Article Figures & Data
Figures
- Figure 1.
2-APB activates recombinant TRPV3 in HEK293 cells. A, Top, Sensitizing whole-cell current responses of TRPV3-transfected HEK293 cells during successive applications of 2-APB (32 μm) and subsequent challenge with heat (+80 mV, upward; –80 mV, downward). Traces were produced by connecting current amplitude (black line) or temperature (gray line) measurements recorded every 2 sec. Middle, Mean current amplitude change normalized to membrane capacitance at +80 mV (upward, filled bars) and –80 mV (downward, open bars) (ΔI±80 mV) after application of 2-APB, at indicated times (n = 5; *p < 10–4; one-way ANOVA). Bottom, I–V traces obtained at times a–e (shown at the top) during repetitive 200 msec voltage ramps from –100 to +100 mV. The dashed line indicates zero current or potential level. V, Membrane potential; I, current. B, ΔI±80 mV recorded from HEK293 cells transfected with mouse TRPV1 (V1), TRPV2 (V2), TRPV3 (V3), and TRPV4 (V4), human TRPV3 (hV3), and pCDNA3 (P3). For V1, V2, and V4, currents were evoked by 100 μm 2-APB (filled bars) followed by heat (open bars; V1, 47°C; V2, 55°C; V4, 43°C) after washout. For V3 and hV3, 2-APB (gray bars) was applied after heat (43°C; open bars). Filled bars for V3 indicate 2-APB addition without previous heat application. n = 6 in each group. Scale bars, V1, 0.5 nA; V2, V4, P3, 50 pA; V3, hV3, 0.1 nA. C, Top, ΔI±80 mV evoked in TRPV3-expressing HEK293 cells at 24°C by different concentrations of 2-APB (–80 mV, squares; +80 mV, circles); n = 6. Bottom, Representative traces from TRPV3-transfected cells at indicated 2-APB concentrations. Dashed lines indicate zero current or potential level.
- Figure 2.
Properties of 2-APB-evoked currents in TRPV3-transfected HEK293 cells. A, 2-APB sensitization of heat-evoked TRPV3 currents in HEK293 cells. Left, Consecutive heat-evoked responses in the absence of 2-APB (top) or with 1 μm 2-APB present during only the second heat challenge (bottom). Right, Mean responses to consecutive heat challenges recorded in the absence (open bars) or presence (filled bars) of 2-APB during the second heat stimulus. For each cell, data were normalized to the peak amplitude of the third heat-evoked response at +80 mV (n = 6; *p < 0.005; **p < 0.0005). B, Representative changes in intracellular Ca2+ in pCDNA3 (left) and TRPV3-transfected (right) HEK293 cells on application of 100 μm 2-APB. Removal of extracellular Ca2+ (0 mm Ca2+, 10 mm EGTA) reversibly decreased the 2-APB-evoked Ca2+ level to or below the prestimulus level. C, Left, Current amplitude changes in TRPV3-transfected HEK293 cells during successive applications of 2-APB (32 μm) with RR (10 μm) present during the indicated time period. Right, ΔI±80 mV at time points a–d, indicated in C. (n = 5; *p < 0.05; **p < 0.005). Corresponding I–V curves are shown at the bottom right. Dashed lines indicate zero current or potential level.
- Figure 3.
Single-channel currents evoked by heat and 2-APB in inside-out patches from TRPV3-transfected HEK293 cells. A, Changes in the open probability of multiple channels (NPo) after heating (left) or addition of 1 μm 2-APB (right). Alternating 540-msec-step pulses to ±60 mV were repeated every 1.5 sec. Dotted lines indicate the closed channel state. Arrowheads indicate multiple-channel open states. At the bottom, a–d and e–h indicate currents recorded at +60 mV and –60 mV, respectively, at the corresponding time points shown at the top. B, Representative single-channel currents evoked by heat (39°C) or 2-APB (1 μm at 25°C) at various voltages. Dashed lines indicate closed channel state. C, I–V relationship of single-channel currents evoked by heat (circles) or 2-APB (squares). n = 5 patches per symbol. Small, filled squares and circles at ±60 mV indicate values derived from linear fit in E at 25 and 39°C, respectively. D, E, Temperature dependence of unitary TRPV3 current amplitude. D illustrates representative currents evoked by 2-APB or heat. In E, unitary current amplitude is plotted as a function of temperature. To facilitate observation of channel openings over a broad temperature range, heat-evoked currents were recorded during both ascending (plus signs) and descending (triangles) phases of heat stimulus. Circles indicate 2-APB-evoked currents recorded at room temperature. Data were derived from eight independent cells. Lines indicate linear fit of heat-evoked current amplitudes (slope = 0.59, Q10 = 2.0 over 29–39°C at +60 mV; slope = 0.63, Q10 = 1.6 at –60 mV).
- Figure 4.
2-APB sensitizes primary keratinocytes to heat. A, Current responses to 100 μm 2-APB plus heat in TRPV4 +/+ keratinocytes. The I–V relationships shown to the right correspond to time points a–c, indicated to the left. B, Augmentation of heat-evoked currents in TRPV4 –/– keratinocytes by 2-APB. Top, Representative current amplitude changes during successive challenges with heat in the presence or absence of 2-APB (100 μm) at ±80 mV. Bottom right, I–V relationships at time points a–d, indicated at the top. Bottom left, ΔI±80 mV before and during three successive heat challenges in the presence (filled bars) or absence (open bars) of 2-APB. Heat-evoked responses in the absence versus the presence of 2-APB were recorded in separate cells (n = 7 for the 2-APB-treated group and 5 for the untreated group; *p < 0.01; **p < 0.05; ***p < 0.005). C, 2-APB (100 μm) applied during heat stimulus enhances heat-evoked currents in TRPV4 –/– keratinocytes. I–V relationships shown to the right were obtained at time points a–c, indicated to the left. D, Effect of RR on currents evoked by 2-APB and heat in TRPV4 –/– keratinocytes. Top, Current traces recorded during challenges with heat and 2-APB (100 μm) with or without RR (10 μm). Bottom, ΔI±80 mV (n = 5; *p < 0.05) (left) and I–V relationships (right) at indicated time points. In all panels, horizontal dashed lines indicate zero current level.
Additional Files
Supplemental data
Supplemental figure
Files in this Data Supplement:
- Supplemental Fig. 1 - Supplemental Figure 1. Supra-additive activation of TRPV3 by RR and 2-APB. Current amplitude changes at +80 mV in response to RR (10 m), 2-APB (left, 3 m; right, 100 m), or both in TRPV3-transfected HEK293 cells (left) or TRPV4 [minus]/[minus] primary keratinocytes (right) are shown. Left, *p < 0.05; **p < 0.01. Right, *p < 0.005; **p < 0.001. n = 6.
