Article Figures & Data
Figures
- Fig. 1.
TRPV4 mediates heat-evoked currents inXenopus oocytes. A–C, Two-electrode voltage-clamp recordings. Shown are representative second (I), fourth (II), and sixth (III) heat-evoked current responses to consecutive heat ramps (from 27 to 45°C in 15 sec, indicated on the scale at the bottom) for water-injected control oocytes (A) and oocytes injected with TRPV4 cRNA (B, C). In C, ruthenium red (horizontal bar, 100 nm) was added 30 sec before the third heat application and washed away for 10 min before the fifth heat application. Inset, Amplitudes of responses II and III relative to response I with (open columns) or without (filled columns) ruthenium red treatment. Data represent the mean ± SEM; n = 4 (*p < 0.01; ***p < 0.001; unpaired t test). D, Representative temperature response profiles evoked by an initial heat stimulus (top) and a second heat stimulus (bottom) in oocytes expressing TRPV4 (V4) or TRPV1 (V1). Heat stimulus ramps went from 22 to 45°C in 15 sec. Currents were normalized to the amplitude at 45°C.E, Effect of suprathreshold temperature fluctuations (bottom) on representative TRPV4-mediated current response (top).
- Fig. 2.
TRPV4 mediates heat-evoked calcium influx in HEK 293 cells. A, Relative [Ca2+]i (indicated by ratio of fura-2 emission at 340/380 nm excitation) in HEK 293 cells transiently transfected with TRPV4 or control vector (four each) after a reduction in osmolarity from 290 to 250 mOsm (horizontal bars) at 24°C. B, C, Relative [Ca2+]i in HEK 293 cells transiently (B) or stably (C) transfected with TRPV4 or control vector (four each) during a heat stimulus from 24 to 40°C in ∼40 sec. D, Comparison of heat-evoked (40°C) increases in fura ratio among cells transiently transfected with control vector (P;n = 5), TRPV4 (V4;n = 12), TRPV1 (V1;n = 3), Drosophila TRPL (dT; n = 3), or human TRPC3 (hT; n = 4) or among stable transformants generated with control vector (P;n = 8), TRPV4 (V4;n = 15), or TRPV1 (V1;n = 13). Data represent the mean ± SEM of the indicated number of coverslips. Comparisons are with controls. *p < 0.05; ***p < 0.001;NS, not significant; unpaired ttest.
- Fig. 3.
Characterization of heat-evoked calcium responses in TRPV4-expressing HEK 293 cells A, Reversible inhibition of heat-evoked (ramp to 40°C; horizontal filled bars) calcium influx in cells stably expressing TRPV4 by ruthenium red (RR; 200 nm; horizontal open bar, top) or by removal of extracellular calcium (horizontal open bar, bottom). Shown at right are relative amplitudes of heat-evoked responses during ruthenium red (hatched bars;n = 3) or calcium-free (open bars;n = 3) challenge (response II/response I) or after return to normal bath solution (response III/response I). Control (filled bars; n = 4) represents three consecutive responses in normal bath solution.B, Temperature response profiles of heat-evoked calcium responses in cells stably transformed with vector (filled squares;n = 3–4), TRPV4 (filled circles; n = 4–11), or TRPV1 (open circles; n = 4–12). Data represent the mean ± SEM of the indicated number of independent microscopic fields. C, Heat-evoked calcium responses in stable TRPV4 transformants (V4) or vector control cells (vector; P) after acclimation (15 min) to 37°C.Left, Representative traces from four cells of each type. Right, Mean ± SEM increase in fura ratio (n = 4). Data represent the mean ± SEM of the indicated number of coverslips. Comparisons are with controls (A, C) or between TRPV4 and TRPV1 (B). *p < 0.05; **p < 0.01; ***p < 0.001; unpaired t test.
- Fig. 4.
TRPV4 mediates heat-evoked currents in HEK 293 cells. A, Representative whole-cell current traces (at −60 mV) from HEK 293 cells stably transformed with TRPV4 or control vector during the temperature ramp indicated at thebottom. B, Representative temperature-response profiles derived from cells stably transformed with TRPV4, TRPV1, or control vector. C, Representative current–voltage relations for heat-evoked responses in cells transformed with TRPV4 (b), TRPV1 (c), or control vector (a). Similar patterns were observed in four cells of each type.
- Fig. 5.
TRPV4-mediated heat-evoked responses are modulated by osmolarity. A, Representative current traces of the second (I), fourth (II), and sixth (III) responses of a TRPV4-expressing oocyte subjected to consecutive heat stimuli (45°C; horizontal open bars). Hypertonic bath solution (410 mOsm; horizontalfilled bar) was applied 1 min before the third heat stimulus, and the oocytes were washed for 2 min after the fourth heat stimulus.Inset, Amplitudes of heat-evoked responses II and III relative to response I in 410 mOsm-treated (open columns) and untreated (filled columns) TRPV4- and TRPV1-expressing oocytes. Data represent the mean ± SEM of four oocytes. B, Representative heat-evoked (40°C, horizontal filled bars) calcium influx responses in HEK 293 cells stably expressing TRPV4. Bath solution osmolarity is indicated. The 300 mOsm low-sodium (300/m) and 250 mOsm solutions contained equivalent NaCl concentrations and differed only by the presence or absence, respectively, of mannitol. In the bottom two traces the experiment was initiated in 300/m solution and was switched to 250 mOsm at the time indicated by the arrows. C, Summary of heat-evoked calcium responses at indicated osmolarities for cells stably transformed with control vector (P; open columns; n = 3–4) or TRPV4 (V4; filled columns;n = 5–9). Data represent the mean ± SEM of the indicated number of coverslips. Comparisons in A andC are with the appropriate 300 mOsm or 300/mcontrols. *p < 0.05; **p < 0.01; ***p < 0.001 (NS, not significant; unpaired t test).
- Fig. 6.
Immunohistological detection of TRPV4 in the anterior hypothalamus and skin keratinocytes. A–D, TRPV4-specific diaminobenzidine immunostaining of the MPA (A, B) and MnPO (C, D) regions of rat hypothalamus. In B and D, anti-TRPV4 was predepleted with antigenic TRPV4 peptide. AC, Anterior commissure;3V, third ventricle. E, F, TRPV4 immunofluorescence in rat plantar skin keratinocytes (arrow) and sweat glands (arrowhead). InF, anti-TRPV4 was predepleted with the antigenic TRPV4 peptide.
