Mammalian TRPV4 (VR-OAC) directs behavioral responses to osmotic and mechanical stimuli in Caenorhabditis elegans

Abstract

All animals detect osmotic and mechanical stimuli, but the molecular basis for these responses is incompletely understood. The vertebrate transient receptor potential channel vanilloid subfamily 4 (TRPV4) (VR-OAC) cation channel has been suggested to be an osmo/mechanosensory channel. To assess its function in vivo, we expressed TRPV4 in Caenorhabditis elegans sensory neurons and examined its ability to generate behavioral responses to sensory stimuli. C. elegans ASH neurons function as polymodal sensory neurons that generate a characteristic escape behavior in response to mechanical, osmotic, or olfactory stimuli. These behaviors require the TRPV channel OSM-9 because osm-9 mutants do not avoid nose touch, high osmolarity, or noxious odors. Expression of mammalian TRPV4 in ASH neurons of osm-9 worms restored avoidance responses to hypertonicity and nose touch, but not the response to odorant repellents. Mutations known to reduce TRPV4 channel activity also reduced its ability to direct nematode avoidance behavior. TRPV4 function in ASH required the endogenous C. elegans osmotic and nose touch avoidance genes ocr-2, odr-3, osm-10, and glr-1, indicating that TRPV4 is integrated into the normal ASH sensory apparatus. The osmotic and mechanical avoidance responses of TRPV4-expressing animals were different in their sensitivity and temperature dependence from the responses of wild-type animals, suggesting that the TRPV4 channel confers its characteristic properties on the transgenic animals' behavior. These results provide evidence that TRPV4 can function as a component of an osmotic/mechanical sensor in vivo.

Fig. 1.

TRPV4 expression directs osmotic and nose touch avoidance in osm-9 mutants. (A) Osmotic avoidance of 1 M fructose or glycerol. (B) Nose touch avoidance. (C) Avoidance of the odorant 2-octanone. Wild-type (w.t.) and osm-9(ky10) animals with or without an ASH::trpv4 transgene, an ASH::trpv4::gfp transgene, or an osm-9::gfp5 transgene were tested. “ASH kill” denotes bilateral laser ablation of the ASH neuron. In all panels, asterisks denote significant differences between the indicated group and the osm-9 group (P < 0.01, one-way ANOVA with Dunnett's posttest analysis). Error bars denote SEM. n = number of animals tested, 10 trials each. (D) TRPV4::GFP expression in the nociceptive ASH neurons. (Left) Lateral view. (Center) Dorsal view. TRPV4::GFP in ASH appears green, and an ODR-1::dsRED fusion protein expressed in the dendrite and weakly in the cilium of the adjacent AWC sensory neuron appears red. Yellow arrow, ASH cilia; blue arrow, base of dendrite. (Scale bar = 5 μm.) (Right) Schematic diagram of C. elegans ASH sensory neuron (red), 1 of 12 amphid sensory neurons (blue) that extend dendrites to the nose, where they terminate in sensory cilia. Two amphids each contain an ASH polymodal nociceptive neuron; only the left amphid is shown. The area depicted in the fluorescent micrographs is highlighted.

Fig. 2.

TRPV4 functions with endogenous C. elegans osmo- and mechanosensory genes. (A) Osmotic avoidance of 1 M fructose or glycerol by single mutants (Left) or double mutants with osm-9 (Right), with (gray) or without (black) the ASH::trpv4 transgene. The first three bars represent the same results depicted in Fig. 1 A. (B) Nose touch avoidance. Strains as are in A. The first three bars represent the same results depicted in Fig. 1B. In both panels, asterisks denote statistically significant differences between the trpv4 transgenic group and the parallel nontransgenic group (P < 0.01, one-way ANOVA with Dunnett's posttest analysis). n = number of animals tested, 10 trials each; w.t., wild type.

Fig. 3.

TRPV4 mutations affect C. elegans behavior and channel function. (A) Schematic drawing of TRPV4 including sites of mutations. ARD, ankyrin-repeat domain; PL, pore-loop domain; ΔN, extent of deleted N-terminal domain; ΔC, extent of deleted C-terminal domain. N and C termini are intracellular. (B) Osmotic avoidance of 1 M fructose or glycerol. Bar graphs depict pooled data of three independent transgenic lines for each mutant. The first three bars represent the same results depicted in Fig. 1 A. (C) Nose touch avoidance. Bar graphs depict the combined results for three independent transgenic lines of each mutant. The first three bars represent the same results depicted in Fig. 1B. For B and C, asterisk denotes significant differences between the indicated group and the osm-9 group (one-way ANOVA with Dunnett's posttest analysis; for B: P < 0.01 except ΔN group and ΔC group, here P < 0.05; for C: P < 0.01 except ΔN group). n = number of animals tested, 10 trials each; w.t., wild type.

Fig. 4.

TRPV4 avoidance behaviors are modulated by stimulus strength and temperature. (A) Nose touch avoidance at different temperatures. (B) Osmotic avoidance of 0.5 M glycerol at different temperatures. (C) Osmotic avoidance of different concentrations of glycerol. The osmotic strength of M13 buffer is 295 mOsmol/liter; i.e., a 0.05 M osmotic stimulus yields a final concentration of 345 mOsmol/liter. Asterisks denote significant differences between osm-9 ASH::trpv4 and wild-type (w.t.) groups (P < 0.05, t test for the 0.08 M group in C, P < 0.01 for all other groups in A–C). n = number of animals tested, 10 trials each.