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OCR for page 19
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-
www.pnas.org/cg 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: cori~itsa.ucsf.edu.
2003 by The National Academy of Sciences of the USA
PNAS 1 November25, 2003 1 vol. 100 1 suppl. 2 1 14531-14536
OCR for page 20
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
20°C 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, www.pnas.org.
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 www.pnas.org/cgi/doi/ 10.1 073/pnas.22356191 00
Liedtke et al.
OCR for page 21
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
OCR for page 22
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
(23°C) and 34-35°C, 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 35°C (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 37°C (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 www.pnas.org/cgi/doi/10. 1 073/pnas.22356191 00
Liedtke et al.
OCR for page 23
A B
* Inn
c, 100 _
ram, 10~ i]
n= 10 1515 1015 15 1010 14 10 1014
RT 35°C RT 35°C
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,
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The Rockefeller University Bio-Imaging Resource Center (Alison
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Representative terms from entire chapter:
osmotic avoidance
