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Colloquium Mammalian TRPV4 (VR-OAC) directs behavioral responses to osmotic and mechanical stimuli in Caenorhabditis eiegans Wolfgang Liedtke*t, David M. Tobint$, Cornelia 1. Bargmannt~, and Jeffrey M. Friedman*ll *Laboratory of Molecular Genetics and IIHoward Hughes Medical Institute, The Rockefeller University, New York, NY 10021; and "Departments of Anatomy and of Biochemistry and Biophysics, Programs in Developmental Biology, Genetics, and Neuroscience, and Howard Hughes Medical Institute, University of California, San Francisco, CA 94143 All animals detect osmotic and mechanical stimuli, but the molec- ular 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 viva, we expressed TRPV4 in Casnorhabditis elegant sensory neurons and examined its ability to generate behavioral responses to sensory stimuli. C elegant ASH neurons function as polymodal sensory neurons that generate a characteristic escape behavior in response to mechanical, osmotic, or olfactory stimuli. These behaviors re- quire 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 avoid- ance behavior. TRPV4 function in ASH required the endogenous C elegant osmotic and nose touch avoidance genes ocr-2, oar-3, osm-10, and gir-1, indicating that TRPV4 is integrated into the normal ASH sensory apparatus. The osmotic and mechanical avoid- ance 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 viva. A nimals perceive and avoid danger through sensory nocicep- ~tion, the detection of noxious stimuli in the external envi- ronment or noxious conditions in internal tissues. Noxious stimuli are polymodal: a stimulus can be recognized as noxious based on its specific chemical composition, as in inflammatory cytokines, based on its physicochemical properties such as pH and osmolarity, based on purely physical properties like pressure or heat, or based on a combination of properties. Some types of nociceptive neurons are intrinsically polymodal, detecting many noxious stimuli, whereas others are specialized to detect partic- ular physical or chemical stimuli (1, 2~. In mammals, dorsal root ganglion neurons sense noxious chemical, thermal, osmotic, acidic, or physical cues, or a combination of such cues (3~. The more specific inner ear hair cells sense sound and acceleration (4), and neurons in circumventricular organs of the brain sense systemic osmotic pressure (5, 6~. Although sensory transduction mechanisms for chemosensation and thermosensation have been extensively characterized, the molecular mechanisms for sensing mechanical or physicochemical stimuli are less well understood. Nociception has been genetically characterized in the inver- tebrates Drosophila melanogaster and Caenorhabditis elegans. In these organisms, mutants with deficits in the response to noxious mechanical, osmotic, and chemical stimuli have been character- i/doi/ 10.1 073/pnas.2235619 100 ized (1, 7). In C. elegans, genetic studies have identified two distinct mechanosensory nociceptive pathways (8, 9). In one mechanosensory pathway, a mechanical stimulus applied to the animal's body elicits a withdrawal response. Members of the mechanically activated channel, degenerin channel, epithelial sodium channel (MEC/DEG/EnaC) family are required for body touch mechanosensation, and are likely to form the mech- anosensory channels in these neurons (8, 10, 11~. In another mechanosensory pathway, an avoidance response to nose touch is mediated by the ciliated ASH sensory neurons in the head (9, 12~. ASH is a polymodal nociceptor: stimulation of the ASH cilia by either light touch, a hyperosmolar solution, or repulsive odorants leads the animal to reverse its direction of movement (13~. Animals with mutations in osm-9 or ocr-2 do not avoid any of these stimuli. The osm-9 and ocr-2 genes encode putative ion channels belonging to the transient receptor potential (TRP) channel superfamily, vanilloid subfamily (TRPV) (14-16) and are proposed to encode the sensory transduction channel in ASH (17~. The extent to which OSM-9 responds directly to sensory stimuli or indirectly to signal transduction pathways is unknown. Interestingly, the Drosophila ortholog of the ocr genes is required for fly hearing, a mechanosensory process (18~. Another TRP family channel, the Drosophila nompC channel, is also thought to encode a mechanosensory channel, and a zebrafish nompC channel is implicated in mechanosensory hair cell function (19, 20~. TRPV4, also known as vanilloid receptor related osmotically activated channel (VR-OAC), OTRPC4, TRP12, or VRL-2, is a vertebrate nonspecific cation channel that is gated by osmotic stimuli in heterologous expression systems (15, 21-25~. TRPV4 belongs to the TRPV subfamily of the TRP ion channel super- family (15, 16, 25, 26) and shows similanty to the C. elegans channels OSM-9 (26% amino acid identity, 44% identity or conservative change) and OCR-2 (24~o identity, 38% identity or conservative change). TRPV4 is also related to the vertebrate vanilloid receptor 1 (VR1; TRPV1), the vanilloid receptor-like channel (VRL-1; TRPV2), and the recently reported TRPV3 (27-32~. TRPV1-3 are cation charlnels that are gated by warm and hot thermal stimuli. This paper results from the Afthur M. Sackler Colloquium of the National Academy of Sciences, "Chemical Communication in a Post-Genomic World," held January 17-19, 2003, at the Arnold and Mabel Beckman Center of the National Academies of Science and Engineering in Irvine, CA. Abbreviations: TRPV, transient receptor potential channel, vanilloid subfamily; VRL-1, vanilloid receptor-like receptor 1; VR1, vanilloid receptor 1. tW.L. and D.M.T. contributed equally to this work. To whom correspondence should be addressed. E-mail: 2003 by The National Academy of Sciences of the USA PNAS 1 November25, 2003 1 vol. 100 1 suppl. 2 1 14531-14536

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The expression of TRPV4 in the circumventricular organs of the vertebrate CNS suggests that this channel could sense systemic osmotic pressure. In transfected cells, TRPV4 is gated by hypotonicity within the physiological range (22, 23), and hypotonicity-gated TRPV4 function is suggested as one mech- anism of pain sensation (33~. However, in the circumventricular organ, hypertonicity is thought to be the physiological stimulus. This difference could result from accessory molecules in osmo- sensitive cells that modify TRPV4 function. TRPV4 is also expressed in vertebrate mechanosensory cells (1~. Most models of mechanosensation propose the existence of multiple acces- sory proteins to mechanosensory channels (6~. A full under- standing of TRPV4 function might best be accomplished in cells with intrinsic mechanosensory or osmosensory function, rather than entirely heterologous cells. To better understand the function of TRPV4 in vivo, we have expressed rat TRPV4 in the ASH neurons of C. elegans. Our results indicate that the rat channel can direct osmotic and mechanosensory behaviors when integrated into the normal ASH sensory transduction apparatus. Methods C elegans Strains and Transgenic Animals. C. elegans was grown at 20C and maintained by using standard methods (34, 35~. Wild-type animals were C. elegans variety Bristol, strain N2. Mutations used in this work were osm-10(n1602> III, glr-1 (k;y1 76J III, osm-9(k~y10) IV, ocr-2(ak47) IV, and odr-3(nl605) ~ All are strong loss-of-function mutations that are believed to represent null alleles. Transgenic arrays used were kyExS75(sra-6::trpv4 elf-2: :GFPJ, kyExS94(sra-6: :trpv4: :GFP oar-1: :dsREDJ, kyEx609 (sra - 6:: trpv4AN elt-2:: GFP), kyEx596 (sra 6: :trpv4 1` C elt-2::GFPJ, kyEx608(sra6::trpv4AN/\C elt-2::GFP', kyEx612(sra-6::trpv4 D672K elt-2::GFPJ, kyExS97 and kyEx605(sra-6::trpv4 M680K elt-2::GFP), kyExS98 and hyEx611 (sra-6::trpv4 D682K elt-2::GFPJ. The coding region of the rat TRPV4 cDNA was expressed under the sra-6 promoter in the C. elegans expression vector pPD49.26 (174. This promoter directs strong expression in the ASH and PVQ neurons, and weak expression in ASI neurons (35~. Germ-line transformation was carried out by injecting DNA at 50 ng/,ul (35) together with the intestinal elt-2::GFP marker at 10 ng/,ul (17~. Transgenic lines were maintained in an osm-9(k;y10) genetic background; kyl0 is a null allele of osm-9 that results from an early stop codon. Details of molecular biology are available upon request. OSM-9::GFP5 lines were as described (344. Neurons were identified for laser ablation by using differential interference contrast optics and a combination of positional and morphological cues as described (36~. Cell kills of ASH were confirmed by bilateral absence of dye-filling with 1,1'-dioctadecyl- 3,3,3',3'-tetramethylindodicarbocyanine perchlorate (DiD). Imaging Animals expressing sra-6::trpv4::gfp, a C-terminally tagged protein, were analyzed with the Deltavision imaging system, which deconvolutes multiple sections of fluorescent micrographs to gen- erate a projection and a 3D reconstruction (37~. Immunohistochemistry of fixed animals was performed by using an antibody raised against the first 445 aa of TRPV4 (a generous gift from Stefan Heller, Harvard University, Boston) and secondary goat anti-rabbit FITC antisera by using described methods (38~. animal had left the drop area was scored as a response (174. For nose touch response, the tip of the nose of a forward-moving animal was touched with a fine hair, and reversal was scored as a response (9, 13~. For chemical avoidance, 2-octanone and 1-octanol were used. A microcapillary containing 5 ,ul of 2-oc- tanone or 1-octanol was placed immediately in front of a freely moving adult animal (17), and reversal within 3 s was scored as a response. For all avoidance assays, 10 trials per animal were recorded and the percentage of positive responses for each animal was compiled per group. Temperature was modulated by using an over-the-counter heat lamp (75-W bulb) at a distance of 10-12 cm from the center of the assay plate under the dissecting microscope. Plate tem- perature was recorded and calibrated with a precision thermom- eter (YSI Temperature, Dayton, OH). For further details, see Supporting Methods, which is published as supporting information on the PNAS web site, Tissue Culture. Cultivation of Chinese hamster ovary (CHO) cells, transfection, and stimulation were carried out as described (22~. For further details, see Supporting Methods. Statistical Analysis. Pairwise comparison was performed by t test, multigroup comparison by ANOVA analysis in combination with Dunnett's posttest analysis. The statistical program PRISM4 for Macintosh was used (GraphPad, San Diego). Results and Diseussion To establish the properties of TRPV4 in a sensory system in vivo, we tested its ability to function within the ciliated ASH sensory neurons of C. elegans in the presence of an osm-9 mutation that eliminates all endogenous ASH functions. osm-9 animals are almost completely defective in their response to noxious hyper- osmotic stimuli, nose touch and several aversive olfactory stim- uli. By contrast, transgenic animals expressing TRPV4 or TRPV4::GFP in ASH neurons avoided both osmotic stimuli (P < 0.01) and nose touch (P ~ 0.01) (Fig. 1A end B and Movies 1-3, which are published as supporting information on the PNAS web site). The osmotic avoidance response of osm-9ASH::trpv4 animals was slightly delayed relative to the endogenous C. elegans behavior; this result is consistent with the relatively slow activation of vertebrate osmosensory channels. To confirm that osmotic avoidance ar~d nose touch avoidance were generated by TRPV4 expression in ASH, the ASH neurons were ablated in osm-9 ASH::trpv4 animals by using a laser microbeam. Ablation of the ASH neurons eliminated osmotic avoidance and nose touch avoidance in the transgenic strain (Fig. 1A end B). The biologically active TRPV4::GFP protein was localized to the cilia of ASH, where mechanical and osmotic stimuli are sensed (Fig. 1D). It was also present in ASH cell bodies, but not in axons or dendrites. Both OSM-9 and OCR-2 are similarly localized to sensory cilia (17~. TRPV4 failed to restore ASH-mediated odorant avoidance of the volatile repellents 2-octanone and 1-octanol to osm-9 mu- tants (Fig. 1C and data not shown). Thus, the TRPV4 channel appears to confer osmosensitivity and mechanosensitivity, but not chemosensitivity, to the ASH neuron in osm-9 mutants. This property is consistent with the expected functions of TRPV4, and different from the normal functions of OSM-9. As a control for this experiment, we tested an osm-9 strain rescued with osm-9::gfp in parallel to the osm-9 ASH::trpv4 and osm-9 ASH::trpv4::~p strains. osm-9 {osm-9::~p]+ animals were res- cued for osmosensation, mechanosensation, and odor sensation, unlike osm-9 ASH::trpv4 animals. The difference between OSM-9 and TRPV4 in odorant response appears to be qualita- tive rather than auantitative. The osm-9 {osm-9::~p)+ strain exn~teo re~at~ve~y weax rescue of nose touch compared with the Behavioral Assays. All behavioral assays were performed by investigators blinded to the genotype of the animals. Osmotic avoidance assays were performed by exposing individual animals to a drop of 1 M fructose or glycerol (39~. A graded series of osmotic stimuli was obtained through serial dilution of 1 M , glycerol. A reversal of more than half a body length before the ~ ~~ ~ ~ - ~ ~ 14532 1 10.1 073/pnas.22356191 00 Liedtke et al.

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A B 100 ~ 0100 ME 848fll~ ~ ~ I ~ 8fl| 0 20 TO 20 L _ n= 35 46 38 7 10 16 19 21 2 5 i,,: ~ ~ ~ ~ hi: ~ ~ ~ ~ ~ o? ~ ~ * * do* C 100 c, ~ 80 .= ~54flL~ 21 16 16 5 6 4 6 4 .~ .~ . ..~ ,. .~ o? o? ~; o? i' ~~ * cilia dendrite ASH sensory neuron imps at led axon 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 ,um.) (Right) Schematic diagram of C elegant 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 noc~cept~ve neuron; only the led amphid is shown. The area depicted in the fluorescent micrographs is highlighted. ASH::trpv4 line (Fig. 1B), presumably because of lower expres- sion of the transgene, yet the line still rescued odorant avoidance (Fig. 1C). Thus, the specific rescue of the osmotic and mechan- ical sensing deficits of osm-9 by TRPV4 represents an intrinsic difference between TRPV4 and OSM-9. TRPV4-expressing animals avoided hyperosmotic stimuli, but in heterologous expression systems, TRPV4-expressing cells are activated only in response to hypoosmolar stimuli (22, 234. This difference suggests that the ASH sensory neuron might contain accessory molecules that allow TRPV4 to sense hyperosmotic stimuli. ocr-2, oar-3, and osm-10 genes are required for endog- enous ASH osmosensory behaviors. ocr-2 encodes another TRPV ion channel subunit, oar-3 encodes a Nonprotein (40), and osm-10 encodes a cytoplasmic protein in the ASH neuron (41~. To assess whether these genes were also required for TRPV4 function in ASH neurons, osm-9ASH::trpv4 transgenic animals were crossed to animals carrying ocr-2, oar-3, and osm-10 mutations. TRPV4 was unable to generate osmosensory behav- iors in ocr-2, oar-3, and osm-10 mutants (Fig. 2~. Thus, endog- enous osmosensory molecules in ASH collaborate with TRPV4 to generate an osmotic response. OCR-2 is required for OSM-9 localization to ASH sensory cilia, but is not required for cilia localization of TRPV4 (Fig. 5, which is published as supporting information on the PNAS web site). This result suggests that the requirement for both OCR-2 and OSM-9 or TRPV4 reflects activity of both channel subunits, and not only their mutual localization to cilia. Endogenous ASH mechanosensation requires ocr-2 and oar-3, but not osm-10. ASH mechanosensation also requires the glr-1 ried~ke et a/. AMPA-type glutamate receptor, which is not required for osmosensation (42, 43~. Mechanosensation mediated by ASH::trpv4 required the function of ocr-2, oar-3, and glr-1 genes (Fig. 2B). As in the endogenous response, glr-1 was not required for ASH::trpv4-mediated osmosensation, and osm-10 was not required for ASH::`rpv4-mediated mechanosensation. Expression of TRPV1 (VR1) does not rescue osmosensation or mechanosensation in osm-9 mutants, but does confer a strong avoidance behavior to capsaicin, a TRPV1 ligand (17~. The capsaicin response of osm-9 ASH::trpvl transgenic animals was retained in ocr-2, oar-3, osm-10, and glr-1 mutant backgrounds. Thus, TRPV4, but not TRPV1, exploits all of the characterized endogenous osmosensory and mechanosensory signaling pro- teins in ASH to generate behavior. If TRPV4 acts as a mechanosensory or osmosensory channel, its behavioral functions should correlate with its ability to function as an ion channel. Three point mutations in the predicted pore-loop domain of TRPV4, D672K, M680K, and D682K were made based on published studies of other family members, TRPV1 (VR1) and TRPV5 (EcaC) (Fig. 3A and Fig. 6, which is published as supporting information on the PNAS web site) (44, 45). The replacement of a methionine with lysine at position 680 (M680K) eliminated the ability of TRPV4 to confer nose touch avoidance and osmotic avoidance to the osm-9 mutant (Fig. 3 B and C). TRPV4M6soK lacked channel activity when expressed in mammalian tissue culture cells stimulated with a TRPV4-activating phorbol ester or hypotonic solution (Fig. 7, which is published as supporting information on the PNAS web site). This observation is consistent with previous PNAS 1 November25, 2003 1 vol. loo 1 suppl. 2 1 14533

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A 100- 80- a' Ct to 60 C) ._ U. o ~ 20 ? ~ .~ ~ . . 1~ ~ ,,,,,. ~ :::: ~ ~ ~ ~ 1~ ~ 6-6 7-8 _ + _ + 0~1 n= 35 in: ~ ~ ,`i B * 100 80 3 60 am, 1 20 O.- n= 16 TRPV4 in' * * ~ h ~ ~ ~ ~ ~6 5-6 5-5 5-5 5-6 {~) J. 1~JL ~ ~ 1 ~ 1 + + + + + ,1 07 0~? ~ OSi ;0 (qua ~? ~ I 1 i, 1 1~ ~ ~" 1 9-2 1 5-7 5-6 _ + _ + _ + ~! 0? ~ ~ Fig. 2. TRPV4 functions with endogenous C elegant 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 statistical Iy 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. studies showing that residue M680 is required for TRPAI ion channel function (44-46~. TRPV4-mediated behaviors were greatly diminished in animals expressing TRPV4 mutations at position 682 (D682K) and at position 672 (D672K) (Fig. 3 B and C). Deletion of the TRPV4 N terminus (residues 1-410), C terminus (residues 741-871), or both N- and C-termini reduced but did not eliminate its behavioral function (Fig. 3 B and C). The I`NliC mutant lacking both N and C termini localized to the ASH cilia, as did the M680K mutant channel (Fig. 8, which is published as supporting information on the PNAS web site). TRPV4 osmotic responses are enhanced at physiological temperatures in transfected mammalian cells (22, 47~. We assessed the temperature-dependence of avoidance behaviors of wild-type and osm-9 ASH::trpv4 animals at room temperature A D672K M680K DC82K (23C) and 34-35C, the upper limit of C. elegans viability. Wild- type animals responded significantly more frequently to nose touch at room temperature, whereas osm-9ASH::trpv4 responded more strongly at 35C (Fig. 4A and Fig. 9, which is published as supporting information on the PNAS web site); a smaller effect was observed for osmotic avoidance (Fig. 4B). Although TRPV4-induced behav- iors appeared to reflect the temperature modulation of TRPV4, TR]PV4 did not generate avoidance behavior to thermal stimuli alone (48, 49), whereas TRlPV1, a channel implicated in noxious heat responses, conferred a significant thermal avoidance response to isotonic M13 buffer at 37C (Fig. 10, which is published as supporting information on the PNAS web site). Thus, the trans- duction of osmotic and mechanical stimuli in osm-9 ASH::opv4 animals bears the molecular signature of TRPV4. Somatosensory B ..,100 80- ._ _ 460 o 40 0 20 ~ 0 ~ n= 35 C * 1 ~ * * 1 ~ * * 1 46 38 8 8 18 " 'a . 10 24 16 16 19 21 10 10 13 9 10 8 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; i`N, extent of deleted N-terminal domain; ~C, 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. 1 B. 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 1~1~1 group and TIC group, here P < 0.05; for C: P < 0.01 except /`N group). n - number of animals tested, 10 trials each; w.t., wild type. 14534 1 1 073/pnas.22356191 00 Liedtke et al.

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A B * Inn c, 100 _ ram, 10~ i] n= 10 1515 1015 15 1010 14 10 1014 RT 35C RT 35C o; ; opt 100 80 ce ._ 60 0 40 con o 20 o * * 1 -A _d _] 10 10 12 12 12 12 10 10 10 10 10 10 20 20 29 10 ] M13 bu~er+O.05M ~ ~.~6 0 i. 1* - - 015 0.08M 0.125M 0.25M O.SM 1M oily jo;~! ~ ~ 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 M 13 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-9ASH::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. perception of mechanical stimuli in humans and seals is modulated by temperature (50-52), and TRPV4 may recapitulate this effect at the molecular level. Vertebrate osmosensation in the central nervous system is exquisitely sensitive to small increases in systemic osmolarity. By contrast, C. elegans osmosensation detects relatively large in- creases in external osmolarity that may be associated with salty or brackish water. This difference in physiological function might predict a difference in the threshold of osmosensory channels in C. elegans and vertebrates. The osmotic avoidance behaviors of wild-type, osm-9, and osm-9ASH::trpv4 animals were assessed by using solutions with different osmotic strengths (Fig. 4C). Wild- type C. elegans responded weakly to increased osmolalities of 0.25 M and below, with a maximal behavioral response at a 1 M osmotic stimulus. By contrast, osm-9 ASH::trpv4 animals re- sponded equally well to 0.125 M, 0.25 M, 0.5 M, and 1 M osmotic stimuli. Thus, the osm-9ASH::trpv4 animals exhibited a signif- icantly lower threshold to hyperosmotic stimuli than wild-type animals, as would be consistent with the high sensitivity of vertebrate osmosensation. TRPV4 allows ASH neurons to respond to hypertonicity, but it responds to hypotonicity when expressed in transfected cells. One possible explanation for this difference is suggested by the properties of the mechanosensitive ion channel gramicidin A, which behaves either as a stretch-inactivated or as a stretch- activated channel depending on the lipid composition of the surrounding lipid bilayer (53~. An alternate possibility is that TRPV4 forms heteromultimeric complexes with C. elegans pro- teins. TRP ion channels can form heteromeric complexes with related family members (17, 25, 54, 55~; notably, the two TRPV channels OCR-2 and OSM-9 may associate in ASH (17~. Expression of rat TRPV4 in ASH rescued the transduction of osmotic and mechanical stimuli in osm-9 but not ocr-2 mutants, whereas rat TRPV1 did not rescue either mutant. These results suggest that TRPV4 and OSM-9 have orthologous functions, 1. Goodman, M. B. & Schwarz, E. M. (2003) Annul Rev. Physiol. 65, 429-452. 2. Gillespie, P. G. & Walker, R. G. (2001) Nature 413, 194-202. 3. Gardner, E. P., Martin, J. H. & Jessell, T. M. (2000) in Principles of Neural Science, ed. Kandel E. R., Schwartz, J. H. & Jessell, T. M. (McGraw-Hill, New York), pp. 430-450. 4. Hudspeth, A. J. (1989) Nature 341, 397-404. 5. Bourque, C. W. & Oliet, S. H. (1997) Annul Rev. Physiol. 59, 601-619. 6. Denton, D. A., McKinley, M. J. & Weisinger, R. S. (1996) Proc. Natl. Acad. Sci. USA 93, 7397-7404. Liedtke et a/. indicating a phylogenetic conservation of function. It is inter- esting that TRPV4 could not rescue the odorant avoidance deficit of osm-9 mutants for the two odors tested. C. elegans senses odorants by using G protein-coupled receptors; it is possible that TRPV4 does not interact with an essential com- ponent of the G protein-coupled receptor signaling pathway, although it does interact with the components required for sensing physical stimuli. Among several models consistent with these results (Fig. 11, which is published as supporting informa- tion on the PNAS web site), the conserved role of TRPV ion channels in osmosensory pathways suggests a central, perhaps direct, role for these channels in osmosensation and mech- anosensation. However, the essential role of the G protein ~ subunit ODR-3 in mechanosensation and chemosensation leaves open the possible involvement of G protein-coupled receptors as mediators or regulators of these sensory signals. Our results provide evidence that mammalian TRPV4 can function as a central component of the sensor for osmotic and mechanical stimuli in ASH sensory neurons in C. elegans. Expression of TRPV4 directs a behavioral response to osmotic and mechanical stimuli in vivo, and specific properties of the behavior are conferred by the mammalian channel. This con- clusion is consistent with findings in trpv4 null mice (56-58), and supports the hypothesis that TRPV4 functions as part of a mammalian osmotic and mechanical sensor. The Rockefeller University Bio-Imaging Resource Center (Alison North, director), provided assistance with imaging, and Jim Hudspeth, Shai Shaham (both from The Rockefeller University), Justin Blau (New York University, New York), Stefan Heller (Harvard University, Bos- ton) and Sebastian Martinek (Pennie Partners, New York) provided valuable suggestions and support. W.L., C.I.B., and J.M.F. were sup- ported by the National Institutes of Health. 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