Mutant TRPV4-mediated toxicity is linked to increased constitutive function in axonal neuropathies

Abstract

Mutations in TRPV4 have been linked to three distinct axonal neuropathies. However, the pathogenic mechanism underlying these disorders remains unclear. Both gain and loss of calcium channel activity of the mutant TRPV4 have been suggested. Here, we show that the three previously reported TRPV4 mutant channels have a physiological localization and display an increased calcium channel activity, leading to increased cytotoxicity in three different cell types. Patch clamp experiments showed that cells expressing mutant TRPV4 have much larger whole-cell currents than those expressing the wild-type TRPV4 channel. Single channel recordings showed that the mutant channels have higher open probability, due to a modification of gating, and no change in single-channel conductance. These data support the hypothesis that a "gain of function" mechanism, possibly leading to increased intracellular calcium influx, underlies the pathogenesis of the TRPV4-linked axonal neuropathies, and may have immediate implications for designing rational therapies.

FIGURE 1.

Physiological localization of wild-type and mutant TRPV4 on the plasma membrane. Confocal microscopy was performed using HeLa cells transfected with plasmids pIRES2-ZsGreen1 containing wtTRPV4 (a–d), TRPV4^R269H (e–h),TRPV4^R315W (i–l), and TRPV4^R316C (m–p). Cells expressing exogenous TRPV4 were labeled by ZsGreen1GFP (c, g, k, and o). TRPV4 is shown by blue (a, e, i, and m) and cadherin by red (b, f, j, and n). Merged images are shown on the right panels (d, h, l, and p). Arrows indicate TRPV4 signals on the plasma membrane. Representative images are provided. For each condition, at least 50 cells were analyzed in more than two independent experiments.

FIGURE 2.

Physiological localization of wild-type and mutant TRPV4 on the plasma membrane. Confocal microscopy was performed using HEK293 cells transfected with plasmids pIRES2-ZsGreen1 containing wtTRPV4 (a–d), TRPV4^R269H (e–h), TRPV4^R315W (i–l), and TRPV4^R316C (m–p). Cells expressing exogenous TRPV4 were labeled by ZsGreen1 GFP (c, g, k, and o). TRPV4 is shown by blue (a, e, i, and m) and cadherin by red (b, f, j, and n). Merged images are shown on the right panels (d, h, l, and p). Arrows indicate TRPV4 signals on the plasma membrane. Representative images are provided. For each condition, at least 50 cells were analyzed in more than two independent experiments.

FIGURE 3.

Physiological localization of wild-type and mutant TRPV4 on the plasma membrane. Confocal microscopy was performed using Neuro2a cells transfected with plasmids pIRES2-ZsGreen1 containing wtTRPV4 (a–d), TRPV4^R269H (e–h), TRPV4^R315W (i–l), and TRPV4^R316C (m–p). Cells expressing exogenous TRPV4 were labeled by ZsGreen1 GFP (c, g, k, and o). TRPV4 is shown by blue (a, e, i, and m) and cadherin by red (b, f, j, and n). Merged images are shown on the right panels (d, h, l, and p). Arrows indicate TRPV4 signals on the plasma membrane. Representative images are provided. For each condition, at least 50 cells were analyzed in more than two independent experiments.

FIGURE 4.

Plasma membrane localization and cytoplasmic retention of TRPV4. Confocal microscopy was performed using HeLa cells transfected with plasmids pIRES2-ZsGreen1 containing wtTRPV4 (a–d), TRPV4^R269H (e–h), TRPV4^R315W (i–l), and TRPV4^R316C (m–p). Cells expressing exogenous TRPV4 were labeled by ZsGreen1 GFP (c, g, k, and o). TRPV4 is shown by blue (a, e, i, and m) and cadherin by red (b, f, j, and n). Merged images are shown on the right panels (d, h, l, and p). Arrows indicate TRPV4 signals on the plasma membrane. Arrowheads indicate cytoplasmic retention of wild-type (a) and mutant TRPV4 (e, i, and m). Representative images are provided. For each condition, at least 50 cells were analyzed in more than two independent experiments.

FIGURE 5.

a, effect of stimulation with 2 μm 4αPDD on internal fluorescence ratio in HeLa cells transfected with either WT-, R269H-, R315W-, or R316C-TRPV4 constructs. Average basal and peak values are given. n>2000 cells for each condition. *, p < 0.0001, indicating significant differences when compared with WT-TRPV4 (two-tailed Student's t test). Error bars, means ± S.E. b, application of 4αPDD induced an increase in intracellular calcium ([Ca²⁺]_i). Mean calcium responses before and during 4αPDD application are given. c, quantification of propidium iodide uptake in HeLa cells expressing wild-type and mutant TRPV4 indicates an increase in cytotoxicity in mutant expressing cells at 48 h that is blocked by the TRP channel blocker RR. Data are averaged from at least three independent experiments. #, p < 0.05, indicating significant differences when compared with WT-TRPV4 (two-tailed Student's t test). Error bars, means ± S.E.

FIGURE 6.

a, effect of stimulation with 2 μm 4αPDD on internal fluorescence ratio in HEK293 cells transfected with either WT-, R269H-, R315W-, or R316C-TRPV4 constructs. Average basal and peak values are given. n>5000 cells for each condition. *, p < 0.0001, indicating significant differences when compared with WT-TRPV4 (two-tailed Student's t test). Error bars, means ± S.E. b, application of 4αPDD induced an increase in intracellular calcium ([Ca²⁺]_i). Mean calcium responses before and during 4αPDD application are given. c, quantification of propidium iodide uptake in HEK293 cells expressing wild-type and mutant TRPV4 indicates an increase in cytotoxicity in mutant expressing cells at 48 h that is blocked by the TRP channel blocker RR. Data are averaged from at least three independent experiments. #, p < 0.05, indicating significant differences when compared with WT-TRPV4 (two-tailed Student's t test). Error bars, means ± S.E.

FIGURE 7.

a, effect of stimulation with 2 μm 4αPDD on internal fluorescence ratio in Neuro2a cells transfected with either WT-, R269H-, R315W-, or R316C-TRPV4 constructs. Average basal and peak values are given. n>9000 cells for each condition. *, p < 0.0001, indicating significant differences when compared with WT-TRPV4 (two-tailed Student's t test). Error bars, means ± S.E. b, application of 4αPDD induced an increase in intracellular calcium ([Ca²⁺]_i). Mean calcium responses before and during 4αPDD application are given. c, quantification of propidium iodide uptake in Neuro2a cells expressing wild-type and mutant TRPV4 indicates an increase in cytotoxicity in mutant expressing cells at 48 h that is blocked by the TRP channel blocker RR. Data are averaged from at least three independent experiments. #, p < 0.05, indicating significant differences when compared with WT-TRPV4 (two-tailed Student's t test). Error bars, means ± S.E.

FIGURE 8.

The density of ruthenium red-sensitive current is higher in HeLa cells expressing mutant TRPV4. Patch clamp recordings were performed from HeLa cells. a and b, currents elicited by slow voltage ramps (600 ms, from −100 to +100 mV, upper traces) in control conditions (black traces) and in the presence of ruthenium red (red traces) in (a) wild-type transfected cells and (b) in cells transfected with the TRPV4^R269H mutant channel. c and d, ruthenium red-sensitive currents from the traces in (a and b) obtained by digital subtraction. e, bar chart summarizing the current density (normalized to membrane capacitance) in cells transfected with either the wild-type or the mutant TRPV4 channel. No differences were detected in other properties of the ruthenium red-sensitive current such as the reversal potential (f).

FIGURE 9.

Cell-attached recordings of wild-type and mutant TRPV4 channels. a, single-channel recordings form a cell expressing wild-type (black trace) and one expressing mutant (red trace) TRPV4 channels. These recordings were performed at Vpip = −100 mV (100 mV depolarization from resting potential). The amplitude histograms in b show that the mutant channels spent more time in the open state. The peak corresponding to the current in the open state was well represented in the mutant channels histogram, but it was necessary to expand the ordinate (inset) to resolve it in the wild-type histogram. c, no change was detected in single channel conductance. Recordings obtained at pipette potentials ranging from −100 to +80 mV in wild type (black traces) and mutant (red traces) TRPV4-expressing cells. The I/V curves obtained from these two cells are shown in the respective I/V plots.

FIGURE 10.

Outside-out recordings of wild-type and mutant TRPV4 channels. a and b, single channel currents recorded at 50 mV from a patch containing wild type (a) and one containing mutant (b) TRPV4 channels. c and d, amplitude histogram of the traces shown in a and b. The vertical axis is cut at 1500 events to allow resolving the open channel currents. The overlaid dotted lines represent Gaussian functions fitted to the experimental data. The arrowheads show the value of unitary current obtained by the fitting. e, bar chart showing the fraction of outside-out patches that showed TRPV4 activity in cells expressing wild-type and mutant channels. f, bar chart showing the increased open probability of mutant channels. Only patches that showed TRPV4 activity were included in the analysis. g, bar chart of the mean open time in wild-type and mutant channels.