465:411-21. doi: 10.1085/jgp.201511454.

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The Xenopus tropicalis orthologue of TRPV3 is heat sensitive.

Liu B , Qin F .

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Thermosensitive members of the transient receptor potential (TRP) family of ion channels (thermal TRP channels) play a crucial role in mammalian temperature sensing. Orthologues of these channels are present in lower vertebrates and, remarkably, some thermal TRP orthologues from different species appear to mediate opposing responses to temperature. For example, whereas the mammalian TRPV3 channel is activated by heat, frog TRPV3 is reportedly activated by cold. Intrigued by the potential implications of these opposing responses to temperature for the mechanism of temperature-dependent gating, we cloned Xenopus laevis TRPV3 and functionally expressed it in both mammalian cell lines and Xenopus oocytes. We found that, when expressed in mammalian cells, the recombinant channel lacks the reported cold sensitivity; rather, it is activated by temperatures >50°C. Furthermore, when expressed in mammalian cells, the frog orthologue shows other features characteristic of mammalian TRPV3, including activation by the agonist 2-aminoethoxydiphenyl borate and an increased response with repeated stimulation. We detected both heat- and cold-activated currents in Xenopus oocytes expressing the recombinant frog TRPV3 channel. However, cold-activated currents were also apparent in control oocytes lacking recombinant TRPV3. Our data indicate that frog TRPV3 resembles its mammalian orthologues in terms of its thermosensitivity and is intrinsically activated by heat. Thus, all known vanilloid receptors are activated by heat. Our data also show that Xenopus oocytes contain endogenous receptors that are activated by cold, and suggest that cold sensitivity of TRP channels established using Xenopus oocytes as a functional expression system may need to be revisited.

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Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: ran trpv3

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	Figure 1. Frog TRPV3 is activated by 2-APB, but not by cold, in HEK293 cells. (A) Representative 2-APB–evoked currents in HEK293 cells expressing frog TRPV3. Holding potential, −60 mV. (B) Dose–response curve of 2-APB. The current density normalized by membrane capacity is shown. The solid line represents a fit by a Hill equation, with Imax = 330 ± 14 pA/pF, nH = 1.15 ± 0.14, and EC50 = 269 ± 28 µM (n = 11). (C and D) Voltage dependence of 2-APB response, showing that the current was outwardly rectified. [2-APB] = 500 µM. n = 5 for current–voltage relationship. (E) Cold evoked no activity in HEK293 cells expressing frog TRPV3, whereas 2-APB produced robust currents before or after cooling in the same cells. Holding potential, −60 mV. (F) Responses evoked by 2-APB were rapidly inhibited by 20 µM RR. (G) Statistic plot of cold responses versus 2-APB responses recorded from the same cells (n = 7). The small decrease in the current upon cooling reflected changes in the baseline activity. Also plotted is the residual 2-APB response after the application of RR, showing the potent inhibition of the current by RR (20 µM RR and 1 mM 2-APB; n = 10). All recordings except cold responses were at room temperature. Error bars represent mean ± SEM.
	Figure 2. Frog TRPV3 is heat sensitive. (A) Representative whole-cell current evoked by temperature jumps in a frog TRPV3–expressing HEK293 cell. Each temperature pulse was 100 ms in duration. The rise time was <0.75 ms. Significant heat-sensitive activity started to occur at 51–54°C. (B) Temperature-response profiles derived from individual recordings (n = 14). Currents were normalized by their maximum responses, respectively. (C and D) Similar responses recorded from excised membrane patches in the outside-out configuration (n = 6). (E) Heat-evoked currents were suppressed by 20 µM RR. Currents in outside-out patches were elicited with a temperature jump to 53°C. RR was applied to patches by perfusion between temperature jumps. The application of RR suppressed the initial current by 94 ± 2% (n = 5). (F) Pre-application of RR to the bath solution prevented activation of heat-sensitive current. Temperature jumps ranging from 32 to 59°C were applied. Representative traces from six recordings. (G) Temperature responses from untransfected control HEK293 cells. Temperature jumps in the range of 30 to 61°C produced no detectable heat response. Representative traces for >10 recordings. Holding potential of −60 mV for all recordings.
	Figure 3. Sensitization of frog TRPV3 by 2-APB. (A) Repetitive 2-APB responses at the same concentration (60 µM), recorded from frog TRPV3–expressing HEK293 cells. The current was increased progressively with repeated stimulations. Representative traces from 11 cells. (B and C) Concentration dependence of frog TRPV3 after sensitization. Solid lines in C are fits to the Hill equation (Imax = 330 ± 10 pA/pF, nH = 1.8 ± 0.26, and EC50 = 31 ± 2 µM; n = 10). Also shown in C is the dose–response curve before sensitization. Error bars represent mean ± SEM.
	Figure 4. Hysteresis of heat activation in frog TRPV3. (A) Current responses upon initial heat stimulation. Each temperature pulse was 100 ms. (B) Current responses to the second stimulation from the same cell. The same temperature jumps were used for both stimulations. (C) Temperature-response profiles before and after hysteresis. Responses from the same cells for the first and the second stimulation are shown (n = 5). (D) Relative change of the maximum response between the first and the second run (n = 5). Recordings were from HEK293 cells at −60 mV. Error bars represent mean ± SEM.
	Figure 5. 2-APB and cold responses in frog TRPV3–expressing Xenopus oocytes. (A) Cold and 500 µM 2-APB both evoked currents in frog TRPV3–injected Xenopus oocytes. Bottom trace plots bath temperature change in time. (B) Representative cold-response profiles from 10 oocytes. The temperature activation threshold was variable from around 20 to 12°C. Currents were normalized to their peak values. Responses during the cooling phase are shown. (C) 2-APB–dependent current was rapidly blocked by 20 µM RR. (D) Cold-evoked current was not rapidly blocked by 20 µM RR. (E) Cold-sensitive current remained when 20 µM RR was included in both wash and cold perfusion buffers. (F) Delocalization of cold- and 2-APB–evoked currents. Cold-sensitive current ran down after initial activation, whereas 2-APB–sensitive current remained. (G) Current response activated by heat in Xenopus oocytes expressing frog TRPV3. Bottom trace shows bath temperature change. (H) Inhibition of heat-evoked currents by 50 µM RR in oocytes. (I) Current response in control oocytes evoked by heat. (J) Temperature-response profiles of heat-activated currents from TRPV3-expressing oocytes. Each trace corresponds to an individual recording. (K) Statistic plot of average current from oocytes containing TRPV3 compared with RR-inhibited current and current of control oocytes. All currents were recorded at a holding potential of −60 mV. Error bars represent mean ± SEM.
	Figure 6. Endogenous cold responses in Xenopus oocytes. (A and B) Cold-sensitive currents recorded in control oocytes (A, uninjected; B, injected with water). (C) Endogenous cold-sensitive current was insensitive to RR block. Both wash and cooling buffers contained 20 µM RR. (D) Cold-response profiles of endogenous cold activity, showing similarly variable temperature activation thresholds as observed in frog TRPV3–expressing oocytes. All currents were recorded at a holding potential of −60 mV.

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