The TRPV3 mutation associated with the hairless phenotype in rodents is constitutively active

Abstract

TRPV3 is a non-selective cation channel activated by warm to hot temperatures. In rodents, TRPV3 is highly expressed in basal keratinocytes of skin and oral/nasal epithelia. TRPV3 knockout mice showed impaired responses to innocuous and noxious heat but otherwise normal appearance and reactions to many sensory modalities. However, point mutations of TRPV3 at Gly573 to Ser and Cys have recently been linked to autosomal dominant hairless phenotypes and spontaneous dermatitis in mice and rats, implicating an important role for TRPV3 in alopecia and skin diseases. Exactly, how the mutations affect TRPV3 function was unexplained. Here, we show that both G573S and G573C mutations of murine TRPV3 are constitutively active in heterologous systems. In HEK 293 cells, expression of the TRPV3 mutants causes cell death. In Xenopus oocytes, the constitutively active mutant channel is irresponsive to thermal and chemical stimuli but it reduces the temperature threshold and enhances the responses to heat and TRPV3 agonists of the wild type channel when they are co-expressed. We conclude that the G573S and G573C substitutions render the TRPV3 channel spontaneously active under normal physiological conditions, which in turn alters ion homeostasis and membrane potentials of skin keratinocytes, leading to hair loss and dermatitis-like skin diseases.

Figure 1. Expression of G573C and G573S mutant of TRPV3 in mammalian cells causes cell death

T-Rex293 cells transfected with GFP-TRPV3 wild type (V3WT) and the mutants (V3G573C and V3G573S) in pcDNA4/TO vector were treated or not with 20 ng/ml doxycycline. Bright field (middle) and fluorescence (sides) images were taken at 26 hrs. Fluorescence images were taken using the same exposure time, gain and other camera settings. Substantial cell death (round-up cells) is seen in both the G573C and G573S expressing cells treated with doxycycline. Only few dead cells are present in untreated cells and cells expressing V3WT.

Figure 2. G573S expression and cell survival are partially rescued by ruthenium red but not by lowering extracellular Ca²⁺

T-Rex293 cells were transfected with GFP-V3WT or GFP-V3G573S as in figure 1 and fusion protein expression was induced by 20 ng/ml doxycycline for 7 hrs. A, phase contrast (middle) and fluorescence (sides) images of cells untreated or treated with 10 μM ruthenium red (RR) for the duration of doxcycline induction. Note, the larger number of normal looking cells and more prominent fluorescence signal in the RR-treated than untreated group. B, images of cells with GFP-V3WT (left) and GFP-V3G573S (right) expression induced in a low Ca²⁺ medium for 8 hrs. Severe cell death and low fusion protein expression are still obvious with the mutant. For both conditions shown in A and B, GFP-V3G573C had a similar effect as GFP-V3G573S (not shown). Fluorescence images were taken using the same exposure time, gain and other camera settings.

Figure 3. Constitutive activity of TRPV3 G573 mutants expressed in Xenopus oocytes

Wild type TRPV3 (WT) and the G573C (G/C) or G573S (G/S) mutants were expressed in oocytes by cRNA injection. Currents were recorded by two-electrode voltage clamp. A, time course of current changes at -100 mV for WT in response to the application of 2APB at the room temperature (22 °C) and to temperature change from 22 °C to 40 °C as indicated. The 2APB-evoked response was blocked by ruthenium red (RR, right panel). Insets show I-V curves obtained by voltage ramps under conditions as indicated. B, similar to A but the oocyte expressed G573C. The basal current was partially blocked by RR (10 μM, right panel). Note the similar response to heat in the presence of RR. The current represents unblocked mutant channel activity as well as leak. The increase is due to normal thermodynamic effect as explained in Fig. 5. C, the response of uninjected oocyte to 2APB and heating. D, summary of basal current at -100 mV for all cells. Data points for individual cells are plotted. Mean values are indicated by the thick bars. Controls (Cntl) are uninjected oocytes.

Figure 4. Ligand-invoked response in oocytes that expressed TRPV3 and the G573 mutants

A, representative current traces at -100 mV for oocytes injected with cRNA for wild type TRPV3 (WT), G573S (G/S), WT plus G/S and uninjected controls (Cntl). Camphor (10 mM) was applied as indicated by the gray bars. Insets show I-V curves at basal (dotted gray lines) and at 20 s after camphor application (solid black lines). B, means ± SEM of currents at -100 mV evoked by 10 mM camphor (open bars) and 300 μM 2APB (gray bars), * p < 0.05 different from control, † p < 0.05 different from WT+G/S.

Figure 5. Heat-evoked activity in oocytes that expressed TRPV3 and the G573S mutant

A, representative current traces at -100 mV for uninjected control (Cntl) and oocytes injected with cRNA for wild type TRPV3 (WT), WT plus G573S (G/S), and G/S alone in response to 300 μM 2APB (open bar), a temperature drop to 10°C, as well as a temperature ramp from 10°C to 40°C as indicated. Note a different scale is used for G/S alone than for other traces. B, I-V relationships for oocytes shown in A at 22°C, 22°C with 2APB, 10°C, and 40°C. C, current vs temperature plots for individual cells grouped by the injected cRNA. Note the large cell-to-cell variation within each group and the different scales used for currents for different groups. D, the same data in C were normalized to current at 40 °C. E, log current vs temperature plot for a representative oocyte that coexpressed WT and G/S. The intersection of the two linear fit lines gives the temperature threshold. Q₁₀ values are determined by the antilog of 10*slope (10¹⁰*^sploe). F, summary of response/basal (means ± SEM) for temperature rise from 22 to 40°C. * p < 0.05 different from control, † p < 0.05 different from WT+G/S.

Figure 6. Partial rescue of G573 mutant expression in mammalian cells by the wild type protein

T-Rex293 cells were cotransfected with GFP-V3WT or GFP-V3G573S together with either the wild type TRPV3 in pcDNA3 or the empty pcDNA3 vector (as a control). Expression of GFP-fusion proteins was induced by 20 ng/ml doxyclycline 16 hrs post transfection. Phase contrast (middle) and fluorescence (sides) images of cells were taken at 8 hrs after induction. Fluorescence images were taken using the same exposure time, gain and other camera settings. Note the more pronounced fluorescence signal with the coexpression of wild type TRPV3 with the G573S mutant even though there are still a large number of round-up cells. Similar results were obtained for GFP-V3G573C (not shown).