Wild-type and brachyolmia-causing mutant TRPV4 channels respond directly to stretch force

Abstract

Whether animal ion channels functioning as mechanosensors are directly activated by stretch force or indirectly by ligands produced by the stretch is a crucial question. TRPV4, a key molecular model, can be activated by hypotonicity, but the mechanism of activation is unclear. One model has this channel being activated by a downstream product of phospholipase A(2), relegating mechanosensitivity to the enzymes or their regulators. We expressed rat TRPV4 in Xenopus oocytes and repeatedly examined >200 excised patches bathed in a simple buffer. We found that TRPV4 can be activated by tens of mm Hg pipette suctions with open probability rising with suction even in the presence of relevant enzyme inhibitors. Mechanosensitivity of TRPV4 provides the simplest explanation of its various force-related physiological roles, one of which is in the sensing of weight load during bone development. Gain-of-function mutants cause heritable skeletal dysplasias in human. We therefore examined the brachyolmia-causing R616Q gain-of-function channel and found increased whole-cell current densities compared with wild-type channels. Single-channel analysis revealed that R616Q channels maintain mechanosensitivity but have greater constitutive activity and no change in unitary conductance or rectification.

FIGURE 1.

Macroscopic currents of rat TRPV4 expressed in oocytes. a, currents from oocytes expressing wild-type TRPV4 3 days after injection of 4 ng of cDNA (upper) and 40 ng of non-conducting M680K TRPV4 (lower) upon voltage steps (from −60 mV hold to −100 to +60 mV test in 20-mV increments) before (left) and 20 min after (right) exposure to 3 μm 4α-PDD. b, peak currents from an oocyte expressing very high levels of wild-type TRPV4 (5 days after injection of 40 ng) upon 100-ms voltage steps (from −20 to +20 mV every 10 s) in response to removal of 100 mm sorbitol from the 250 mosm bath solution (open bars) and the addition of 3 μm ruthenium red (RuR; filled bar). The inset shows the raw traces from which peak currents were assessed. (Only every fifth trace from time a to a′ is displayed for clarity.) hypo, hypotonic; iso, isotonic. c, typical currents from oocytes injected with 4 ng of R616Q cRNA (upper) or 4 or 40 ng of wild-type cRNA (lower). Injections of R616Q RNA above 4 ng killed the oocytes. Tests were performed 3 days after injection with the voltage steps as in a but with shorter durations. d, expressed currents (assessed at +60 mV) of 15 wild type-expressing oocytes (●) and 15 R616Q-expressing oocytes (○) injected with different amounts of cRNA examined 66–72 h after injection.

FIGURE 2.

Spontaneous microscopic TRPV4 currents. a, spontaneous single-channel activities from a patch excised from an oocyte highly expressing the wild type (left) and a typical one expressing R616Q (right) examined at +100 mV (upper) or −100 mV (lower). Segments of the recordings are displayed at a faster time base showing discrete opening and closing events. C marks the closed levels. b, I-V plots showing the unitary conductances of the wild type (●) and R616Q (○). c, nP_o-V plot from a typical wild-type TRPV4-expressing patch showing activation by positive voltages.

FIGURE 3.

Direct activation of wild-type TRPV4 by membrane stretch. a, sample of raw traces of average quality from three patches excised from three different oocytes, showing activation by 60-mm Hg suctions applied to excised patches held at +50 mV under a patch clamp. The upper traces are displayed at a faster time base to show unitary transitions between closed (C) and open (O1 and O2) levels. b, sample traces from a high-quality wild-type patch subjected to a range of pipette suctions recorded with a pipette solution of 98 mm KCl, 1 mm MgCl₂, and 20 mm Na⁺ citrate, pH 4.5. c, plot of nP_o versus suction of the wild type (filled symbols) and R616Q (open symbols). nP_o values from three patches each are normalized to that at 60 mm Hg. Different symbols are different typical patches chosen to show the trend as well as variability; curves are segments of symmetric sigmoidal fits overlayed by eye.

FIGURE 4.

BPB inhibits both the hypotonic and 4α-PDD responses in intact oocytes. Unexposed oocytes (a and b) or those incubated for 5–9 h in 100 μm BPB (c and d) were subjected to 1-s voltage steps of between −100 and 60 mV either before (circles) or after (squares) exposure to hypotonicity (hypo; as described in the legend to Fig. 1) for 10 min (a and c) or to 3 μm 4α-PDD for 20 min (b and d). BPB was present during experimentation as well in c and d. Plotted are peak currents versus test potentials. e shows the relative increase in the peak response at 60 mV in the absence (white bars) or presence (as in c and d; gray bars) of BPB to hypotonicity (left) or 4α-PDD (right) (mean ± S.E., n = 4). Note that the apparent smaller response to hypotonicity in e compared with that shown in Fig. 1b reflects that it was assessed at 60 mV here as opposed to 20 mV in Fig. 1b, and hypotonicity appears to cause a slight leftward shift in the G-V relationship, as can be seen in a.

FIGURE 5.

Enzyme inhibitors do not alter TRPV4 mechanosensitivity in excised patches. Five patches were excised from each of two R616Q-expressing oocytes held at +50 mV before and another 5 × 2 patches excised after 30 min of incubation in the presence of 200 μm BPB (upper panels). a and b show sample traces of the response to suctions. c shows nP_o increases by 60-mm Hg suctions in five different patches from one oocyte (black lines) and five from a second oocyte (gray lines) before the BPB treatment. d shows nP_o increases by the same suctions of five new patches from the first oocyte (black lines) and five others from the second oocyte (gray lines) sampled after the BPB treatment. e summarizes the -fold increase in nP_o from 0- to 60-mm Hg suction. (Those with nP_o ∼ 0 without suction are excluded in the calculation.) f–j show parallel experimental results with 100 μm 17-octadecynoic acid (17-ODYA).