Article
A Cold-Sensing Receptor Encoded by a Glutamate
Receptor Gene
Graphical Abstract
Highlights
d C. elegans genetic screen identifies the glutamate receptor
GLR-3 as a cold receptor
d GLR-3 senses cold in a peripheral sensory neuron to trigger
cold-avoidance behavior
d GLR-3 is a metabotropic cold receptor requiring G proteins
to transmit cold signals
d The mouse GLR-3 homolog GluK2 acts in DRG sensory
neurons to mediate cold sensation
Authors
Jianke Gong, Jinzhi Liu,
Elizabeth A. Ronan, ..., Bo Duan,
Jianfeng Liu, X.Z. Shawn Xu
Correspondence
jfliu@mail.hust.edu.cn (J.L.),
shawnxu@umich.edu (X.Z.S.X.)
In Brief
An evolutionarily conserved cold sensor,
which is encoded by a glutamate receptor
gene, transmits cold signals through
G-protein signaling, independent of its
channel function.
Gong et al., 2019, Cell 178, 1375–1386
September 5, 2019 ª 2019 Elsevier Inc.
https://doi.org/10.1016/j.cell.2019.07.034
Article
A Cold-Sensing Receptor Encoded
by a Glutamate Receptor Gene
Jianke Gong,1,2,3,9 Jinzhi Liu,1,2,3,9 Elizabeth A. Ronan,2,3,9 Feiteng He,1,2,3,9 Wei Cai,2,3 Mahar Fatima,4
Wenyuan Zhang,1,2,3 Hankyu Lee,4 Zhaoyu Li,2,3,8 Gun-Ho Kim,7 Kevin P. Pipe,5,6 Bo Duan,4 Jianfeng Liu,1,*
and X.Z. Shawn Xu2,3,10,*
1College of Life Science and Technology, Key Laboratory of Molecular Biophysics of MOE, and International Research Center for Sensory
Biology and Technology of MOST, Huazhong University of Science and Technology, Wuhan, Hubei 430074, China
2Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA
3Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA
4Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA
5Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
6Department of Electrical Engineering and Computer Science, University of Michigan, Ann Arbor, MI 48109, USA
7Department of Mechanical Engineering, Ulsan National Institute of Science and Technology, 50 UNIST-gil, South Korea
8Present address: Queensland Brain Institute, University of Queensland, St. Lucia QLD 4072, Australia
9These authors contributed equally
10Lead Contact
*Correspondence: jfliu@mail.hust.edu.cn (J.L.), shawnxu@umich.edu (X.Z.S.X.)
https://doi.org/10.1016/j.cell.2019.07.034
SUMMARY
In search of the molecular identities of cold-sensing
receptors, we carried out an unbiased genetic screen
for cold-sensing mutants in C. elegans and isolated a
mutant allele of glr-3 gene that encodes a kainatetype
glutamate receptor. While glutamate receptors
are best known to transmit chemical synaptic signals
in the CNS, we show that GLR-3 senses cold in the
peripheral sensory neuron ASER to trigger coldavoidance
behavior. GLR-3 transmits cold signals
via G protein signaling independently of its glutamate-gated
channel function, suggesting GLR-3
as a metabotropic cold receptor. The vertebrate
GLR-3 homolog GluK2 from zebrafish, mouse, and
human can all function as a cold receptor in heterologous
systems. Mouse DRG sensory neurons express
GluK2, and GluK2 knockdown in these neurons
suppresses their sensitivity to cold but not cool temperatures.
Our study identifies an evolutionarily
conserved cold receptor, revealing that a central
chemical receptor unexpectedly functions as a thermal
receptor in the periphery.
INTRODUCTION
The ability to sense cold is essential for life. Cold temperatures
trigger profound physiological and behavioral responses in
nearly every organism (Bandell et al., 2007; Schepers and Ringkamp,
2010). For example, cold stimuli, particularly noxious
cold, are not only life-threatening, but also cause severe tissue
damage and evoke pain in animals and humans (Foulkes and
Wood, 2007). To survive, organisms have evolved exquisite thermosensory
systems to detect and react to cold temperatures
(Bandell et al., 2007; Schepers and Ringkamp, 2010). Molecular
cold sensors are a central player in cold sensation (Bandell et al.,
2007; Schepers and Ringkamp, 2010). These cold receptors,
which are expressed in cold-sensitive neurons/cells in the periphery,
sense cold temperatures and relay the signals to the
central nervous system (CNS) to trigger nocifensive behaviors
and elicit pain (Schepers and Ringkamp, 2010).
Despite decades of intensive research, little is known about
the molecular mechanisms underlying cold sensation. Thus far,
only one cold receptor, TRPM8, which is a TRP family channel,
has been verified both in vivo and in vitro in mammals. TRPM8
senses cool temperatures with an activation threshold at
$26
C and mediates cool sensation in mice (Bautista et al.,
2007; Dhaka et al., 2007; McKemy et al., 2002; Peier et al.,
2002). Some other mammalian TRP channels (e.g., TRPA1)
have also been suggested as a cold receptor, but their function
in thermosensation appears complex and remains to be defined
(Moparthi et al., 2016; Story et al., 2003; Vandewauw et al., 2018;
Winter et al., 2017). As such, the molecular identities of cold receptors
remain largely elusive. Because animals and humans are
clearly capable of sensing temperatures below 26
C, and
TRPM8 knockout mice show robust responses to noxious cold
(Bautista et al., 2007; Dhaka et al., 2007), unknown cold receptors,
particularly those sensing noxious cold, must exist but
remain to be identified.
Glutamate receptors, such as kainate, AMPA, and NMDA
receptors, are glutamate-gated ion channels that are primarily
expressed in the brain. These chemical-sensing receptors transmit
chemical signals between neurons and mediate the majority
of excitatory chemical synaptic transmission in the CNS (Traynelis
et al., 2010). These receptors also mediate synaptic plasticity
in the CNS, which underlies learning and memory (Traynelis
et al., 2010). Dysfunction of glutamate receptors leads to a
wide variety of CNS disorders, ranging from epilepsy, ischemic
stroke, and neurodegeneration (e.g., Alzheimer’s disease and
Parkinson’s disease), to mental retardation (Bowie, 2008).
Cell 178, 1375–1386, September 5, 2019 ª 2019 Elsevier Inc. 1375
Together, these highlight a fundamental role of glutamate receptors
in the function and organization of the CNS.
Here, we designed an unbiased, activity-based genetic screen
for cold receptors in C. elegans, a model organism widely used
for the study of sensory biology (Bargmann, 2006; Garrity et al.,
2010; Goodman, 2006; Ward et al., 2008). To do so, we employed
a real-time PCR thermocycler, which allowed us to conduct a high
throughput genetic screen for mutants defective in cold sensation
using live animals.We identify GLR-3,a kainate-typeglutamate receptor
homolog, as a cold receptor. GLR-3 senses cold temperatures
in the sensory neuron ASER to trigger cold-avoidance
behavior, indicating that GLR-3 functions in the peripheral nervous
system to mediate cold sensation. Heterologous expression of
GLR-3 in mammalian cell lines confers cold sensitivity, suggesting
that it is sufficient to function as a cold receptor. The activation
threshold of GLR-3 is below 20
C, suggesting that it mainly senses
noxious cold rather than cool temperatures. Surprisingly, GLR-3
functions as a metabotropic cold receptor rather than a typical
temperature-gated ion channel, and its role in cold sensation is independent
of its glutamate receptor function. The GLR-3 homolog
GluK2 from fish, mouse, and human can all function as a cold receptor
in vitro, and mouse GluK2 can functionally substitute
for GLR-3 in vivo. Mouse GluK2 is expressed in dorsal root ganglion
(DRG) sensory neurons, and knockdown of GluK2 in DRG
neurons suppresses the sensitivity of these sensory neurons to
cold but not cool temperatures. Our studies identify an evolutionarily
conserved cold receptor. As glutamate receptors are best
known to transmit chemical signals across synapses in the CNS,
our studies also present a striking case where a central chemical
receptor functions as a thermal receptor in the periphery.
RESULTS
Designing an Unbiased, Activity-Based Genetic Screen
for Cold Receptors
Previous efforts to identify cold receptors mostly focused on the
use of candidate gene approaches but not unbiased genetic
screens (Bandell et al., 2007; Castillo et al., 2018). Those cold receptors
that do not fall into the category of known thermosensors
(e.g., TRP family channels) would thus have escaped detection.
To overcome this difficulty, one approach is to design and
conduct an unbiased screen, which has the potential to uncover
new types of cold receptors.
Thus, we sought to design a forward genetic screen for mutants
defective in cold sensation in C. elegans, an organism
widely used as a genetic model for sensory biology. Traditionally,
behavioral assays are employed to conduct such a genetic
screen in C. elegans. Despite its convenience, this type of screen
lacks specificity, as the phenotype in isolated mutants might
simply result from defects in sensory processing by downstream
neural circuits rather than in cold sensing by sensory neurons/
cells. We thus decided to design an activity-based genetic
screen by directly targeting sensory neurons/cells.
We previously reported that worm intestinal cells directly sense
cold (Xiao et al., 2013). Cooling induces robust calcium response
in the intestine dissected out of the worm, shown by calcium imaging
using the genetically encoded calcium sensor GCaMP
(Xiao et al., 2013). The intestine is the largest worm tissue
composed of 20 epithelial cells (McGhee, 2007). The large size
of the intestine makes it possible to conduct a high throughput
activity-based screen by monitoring cooling-triggered calcium
response in the intestine. To design such a screen, we considered
using a real-time PCR thermal cycler that is designed for
amplifying and quantifying cDNA (Figure 1A). This type of equipment
can rapidly and precisely cool/heat the sample while monitoring
changes in the fluorescence (e.g., GCaMP) level of the
sample in real time. This feature and the convenience that worms
can readily fit into PCR tubes motivated us to employ such a thermal
cycler to conduct a high throughput activity-based screen
using live worms (Figure 1A). Though TRPA-1 contributes to
cold-evoked calcium response in the intestine, robust cold
response persists in trpa-1 mutant worms (Xiao et al., 2013), indicating
the presence of unknown cold receptor(s). We thus performed
a large scale chemical mutagenesis screen for such unknown
cold receptor(s) using this strategy (Figure 1A).
glr-3 Mutants Show a Strong Defect in Cooling-Evoked
Calcium Response
After screening >30,000 F2 strains, we recovered 11 mutants.
We focused on xu261, a mutant allele that showed a strong
phenotype in cooling-evoked calcium response (Figure 1A). By
whole-genome sequencing, we mapped the mutation to the
glr-3 gene that encodes a kainate-type glutamate receptor
(Brockie et al., 2001; Figure 1A).
To further characterize GLR-3, we performed standard calcium
imaging experiments by fluorescence microscopy. We
found that glr-3 null mutant worms (i.e., glr-3(tm6403)) displayed
a severe defect in cooling-evoked calcium increase in the intestine,
a phenotype that was rescued by transgenic expression of
wild type glr-3 gene in the intestine using an intestine-specific
promoter (Figures 1B and 1C). glr-3(xu261) mutant worms also
displayed a similar phenotype (Figure S1). These results identify
a key role for GLR-3 in mediating cooling-evoked cold response
in the intestine.
The Sensory Neuron ASER Is Cold-Sensitive and
Requires GLR-3 to Sense Cold
We next set out to characterize the in vivo functions of GLR-3 in
cold sensation. GLR-3 has been reported to be expressed in central
neurons (i.e., RIA interneurons in the head) (Brockie et al.,
2001). Using a longer promoter, we found that GLR-3 was also expressed
in the intestine (Figure 1D), as well as the sensory neuron
ASER (Figure 1E). The intestinal expression of GLR-3 is consistent
with our finding that GLR-3 mediates cooling-evoked calcium
response in the intestine. Because we are more curious about
the potential role of GLR-3 in regulating sensory behavior, we
decided to focus on its function in the nervous system.
The expression of GLR-3 in the sensory neuron ASER suggests
that GLR-3 may regulate sensory behavior in response
to cold. While ASER was originally identified as a chemosensory
neuron (Ortiz et al., 2009; Pierce-Shimomura et al., 2001), the
fact that GLR-3 is expressed in ASER suggests that this chemosensory
neuron may also be cold-sensitive. Indeed, cooling
evoked robust calcium response in ASER (Figures 2A, 2B,
S2A, and S2B). glr-3 mutant worms exhibited a severe defect
in this cooling response, a phenotype that was rescued by
1376 Cell 178, 1375–1386, September 5, 2019
transgenic expression of wild type glr-3 gene in ASER using an
ASER-specific promoter (Figures 2A and 2B). By contrast, loss
of GLR-3 did not affect salt-evoked (NaCl) calcium response in
ASER neuron (Figures 2C and 2D), indicating that the chemosensory
function of ASER neuron was not compromised in glr-3
mutant worms. As a control, che-2 mutant worms, which are
known to be defective in chemosensation (Perkins et al., 1986),
showed a severe defect in salt-evoked calcium transients in
ASER (Figures 2C and 2D). Thus, GLR-3 acts in ASER neuron
to mediate cold sensation. These results suggest that ASER is
a cold-sensitive neuron.
Nevertheless, it remains possible that the observed coolingevoked
response in ASER neuron might arise from another
sensory neuron (or neurons) via neurotransmission cell-nonautonomously.
To exclude this possibility, we repeated the
calcium imaging experiment in unc-13 and unc-31 mutant backgrounds,
in which secretion of neurotransmitters and neuropeptides
from synaptic vesicles (SV) and dense-core vesicles (DCV)
is abolished, respectively (Richmond et al., 1999; Speese et al.,
2007). Cooling-evoked calcium response in ASER neuron persisted
in unc-13 and unc-31 mutant worms (Figures 2E and
2F). Thus, ASER neuron can sense cold without inputs from other
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WT
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glr-3::sl2::yfpD E
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***
ASER
Intestine
RIA
Figure 1. An Unbiased Activity-Based
Genetic Screen Identifies a Key Role for
GLR-3 in Cold Sensation
(A) Schematic describing the design of the screen.
Worms carrying a transgene co-expressing
GCaMP3 and DsRed in the intestine were mutagenized
with EMS and their progeny was screened
using a real-time PCR thermocycler for candidates
defective in cooling-evoked calcium increase in
the intestine. Sample traces were shown to the
right, highlighting glr-3(xu261) mutant. The temperature
was cooled from 23
C to 10
C.
(B and C) Calcium imaging shows that the
glr-3(tm6403) deletion mutant exhibited a severe
defect in cooling-evoked calcium response in the
intestine. This phenotype was rescued by expressing
wild type glr-3 gene specifically in the
intestine using the ges-1 promoter. (B) Sample
traces. (C) Bar graph. Error bars, SEM n R 13.
***p < 0.0001 (ANOVA with Bonferroni test).
(D and E) glr-3 is expressed in the intestine and the
ASER sensory neuron, in addition to the central
interneuron RIA. A genomic DNA fragment encompassing
3.2 kb of 50
UTR and the entire coding
region of glr-3 was used to drive YFP expression.
(D) Low magnification image. (E) High magnification
image highlighting neuronal expression in the
head. (D) and (E) are two independent images
taken with different objectives. Arrow heads point
to ASER and RIAR (RIAL not visible on this focal
plane). Scale bars, 100 mm (D); 20 mm (E).
See also Figure S1.
neurons, providing further evidence supporting
that ASER is a cold-sensitive
neuron.
As GLR-3 is a member of the glutamate
receptor family, we also examined mutant
worms lacking eat-4, a gene that encodes
the sole vesicular glutamate transporter in the worm genome
(Lee et al., 1999). eat-4 mutant worms, which are devoid of glutamate
signaling, displayed normal cooling-evoked response in
ASER neuron (Figures 2E and 2F), providing additional evidence
that ASER is a cold-sensitive neuron. This experiment also demonstrates
that the role of GLR-3 in ASER cold sensation is independent
of its function as a glutamate receptor.
GLR-3 Acts in ASER Neuron to Mediate Cold-Avoidance
Behavior
What would be the behavioral consequences following GLR-3mediated
activation of ASER neuron by cold? Activation of
ASER neuron is known to trigger backward movement (reversals)
followed by turns, a type of avoidance response (Suzuki et al.,
2008). We first used a swimming assay to quantify turns. We
found that cooling triggered turns in wild type worms (Figure 2G).
glr-3 mutant worms exhibited a severe defect in this cold-avoidance
behavior (Figure 2G), while no such defect was detected
in trpa-1 mutant worms (Figure S2G). Transgenic expression of
wild type glr-3 gene specifically in ASER neuron rescued this
behavioral phenotype (Figure 2G). By contrast, glr-3 mutant
worms exhibited normal salt chemotaxis behavior compared to
Cell 178, 1375–1386, September 5, 2019 1377
wild type worms, though che-2 mutant worms were severely
defective (Figure 2H). To provide additional evidence, we sought
to develop another cold-avoidance behavioral assay. We examined
whether cold stimuli can trigger avoidance response in
worms crawling on the surface of an agar plate by cooling the
air near the worm head with a cold probe. Indeed, cooling triggered
backward movement (reversals) in wide-type worms (Figure
S2H), and this avoidance behavioral response was defective
in glr-3 mutant worms, a phenotype that was rescued by transgenic
expression of wild type glr-3 gene specifically in ASER
neuron (Figure S2H). As the swimming assay allows us to deliver
cold stimuli to the worm with greater ease, we decided to focus on
this assay for further characterizations. These behavioral data,
together with calcium imaging results, demonstrate that GLR-3
acts in ASER sensory neuron to mediate cold sensation in vivo,
suggesting GLR-3 as a cold receptor.
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GCaMP∆F/F(fold)
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***
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WT
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eat-4
unc-13
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***
GCaMP∆F/F(fold)
21oC
18oC
Turningrate(Hz)
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glr-3
WT
Time (sec)
ASER::glr-3; glr-3
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glr-3
che-2
50 mM NaCl
0 mM NaCl
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GCaMP∆F/F(fold)
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ChemotaxisIndex
ns
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ns
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Figure 2. GLR-3 Acts in ASER Neuron to
Mediate Cold Sensation and Cold-Avoidance
Behavior
(A and B) ASER neuron is cold-sensitive and requires
GLR-3 for cold sensation. Cooling evoked
robust calcium response in ASER neuron, which is
defective in glr-3(tm6403) mutant worms. Transgenic
expression of wild type glr-3 cDNA in ASER
using the ASER-specific gcy-5 promoter rescued
the mutant phenotype. (A) Sample traces. (B) Bar
graph. Error bars, SEM n R 12. ***p < 0.0001
(ANOVA with Bonferroni test).
(C and D) Loss of GLR-3 does not affect the
sensitivity of ASER neuron to salt. glr-3(tm6403)
mutant worms showed normal response to NaCl
gradient compared to WT, while che-2(e1033)
mutant worms were defective in this response.
(C) Sample traces. (B) Bar graph. Error bars, SEM
n R 11. ***p < 0.0001 (ANOVA with Bonferroni
test).
(E and F) Cooling-evoked calcium response persists
in unc-13, unc-31, and eat-4 mutant backgrounds.
(E) Sample traces. (F) Bar graphs. Error
bars, SEM n R11. ***p < 0.0001 (ANOVA with
Bonferroni test).
(G) GLR-3 mediates cooling-evoked avoidance
behavior in ASER neuron. Cooling triggered turns
in worms during swimming, and glr-3 mutant
worms showed a strong defect in this behavioral
response, which was rescued by transgenic
expression of glr-3 cDNA in ASER. Error bars, SEM
n R 15.
(H) glr-3(tm6403) mutant worms show normal salt
chemotaxis behavior, while che-2(e1033) mutant
worms are defective in this behavior. The chemotaxis
assay was run in triplicates, and the experiment
was repeated four times. Error bars: SEM.
***p < 0.0001 (ANOVA with Bonferroni test).
See also Figure S2.
GLR-3 Can Function as a Cold
Receptor
While our data show that GLR-3 is
required for cold sensation in vivo,
they do not constitute sufficient evidence
supporting that GLR-3 is a cold
receptor, as the observed effect might be indirect, which
could potentially be contributed by a different protein. To
demonstrate that a candidate protein can function as a cold
receptor, one of the most commonly employed approaches
is to express it in cold-insensitive heterologous cells to test
whether it can confer cold sensitivity to these otherwise
cold-insensitive cells (Bandell et al., 2007; Castillo et al.,
2018). To this end, we first ectopically expressed GLR-3 in
worm body-wall muscle cells that are commonly used as a
vehicle to express exogenous proteins, particularly membrane
receptors (Gong et al., 2016; Wang et al., 2012b). While bodywall
muscle cells were insensitive to cold, ectopic expression
of GLR-3 in muscle cells conferred cold sensitivity to these
otherwise cold-insensitive cells (Figures 3A, 3B, S2C, and
S2D), providing strong evidence that GLR-3 is sufficient to
function as a cold receptor.
1378 Cell 178, 1375–1386, September 5, 2019
To gather further evidence, we expressed GLR-3 in mammalian
cell lines. Transfection of GLR-3 in CHO cells conferred
cold sensitivity to these cells (Figures 3C, 3D, S2E, and S2F).
Transfection of GLR-3 in COS-7 and HeLa cells also yielded a
similar result (Figures S3A–S3D). These heterologous expression
data provide further evidence that GLR-3 can function as a cold
receptor.
The Mouse GLR-3 Homolog GluK2 Can Functionally
Substitute for Worm GLR-3 In Vivo and Function as a
Cold Receptor In Vitro
Having characterized GLR-3 both in vivo and in vitro, we then
wondered if the role of GLR-3 in cold sensation is evolutionarily
conserved. In mouse, GluK2 is a close homolog of GLR-3 and is
also a kainate-type glutamate receptor. We expressed mouse
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mGluK2 Fura-2∆R/R(fold)
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GCaMP∆F/F(fold)
GCaMP∆F/F(fold)
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WT
Time (sec)
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ASER ASER
***
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Figure 3. GLR-3 and Mouse GluK2 Function
as a Cold Senor In Vitro, and Mouse GluK2
Can Functionally Substitute for GLR-3 in
Cold Sensation In Vivo
(A and B) Ectopic expression of GLR-3 and mouse
GluK2 in worm muscle cells confers cold-sensitivity
to these cells. GLR-3 and mouse GluK2 were
expressed as a transgene in worm body-wall
muscles using the myo-3 promoter. Cooling
evoked calcium response in muscle cells of
transgenic worms. All genotypes carried a transgene
expressing GCaMP6. (A) Sample traces. (B)
Bar graph. Error bars, SEM n R 11. ***p < 0.0001
(ANOVA with Bonferroni test).
(C and D) Heterologous expression of GLR-3 and
mouse GluK2 in CHO cells confers cold sensitivity
to these cells. GLR-3 and mouse GluK2 were
transfected into CHO cells, and cooling evoked
calcium response in these cells as shown by
Fura-2 imaging. (C) Sample traces. (D) Bar graph.
Error bars, SEM n R 20. ***p < 0.0001 (ANOVA with
Bonferroni test).
(E and F) Mouse GluK2 can functionally substitute
for GLR-3 in cold sensation in ASER neuron.
Transgenic expression of mouse GluK2 in ASER
neuron of glr-3 mutant worms rescued the glr-3
mutant phenotype in cooling-evoked calcium
response in ASER neuron. (E) Sample traces. (F)
Bar graph. Error bars, SEM n R 12. ***p < 0.0001
(ANOVA with Bonferroni test).
(G) Mouse GluK2 can functionally substitute for
GLR-3 in cold-avoidance behavior mediated by
ASER neuron. Transgenic expression of mouse
GluK2 in ASER neuron of glr-3 mutant worms
rescued the glr-3 mutant phenotype in cooling-
evokedavoidancebehavior.Errorbars,SEMnR15.
See also Figures S2 and S3.
GluK2 as a transgene in ASER neuron
and found that mouse GluK2 can restore
cooling-evoked calcium response in
ASER neuron of glr-3 mutant worms (Figures
3E and 3F). In addition, mouse GluK2
expression in ASER also rescued the
cold-avoidance behavioral phenotype in
glr-3 mutant worms (Figure 3G). Thus,
mouse GluK2 can functionally substitute for GLR-3 in cold
sensation in vivo, suggesting that mouse GluK2 has the potential
to function as a cold receptor.
We also expressed mouse GluK2 as a transgene in worm
muscle cells, and found that it conferred cold sensitivity to
these cells (Figures 3A, 3B, S2C, and S2D). Heterologous
expression of mouse GluK2 in CHO cells also conferred cold
sensitivity (Figures 3C, 3D, S2E, and S2F). Cooling-evoked
calcium response mediated by GluK2 and GLR-3 appears to
primarily result from calcium influx (Figures S3E and S3F). As
was the case with GLR-3, transfection of mouse GluK2 in
COS-7 cells and HeLa cells also conferred cold sensitivity to
these cells (Figures S3A–S3D). For simplicity, we focused
on using CHO cells as our main heterologous expression
system for further characterization of GLR-3/GluK2 in vitro.
Cell 178, 1375–1386, September 5, 2019 1379
In summary, heterologous expression data demonstrate that
mouse GluK2 can function as a cold receptor in vitro, suggesting
that the role of GLR-3/GluK2 as a cold receptor might be
evolutionarily conserved.
GLR-3/GluK2 Mainly Senses Cold but Not Cool
Temperatures
To further characterize GLR-3/GluK2, we determined its activation
threshold. TRPM8 is a cool sensor with an activation
threshold at $26
C (Bandell et al., 2007; McKemy et al., 2002).
Indeed, cooling from 32
C to 20
C evoked robust calcium
response in CHO cells transfected with TRPM8 (Figures 4A
and 4B). As TRPM8 is also a menthol receptor, we tested
menthol on these cells. As predicted, menthol activated
TRPM8 but not GLR-3 or mouse GluK2 in CHO cells (Figures
4A and 4B). By contrast, cooling from 32
C to 20
C triggered little,
if any, calcium response in CHO cells expressing GLR-3 or
mouse GluK2 (Figures 4A and 4B). This indicates that GLR-3/
GluK2 was activated at a temperature much lower than
TRPM8, and the activation threshold of GLR-3/GluK2 is below
20
C. Indeed, we estimated that the activation threshold for
GLR-3/GluK2 in CHO cells was $18
C (18.2 ± 0.14 for GLR-3;
18.3 ± 0.16 for mGluK2; n = 20). Similar results were obtained
in ASER neuron in vivo (18.9 ± 0.14; n = 15; Figure S4), as well
as in muscle cells ectopically expressing GLR-3 and mouse
GluK2 (18.4 ± 0.16 for GLR-3; 18.5 ± 0.18 for mGluK2; n = 15).
These observations together suggest GLR-3/GluK2 as a noxious
cold receptor.
The Cold Sensitivity of GLR-3/GluK2 Is Independent of
Its Channel Activity In Vitro and In Vivo
We made the surprising observation that while glutamate can
activate mouse GluK2 in CHO cells as predicted, it cannot activate
GLR-3 using the calcium imaging assay (Figures 4A and
4B). We thus recorded these CHO cells by whole-cell patchclamping.
Glutamate evoked a typical glutamate-gated current
in CHO cells expressing mouse GluK2 (Figures S5D and S5I),
but not GLR-3 (Figures S5H and S5I), indicating that GLR-3
lacked glutamate-gated channel activity, even though it can
function as a cold receptor in these cells (Figures 3C and 3D).
This surprising finding suggests that the observed cold response
mediated by GLR-3/GluK2 may be independent of its channel
function. To test this model, we generated two channel-dead
mutants of mouse GluK2: M620R and Q622R (Figure S5A),
which mutated residues adjacent to the Q/R RNA editing site
in the channel pore region (Dingledine et al., 1992; Robert
et al., 2002). As reported previously (Dingledine et al., 1992; Robert
et al., 2002), these two point mutations abolished glutamategated
current of mouse GluK2 in CHO cells (Figures S5E, S5F,
and S5I). Strikingly, these two channel-dead GluK2 variants
were still cold-sensitive in these cells (Figures 5A and 5C).
Although GLR-3 lacked glutamate-gated channel activity in our
assay, we introduced similar point mutations (M582R and
Q584R) to disrupt any potential channel activity (Figure S5A),
and found that these two channel-dead GLR-3 variants also retained
normal cold sensitivity when expressed in CHO cells (Figures
5B and 5C). These results demonstrate that the cold sensitivity
of GLR-3/GluK2 is independent of its channel function
in vitro.
To obtain further evidence, we tested the two channel-dead
GLR-3/GluK2 variants in vivo. We found that both GLR-3 and
mouse GluK2 channel-dead variants can still function as a cold
receptor in ASER neuron, as transgenic expression of these variants
restored cooling-evoked calcium response in ASER neuron
of glr-3 mutant worms (Figures 5D–5F). Furthermore, ectopic
expression of these channel-dead variants in worm muscle cells
can still confer cold-sensitivity to these cells (Figures 5G–5I). We
thus conclude that the cold sensitivity of GLR-3/GluK2 is independent
of its channel function both in vitro and in vivo.
We also explored the converse scenario: can we identify mutations
that disrupt the cold sensitivity of GLR-3/GluK2 but not its
glutamate-gated channel function? The glr-3(xu261) mutant isolated
from our genetic screen carried a P121L missense mutation
(Figure S5B). This point mutation abolished the cold sensitivity
of GLR-3 (Figures 5J and 5L). As GLR-3 did not exhibit
channel activity on its own, we tested our model in mouse
GluK2. Because this P residue is not found in mouse GluK2,
we mutated an adjacent P residue P151 to L in the mouse protein
(Figure S5B). These P residues are located in the N-terminal ATD
domain of GLR-3/GluK2 (Traynelis et al., 2010). P151L mutation
disrupted the cold sensitivity of mouse GluK2 in CHO cells (Figures
5K and 5L), and a corresponding P130L mutation in GLR-3
exerted a similar effect (Figures 5J and 5L). This demonstrates
that the N-terminal ATD domain is required for the cold-sensitivity
of GLR-3/GluK2. Strikingly, despite its lack of cold sensitivity,
GluK2(P151L) retained glutamate-evoked channel activity
in calcium imaging assay (Figures 5K and 5L). Whole-cell
-0.4
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2.4
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3
A
Fura-2∆R/R(fold)
TRPM8
mGluK2
GLR-3
Vector
32℃
20℃
100 s
CHO
Fura-2∆R/R(fold)
32℃ 20℃ glutamate menthol
CHO
B
***
ns ns ns ns ns ns
***
***
Figure 4. The Sensitivity of GLR-3/GluK2 and TRPM8 to Cold,
Glutamate, and Menthol
(A and B) Cooling from 32
C to 20
C activated TRPM8 but not GLR-3 or mouse
GluK2 when transfected in CHO cells. Mouse GluK2, but not GLR-3 or TRPM8,
can be activated by glutamate (10 mM), while TRPM8 was also sensitive to
menthol (100 mM) but not glutamate (10 mM). (A) Sample traces. (B) Bar graph.
Error bars, SEM n R 20. ***p < 0.0001 (ANOVA with Bonferroni test).
See also Figures S4 and S5.
1380 Cell 178, 1375–1386, September 5, 2019
recording also showed that GluK2(P151L) retained glutamategated
current, although its kinetics were altered (Figures S5G
and S5I). This is consistent with the notion that the ATD domain
of glutamate receptors is not required for their channel function
and only plays a modulatory role (Traynelis et al., 2010). Thus,
it appears that the cold-sensing and glutamate-gated channel
functions of GLR-3/GluK2 can be dissociated and may require
distinct domains. Together, our results suggest that GLR-3/
GluK2 may function as a receptor-type cold sensor rather than
a cold-activated channel.
GLR-3/GluK2 Functions as a Metabotropic Cold
Receptor and Requires G Protein Signaling to Transmit
Cold Signals In Vitro and In Vivo
Interestingly, mammalian kainate receptors have been reported
to possess both metabotropic and ionotropic functions (Rodrı´guez-Moreno
and Lerma, 1998; Rozas et al., 2003; Valbuena
and Lerma, 2016). When acting as metabotropic receptors,
mGluK2
0
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100 s
23℃
G H I
D E F
GCaMP∆F/F(fold)
WT
Muscle::mGluK2
Muscle::mGluK2(M620R)
Muscle::mGluK2(Q622R)
23℃
GCaMP∆F/F(fold)
GCaMP∆F/F(fold)
Fura-2∆R/R(fold)
23℃
Fura-2∆R/R(fold)
Fura-2∆R/R(fold)
10℃
100 s
10℃ 10℃
100 s
CHO CHO
CHO
ASER
Muscle
23℃
glr-3
ASER::mGluK2; glr-3
ASER::mGluK2(M620R); glr-3
ASER::mGluK2(Q622R); glr-3
GCaMP∆F/F(fold)
10℃
100 s
ASER
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GCaMP∆F/F(fold)
glr-3
ASER::glr-3; glr-3
ASER::glr-3(M582R); glr-3
ASER::glr-3(Q584R); glr-3
23℃ 100 s
10℃
ASER
Muscle
-0.5
0
0.5
1
1.5
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GCaMP∆F/F(fold)
WT
Muscle::glr-3
Muscle::glr-3(M582R)
Muscle::glr-3(Q584R)
23℃
10℃
100 s
Muscle
-0.4
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2
*** *** *** ***
***
***
***
***
***
***
***
***
mGluK2 GLR-3
mGluK2GLR-3
GLR-3 mGluK2
*** *** ***
***
*** ***
A B CVector
GLR-3
GLR-3(M582R)
GLR-3(Q584R)
Vector
mGluK2
mGluK2(M620R)
mGluK2(Q622R)
-0.2
0
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0.6
0.8
1
1.2
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1.6
CHO
Fura-2∆R/R(fold)
23℃
10℃
100 s
GLR-3
Vector
GLR-3 (P121L)
GLR-3 (P130L)
-0.5
0
0.5
1
1.5
2
2.5
23℃
10℃
100 s
glutamate
CHO
Vector
mGluK2(P151L)
mGluK2
-0.2
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1
1.2
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Fura-2∆R/R(fold)
Fura-2∆R/R(fold)
glutamatecoldCHO
GLR-3
***
*** ***
***
J K L
mGluK2
-0.4
0
0.4
0.8
1.2
1.6
Figure 5. The Cold Sensitivity of GLR-3/
GluK2 Is Independent of Its Channel Function
(A–C) Channel-dead GLR-3/GluK2 variants show
normal cold sensitivity in CHO cells. The two
channel-dead variants of mouse GluK2 (A), as well
as GLR-3 (B), showed similar cold sensitivity
compared to wild type GluK2/GLR-3 when transfected
in CHO cells. (A and B) Sample trances. (C)
Bar graph. Error bars, SEM n R 20. ***p < 0.0001
(ANOVA with Bonferroni test).
(D–F) Channel-dead GLR-3/GluK2 variants show
normal cold sensitivity in ASER neuron in vivo. The
two channel-dead variants of GLR-3 (D), as well as
mouse GluK2 (E), showed similar cold sensitivity
compared to wild type GluK2/GLR-3 when
expressed as a transgene in ASER neuron of glr-3
mutant worms. (D and E) Sample traces. (F) Bar
graph. Error bars, SEM n R 12. ***p < 0.0001
(ANOVA with Bonferroni test).
(G–I) Channel-dead GLR-3/GluK2 variants show
normal cold sensitivity in worm muscle cells. The
two channel-dead variants of GLR-3 (G), as well as
mouse GluK2 (H), showed similar cold sensitivity
compared to wild type GluK2/GLR-3 when expressed
as a transgene in worm muscle cells.
(G and H) Sample traces. (I) Bar graph. Error bars,
SEM n R 12. ***p < 0.0001 (ANOVA with Bonferroni
test).
(J–L) Cold-insensitive GLR-3/GluK2 mutants
display glutamate-gated channel activity. (J) GLR-
3(P121L) and GLR-3(P130L) were no longer sensitive
to cold. (K) GluK2(P151L) was insensitive to
cold but sensitive to glutamate. (L) Bar graph. Error
bars, SEM n R 22. ***p < 0.0001 (ANOVA with
Bonferroni test).
See also Figure S5.
they transmit glutamate signals via G proteins
(Rodrı´guez-Moreno and Lerma,
1998; Rozas et al., 2003; Valbuena and
Lerma, 2016). In this case, these kainate
receptors act as ‘‘GPCR-like’’ glutamate
receptors rather than glutamate-gated
channels (Rodrı´guez-Moreno and Lerma, 1998; Rozas et al.,
2003; Valbuena and Lerma, 2016). This led us to hypothesize
that GLR-3/GluK2 may also function as a ‘‘GPCR-like’’ cold receptor.
If so, GLR-3/GluK2 should depend on G proteins to
transmit cold signals. To test this, we examined mSIRK, a membrane-permeable
peptide that dissociates Ga from Gbg without
stimulating its GTP-binding activity, thereby inhibiting receptormediated
activation of G proteins (Goubaeva et al., 2003).
mSIRK abolished cooling-evoked calcium response in CHO cells
expressing GLR-3 or mouse GluK2 (Figures 6A–6D). Pertussis
toxin (PTX), a Gi/o inhibitor (Gierschik, 1992), also blocked the
cold sensitivity of GLR-3 and mouse GluK2 in CHO cells (Figures
6A–6D). By contrast, the Gq/11 inhibitor, YM-254890 (Takasaki
et al., 2004), had no effect on the cold sensitivity of GLR-3/
GluK2 (Figures 6A–6D). As a control, this Gq/11 inhibitor can
block bradykinin-evoked calcium response (Figures S6A and
S6B), which was mediated by endogenous Gq/11-coupled bradykinin
receptors in CHO cells (Pauwels and Colpaert, 2003),
Cell 178, 1375–1386, September 5, 2019 1381
whereas the Gi/o inhibitor PTX had no effect on bradykininevoked
response (Figures S6A and S6B). These results demonstrate
that GLR-3/GluK2 transmits cold signals via Gi/o signaling
in vitro.
Encouraged by our in vitro results, we next tested whether
GLR-3 transmits cold signals via Gi/o signaling in vivo. ASER
neuron expresses at least two Gi/o genes: goa-1 and gpa-3
(Jansen et al., 1999). Mutations in goa-1, but not gpa-3, led to
a severe defect in cooling-evoked calcium response in ASER
neuron (Figures 6E and 6F), a phenotype that was rescued by
transgenic expression of wild type goa-1 gene specifically in
ASER neuron (Figures 6E and 6F), suggesting that GLR-3 transmits
cold signals via Gi/o signaling in vivo. A similar result was
obtained with cooling-evoked calcium response in the intestine
(Figures S6C and S6D). Taken together, both of our in vitro and
in vivo results support the model that GLR-3/GluK2 functions
as a metabotropic, rather than an ionotropic, cold receptor,
and does so by transmitting cold signals via Gi/o proteins. This
0
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GCaMP∆F/F(fold)
23℃
GCaMP∆F/F(fold)
10℃
100 s
ASER
ASER
***
ns
***
Fura-2∆R/R(fold)
Vector
GLR-3
GLR-3 + YM-254890
CHO
GLR-3 + mSIRK
GLR-3+ PTX
A B
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*** ***
ns ns
CHO
23℃
10℃ 100 s
gpa-3; goa-1
WT
gpa-3
goa-1
ASER::goa-1; goa-1
ns
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Fura-2∆R/R(fold)
23℃
Vector
mGluK2
mGluK2 + YM-254890
Fura-2∆R/R(fold)
mGluK2 + mSIRK
mGluK2+ PTX
10℃
100 s
CHO
CHO
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1
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1.4
***
***
ns
ns
C D
Fura-2∆R/R(fold)
Figure 6. GLR-3 Relies on G Protein
Signaling to Transmit Cold Signals
(A and B) G protein inhibitors blocked the cold
sensitivity of GLR-3 in CHO cells. The pan-G protein
inhibitor mSIRK (50 mM), the Gi/o inhibitor PTX
(100 ng/mL), but not the Gq/11 inhibitor YM-
254890 (10 mM), blocked cooling-evoked calcium
response mediated by GLR-3 in CHO cells. (A)
Sample traces. (B) Bar graph. Error bars, SEM n
R 20. ***p < 0.0001 (ANOVA with Bonferroni test).
(C and D) G protein inhibitors blocked the cold
sensitivity of mouse GluK2 in CHO cells. The
pan-G protein inhibitor mSIRK (50 mM), the Gi/o
inhibitor PTX (100 ng/mL), but not the Gq/11 inhibitor
YM-254890 (10 mM), blocked coolingevoked
calcium response mediated by mouse
GluK2 in CHO cells. (C) Sample traces. (D) Bar
graph. Error bars, SEM n R 20. ***p < 0.0001
(ANOVA with Bonferroni test).
(E and F) The cold sensitivity of ASER neuron requires
the Gi/o protein gene goa-1. goa-1(n1134)
mutant worms showed a strong defect in coolingevoked
calcium response in ASER neuron, a
phenotype that was rescued by transgenic
expression of wild type goa-1 gene specifically in
ASER neuron. (E) Sample traces. (F) Bar graph.
Error bars, SEM n R 11. ***p < 0.0001 (ANOVA with
Bonferroni test).
See also Figure S6.
identifies GLR-3/GluK2 as a previously
unknown type of cold receptor.
Mouse DRG Neurons Express GluK2
and Knockdown of GluK2 in DRG
Neurons Suppresses Their
Sensitivity to Cold but Not Cool
Temperatures
Mammalian glutamate receptors are best
known to function in the brain to regulate
synaptic transmission and plasticity. Interestingly,
some of these receptors are also
expressed in the peripheral nervous system such as DRG sensory
neurons but with an unknown function (Coggeshall and Carlton,
1998; Huettner, 1990). We thus wondered if GluK2 is expressed
in DRG neurons. These primary sensory neurons detect somatosensory
and painful stimuli in the periphery, including temperature
cues. Using RNAscope assay, we detected GluK2 mRNA transcripts
in mouse DRGs (Figures 7A and 7B). Approximately
13.6% (140/1031) DRG neurons expressed GluK2. Among
them, most were small-diameter neurons (75.0%, 105/140), while
a small percentage (24.2%, 34/140) were medium-diameter neurons.
This data suggests that GluK2 is expressed in DRG neurons.
We next knocked down GluK2 expression in cultured DRG
neurons with siRNA (Figure 7C). While knockdown of GluK2
did not affect the sensitivity of DRG neurons to cool temperatures
(cooling to 22
C) (Figures 7D and 7F; 30/699 control cells
versus 26/598 siRNA cells responded), it greatly reduced the
sensitivity of these sensory neurons to cold temperatures
(cooling to 10
C) (Figures 7E and 7F; 131/699 control cells versus
1382 Cell 178, 1375–1386, September 5, 2019
45/598 siRNA cells responded; c2
test, p < 0.0001). This is
consistent with our data that GluK2 primarily senses cold but
not cool temperatures. These observations suggest that GluK2
may sense cold temperatures in mouse DRG neurons.
The Fish and Human GLR-3/GluK2 Homologs Can Also
Function as a Cold Receptor In Vitro
Our observation that both GLR-3 and mouse GluK2 can function
as a cold receptor prompted us to ask how deeply the role of GLR-
3/GluK2 as a cold receptor is conserved across the phylogeny.
We thus tested the zebrafish and human homologs of GluK2.
Transfection of both zebrafish and human GluK2 in CHO cells
conferred cold sensitivity to these cells (Figures 7G and 7H), suggesting
that they both can function as a cold receptor in heterologous
systems. Thus, GLR-3/GluK2 from worms, zebrafish, mice,
and humans all have the capacity to function as a cold receptor,
suggesting that the function of GLR-3/GluK2 in cold sensation
might be evolutionarily conserved from worms to humans.
DISCUSSION
Despite decades of intensive research, little is known about the
molecular mechanisms underlying cold sensation. Previous efforts
to identify cold sensors using candidate gene approaches
have not been very fruitful, although this approach is highly successful
in identifying heat sensors such as thermo-TRP channels
(Bandell et al., 2007; Castillo et al., 2018). To overcome this technical
difficulty, in the current study, we designed an unbiased,
activity-based genetic screen for cold-sensing receptors in
C. elegans using a real-time PCR thermocycler. We showed
that GLR-3, a member of the kainate-type of glutamate receptors,
surprisingly functions as a cold receptor.
GLR-3 possesses several interesting features. First, its activation
threshold is lower than that of TRPM8 (i.e., $18
C versus
$26
C), which is close to the range of noxious cold, suggesting
that GLR-3 may primarily sense noxious cold rather than cool
temperatures. Second, unlike other thermal receptors that are
temperature-gated ion channels (Bandell et al., 2007; Castillo
et al., 2018), GLR-3 functions as a metabotropic thermal receptor.
This is supported by the observation that channel-dead
GLR-3 and mouse GluK2 variants show normal cold sensitivity.
In addition, although worm GLR-3 is cold-sensitive, it does not
display any detectable channel activity. Furthermore, both
GLR-3 and mouse GluK2 rely on G proteins to transmit cold signals.
Another interesting feature of GLR-3 is that glutamate is not
required for this receptor to sense cold in vivo, as eat-4 mutant
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1.8 CHO
G H
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2
Fura-2∆R/R(fold)
CHO
Vector
fish Gluk2
human Gluk2
Fura-2∆R/R(fold)
***
***
23℃
10℃
100 s
-0.2
0
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0.8
Fura-2∆R/R(fold)
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0
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Fura-2∆R/R(fold)
22oC
35℃ 22oC
10oC
A B
F
0
5
10
15
20
25
%RespondingDRGneurons
Cool
Cold
***
E
ns
Cool Cold
0
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1
1.2
RelativemRNAlevel
mGluK2 mRNA
***
DRG
DRG DRG
100 s
DRG
GluK2 / DAPI Negative Control / DAPI
D
C Figure 7. Mouse DRG Neurons Express
GluK2 and Knockdown of GluK2 in DRG
Neurons Suppresses Their Sensitivity to
Cold but Not Cool Temperatures
(A and B) Mouse DRG neurons express GluK2
mRNA transcripts. (A) In situ hybridization using
RNAscope assay detected GluK2 mRNA signals
(puncta) in DRG tissue. The dotted ovals denote
the shape of the soma of the neurons expressing
GluK2 transcripts. Only those neurons with at least
4 puncta inside the soma were scored positive, as
the background signals detected by the control
probe varied between 0–2 puncta/cell. The inset
image in (A) highlights one GluK2-positive neuron.
Green, GluK2 transcripts. Blue, DAPI. (B) Negative
control probe staining (provided by Advanced Cell
Diagnostics). Scale bar, 50 mm.
(C) Real-time qPCR analysis shows that siRNA
knockdown of GluK2 in mouse DRG neurons
greatly reduced the mRNA level of GluK2. qPCR
reactions were run in triplicates. The experiment
was repeated five times. Error bars, SEM.
***p < 0.0001 (t test).
(D–F) siRNA knockdown of GluK2 in mouse DRG
neurons greatly reduces the sensitivity of these
neurons to cold but not cool temperatures. (D)
Sample trace showing calcium response to cool
temperature (cooling to 22
C). (E) Sample trace
showing calcium response to cold temperature
(cooling to 10
C). (F) Bar graph. Sample sizes were
shown on top of each bar. ***p < 0.0001 (c2
test).
(G and H) Fish and human GluK2 can also function
as a cold receptor in vitro. Heterologous expression
of fish and human GluK2 in CHO cells confers
cold sensitivity to these cells. (G) Sample traces.
(H) Bar graph. Error bars, SEM n R 20.
***p < 0.0001 (ANOVA with Bonferroni test).
Cell 178, 1375–1386, September 5, 2019 1383
worms, which are devoid of glutamate signaling, show normal
cold sensitivity in ASER neuron. GLR-3 and mouse GluK2 can
also sense cold in the absence of glutamate in heterologous systems.
This suggests that the cold-sensing activity of GLR-3/
GluK2 is independent of its glutamate receptor function. Furthermore,
mutations in the N-terminal ATD domain of GluK2, which
disrupt the cold sensitivity of mouse GluK2, spare its glutamate-gated
channel activity, suggesting that the functions of
cold-sensing and glutamate-sensing of GluK2 could be dissociated
and may require distinct domains. We thus propose that
some glutamate receptors, such as GLR-3/GluK2, are multifunctional,
acting as a chemical receptor in the CNS but functioning
as a thermal receptor in the periphery.
As a metabotropic cold receptor, GLR-3 requires G proteins to
transmit cold signals, which would presumably lead to the activation
of downstream transduction channels and hence cooling-evoked
calcium response. Indeed, cooling-activated calcium
response mediated by GLR-3/GluK2 primarily results
from calcium influx (Figures S3E and S3F), suggesting the presence
of downstream transduction channel(s). The identity of the
transduction channel(s) acting downstream of GLR-3 and G proteins
is currently unknown. Future studies are needed to address
this question, as well as to identify the detailed transduction
mechanisms. We found that a Gi/o protein is required for
GLR-3 to transduce cold signals. Although Gi/o is known to
trigger inhibitory signaling, interestingly, it can also be coupled
to excitatory signaling mediated mostly by its Gbg subunits
(Neves et al., 2002). Notably, in addition to channels, G proteins
can also regulate many other types of effectors, such as enzymes,
transporters, transcriptional machinery, mobility/
contractibility machinery, secretory machinery, etc. (Neves
et al., 2002). In this regard, a metabotropic thermal receptor
would be more functionally versatile and may potentially regulate
a wider range of cellular processes. GLR-3 thus represents a
previously unknown class of thermal receptor.
Glutamate receptors are chemical-sensing receptors that are
well known to mediate chemical synaptic transmission/plasticity
in the CNS. Our results reveal an unexpected case where a central
chemical receptor functions as a thermal receptor in the periphery.
As glutamate receptors are evolutionarily conserved, this raises
the intriguing possibility that in addition to sensing chemicals
(i.e., glutamate), one ancestral function of glutamate receptors
might be to sense non-chemical cues such as temperature. Interestingly,
some insect chemical-sensing ionotropic receptors (IRs)
regulate thermosensation (Budelli et al., 2019). It would be interesting
to test whether they can sense temperature.
Another interesting observation is that the vertebrate GLR-3
homolog GluK2 from mouse, human, and zebrafish can all function
as a cold receptor in heterologous systems. Mouse GluK2
can also functionally substitute for worm GLR-3 in cold sensation
in vivo. In addition to the brain, we found that mouse GluK2 is expressed
in DRG neurons in the periphery, and knockdown of
GluK2 in DRG neurons greatly reduced the sensitivity of these
primary sensory neurons to cold but not cool temperatures,
raising the possibility that GluK2 may function as a cold receptor
in DRG neurons in mammals. We thus propose that the role of
GLR-3/GluK2 as a cold receptor may be evolutionarily
conserved from worms to humans.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d LEAD CONTACT AND MATERIALS AVAILABILITY
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
d METHOD DETAILS
B Molecular biology and genetics
B Genetic screen
B C. elegans calcium imaging and behavioral assays
B Cell culture and calcium imaging
B Mouse DRG neuron culture, transfection, qPCR, and
calcium imaging
B Mouse DRG in situ hybridization (RNAscope)
B Electrophysiology
d QUANTIFICATION AND STATISTICAL ANALYSIS
d DATA AND CODE AVAILABILITY
ACKNOWLEDGMENTS
We thank Min Guo, Xi Lan, Jie Liu, Rebekah Ronan, Bonnie Zeng, Seth
Kattapong-Graber, and Jonathan Ronan for technical assistance, and Bing
Ye for comments. Some strains were obtained from the CGC and Japan
knockout consortium. E.A.R. was supported by an NIA T32 training grant.
Jianfeng Liu received funding support from the MOST and NSFC
(2018YFA0507003, 31420103909, 81872945, and 81720108031) and the
111 project of the MOE (B08029). G.-H.K. received funding support from the
National Research Foundation of Korea (NRF-2017R1A4A1015564). B.D.
received funding support from the NINDS. X.Z.S.X. received funding support
from the NIGMS and NIA.
AUTHOR CONTRIBUTIONS
J.G. and E.A.R. performed in vivo experiments and analyzed the data. J.G. and
F.H. performed in vitro experiments and analyzed the data. Jinzhi Liu generated
nearly all the reagents. W.C., M.F., H.L., and B.D. performed mouse DRG experiments
and analyzed the data. W.Z. assisted J.G. in performing some experiments.
Z.L. helped identify GLR-3 expression pattern. G.-H.K. and K.P.P.
contributed to the design and fabrication of cooling devices. J.G., E.A.R., Jianfeng
Liu, and X.Z.S.X. wrote the paper with input from other authors.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: March 10, 2019
Revised: May 31, 2019
Accepted: July 17, 2019
Published: August 29, 2019
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1386 Cell 178, 1375–1386, September 5, 2019
STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER
Bacterial and Virus Strains
E. coli:OP50 Caenorhabditis Genetics
Center
OP50
Chemicals, Peptides, and Recombinant Proteins
TRIzol LS Reagent Thermo Fisher Scientific 10-296-010
Power SYBR Green Thermo Fisher Scientific 4367659
M-MLV Reverse Transcriptase Thermo Fisher Scientific 28025013
RNAscope 3-plex Negative Control Probe Advanced Cell Diagnostics 320871
RNAscope 3-plex Positive Control Probe Advanced Cell Diagnostics 320881
RNAscope Probe Mm-Gluk2 Advanced Cell Diagnostics 438781
L-Glutamic acid Sigma 49449
Lipofectamine 2000 Thermo Fisher Scientific 11668-019
Fura-2 AM Thermo Fisher Scientific F1221
Pluronic F-127 Sigma P2443
mSIRK Millipore 371818
YM-254890 WAKO-Chemicals 257-00631
Pertussis Toxin Thermo Fisher Scientific PHZ1174
Papain Sigma 10108014001
Collagenase/Dispase Sigma 11097113001
4D-NucleofectorTM
X Kit Lonza V4XP-3024
Critical Commercial Assays
TruSeq Nano DNA LT Sample Preparation Kit – Set A Illumina FC-121-4001
TruSeq Nano DNA LT Sample Preparation Kit – Set B Illumina FC-121-4002
Experimental Models: Cell Lines
CHO ATCC CCL-61
COS-7 ATCC CRL-1651
HeLa ATCC CCL-2
Experimental Models: Organisms/Strains
C. elegans: Strain N2 var. Bristol: wild-type Caenorhabditis Genetics
Center
WB Strain: N2
C. elegans: glr-3(tm6403) Shohei Mitani WB Strain: tm6403
C. elegans: xuIs189[Plfe-2::GCaMP3.0+Plfe-2::DsRed] This paper TQ3700
C. elegans: xuIs189[Plfe-2::GCaMP3.0+Plfe-2::DsRed]; glr-3(tm6403) This paper TQ5945
C. elegans: xuIs189[Plfe-2::GCaMP3.0+Plfe-2::DsRed]; glr-3(xu261) This paper TQ4308
C. elegans: xuIs189[Plfe-2::GCaMP3.0+Plfe-2::DsRed]; xuEx2021[Pges-1::glr-
3(gDNA)::SL2::CFP]; glr-3(tm6403)
This paper TQ5946
C. elegans: xuEx2250[Pges-1::glr-3::SL2::CFP] This paper TQ6409
C. elegans: xuEx2021[Pges-1::glr-3(gDNA)::SL2::CFP]; glr-3(tm6403) This paper TQ5947
C. elegans: xuEx3000[Pgcy-5::GCaMP6(f)] This paper TQ8078
C. elegans: xuEx2902[glr-3(7kb)::sl2::YFP] This paper TQ7749
C. elegans: xuEx2994[Pgcy-5::glr-3(cDNA)::sl2::CFP]; glr-3(tm6403) This paper TQ8072
C. elegans: xuEX3000[Pgcy-5::GCaMP6(f)]; xuEx2994[Pgcy-5::glr-3(cDNA)::sl2::CFP];
glr-3(tm6403)
This paper TQ8138
C. elegans: xuEx3144[Pgcy-5::mGluK2::sl2::CFP]; glr-3(tm6403) This paper TQ8700
C. elegans: xuEx2383[Pmyo-3::GCaMP6(f)]; xuEx3143[Pmyo-3::mGluk2::sl2::CFP] This paper TQ8832
(Continued on next page)
Cell 178, 1375–1386.e1–e5, September 5, 2019 e1
Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
C. elegans: xuEx3000[Pgcy-5::GCaMP6(f)]; xuEx3144[Pgcy-5::mGluk2::sl2::CFP]; glr-
3(tm6403)
This paper TQ8869
C. elegans: xuEx3000[Pgcy-5::GCaMP6(f)]; unc-31(e169) This paper TQ9038
C. elegans: xuEx3000[Pgcy-5::GCaMP6(f)]; unc-13(e51) This paper TQ9040
C. elegans: xuEx3000[Pgcy-5::GCaMP6(f)]; eat-4(ky5) This paper TQ9045
C. elegans: xuEx2383[Pmyo-3::GCaMP6(f)]; xuEX3175[Pmyo-3::glr-3(cDNA)::sl2::CFP] This paper TQ9174
C. elegans: xuEx2383[Pmyo-3::GCaMP6(f)]; xuEx3182[Pmyo-3::glr-3(M582R)::sl2::CFP] This paper TQ9175
C. elegans: xuEx2383[Pmyo-3::GCaMP6(f)]; xuEx3184[Pmyo-3::glr-3(Q584R)::sl2::CFP] This paper TQ9177
C. elegans: xuEX3000[Pgcy-5::GCaMP6(f)]; xuEx3186[Pgcy-5::glr-3(M582R)::sl2::CFP];
glr-3(tm6403)
This paper TQ9179
C. elegans: xuEx3000[Pgcy-5::GCaMP6(f)]; xuEx3188[Pgcy-5::glr-3(Q584R)::sl2::CFP];
glr-3(tm6403)
This paper TQ9180
C. elegans: xuEx2383[Pmyo-3::GCaMP6(f)]; xuEx3191[Pmyo-3::mGluk2(Q622R)::
sl2::CFP]
This paper TQ9228
C. elegans: xuEx2383[Pmyo-3::GCaMP6(f)]; xuEx3197[Pmyo-3::mGluk2(M620R)::
sl2::CFP]
This paper TQ9269
C. elegans: xuEx3000[Pgcy-5::GCaMP6(f)]; xuEx3193[Pgcy-5::mGluk2(Q622R)::sl2::
CFP]; glr-3(tm6403)
This paper TQ9227
C. elegans: xuEx3000[Pgcy-5::GCaMP6(f)]; xuEx3200[Pgcy-5::mGluk2(M620R)::sl2::
CFP]; glr-3(tm6403)
This paper TQ9271
C. elegans: xuEx3000[Pgcy-5::GCaMP6(f)]; gpa-3(pk35) This paper TQ9289
C. elegans: xuEx3000[Pgcy-5::GCaMP6(f)]; goa-1(n1134) This paper TQ9290
C. elegans: xuEx3000[Pgcy-5::GCaMP6(f)]; gpa-3(pk35); goa-1(n1134) This paper TQ9291
C. elegans: xuEx3000[Pgcy-5::GCaMP6(f)]; che-2(e1033) This paper TQ9667
C. elegans: xuEx3251[Pgcy-5::goa-1(cDNA)::sl2::mcherry2]; xuEx3000[Pgcy-5::
GCaMP6(f)]; goa-1(n1134)
This paper TQ9625
C. elegans: xuIs259 [Plfe-2::GCamp3.0+Plfe-2::DsRed]; goa-1(n1134) This paper TQ9671
C. elegans: xuIs259 [Plfe-2::GCamp3.0+Plfe-2::DsRed]; glr-3(xu261) This paper TQ9741
C. elegans: xuIs259 [Plfe-2::GCamp3.0+Plfe-2::DsRed]; trpa-1(ok999); glr-3(tm6403) This paper TQ9740
Oligonucleotides
siRNA targeting sequence: GluK2 #1:ACGCAGATTGGTGGCCTTATA This paper N/A
siRNA targeting sequence: GluK2 #2:AAGGTACAATCTTCGACTTAA This paper N/A
siRNA targeting sequence: GluK2 #3:TCGCTTCATGAGCCTAATTAA This paper N/A
siRNA targeting sequence: GluK2 #4:TACAGGCAGAATTACATTTAA This paper N/A
Primer: For qPCR mGluK2: fwd: GCGCAACCATGACGTTTTTCAAG This paper N/A
Primer: For qPCR mGluK2: rev: CCATGGGAGTGCCAACACCATAG This paper N/A
Primer: For glr-3 promoter: fwd: TTAATTCACATTCCATCGGAAAAATC This paper N/A
Primer: For glr-3 promoter: rev: GCCTAGTGACTCGGTCTCTACTCGTA This paper N/A
Recombinant DNA
Plasmid: Pglr-3::sl2::YFP This paper pSX2074
Plasmid: Pgcy-5::glr-3(cDNA):sl2::CFP This paper pSX2101
Plasmid: Pmyo-3::glr-3(cDNA)::sl2::mcherry This paper pSX2120
Plasmid: PcDNA3.1-Flag-glr-3(cDNA) This paper pSX2177
Plasmid: PcDNA3.1+N-DYK-hGluK2 GenScript OHu23233
Plasmid: PcDNA3.1+N-DYK -mGluK2 GenScript OMu13059
Plasmid: PcDNA3.1+N-DYK -fGluK2 GenScript ODa42289
Plasmid: Pmyo-3::mGluK2::sl2::CFP This paper pSX2191
Plasmid: Pgcy-5::mGluK2::sl2::CFP This paper pSX2192
Plasmid: PcDNA3.1-Flag-glr-3(cDNA,M582R) This paper pSX2791
(Continued on next page)
e2 Cell 178, 1375–1386.e1–e5, September 5, 2019
LEAD CONTACT AND MATERIALS AVAILABILITY
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, X.Z.
Shawn Xu (shawnxu@umich.edu). This study has generated plasmids and C. elegans strains, which are listed in the Key Resources
Table. These reagents will be made available upon request.
EXPERIMENTAL MODEL AND SUBJECT DETAILS
C. elegans strains were maintained at 20
C on nematode growth medium (NGM) plates seeded with OP50 bacteria unless otherwise
specified. Transgenic lines were generated by injecting plasmid DNA directly into hermaphrodite gonad. Mutant strains and integrated
transgenic strains were outcrossed at least six times before use.
CHO cells were cultured in DMEM/F12 media with 10% FBS (heat-inactivated) at 37
C under 5% CO2. DMEM media with 10%
FBS (heat-inactivated) were used to culture COS-7 and HeLa cells. These cell lines were obtained from the ATCC. See Key Resources
Table for details.
METHOD DETAILS
Molecular biology and genetics
For the experiments involving transgenes, two to three independent transgenic lines were tested to confirm the results. glr-3 cDNA
was cloned by RT-PCR from total RNA isolated from WT (N2) worms. The expression of the transgene was verified by CFP, YFP or
mCherry expression, which was driven by SL2 from the same transcript. Mouse, human and zebrafish GluK2 cDNA plasmids were
obtained from GenScript (all in the pcDNA3.1+N-DYK vector). See Key Resources Table for plasmid information.
Genetic screen
EMS was used to mutagenize worms carrying a transgene co-expressing GCaMP and DsRed, which enables ratiometric detection of
changes in GCaMP fluorescence by a real-time qPCR thermocycler (ABI 7500). Worms from each F2 plate were washed with M9
buffer and transferred to an unseeded NGM plate to clean off bacteria. Five individual worms from each strain were picked to a
well of a 96-well plate with 20 mL recording buffer (10 mM HEPES at pH 7.4, 5 mM KCl, 145 mM NaCl, 1.2 mM MgCl2, 2.5 mM
CaCl2, 10 mM glucose). PCR plates were loaded onto a real-time qPCR thermocycler and cooled from 23
C to 10
C. Those candidates
that failed to respond to cooling were recovered and outcrossed to the parental strain for six times. Both the parental and
mutant strains were then subjected to whole-genome sequencing (WGS). Analysis of the sequencing data was done as described
(Minevich et al., 2012). By comparing the WGS data between parental and mutant strain, we obtained a density map of single nucle-
Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Plasmid: PcDNA3.1-Flag-glr-3(cDNA,Q584R) This paper pSX2792
Plasmid: PcDNA3.1-Flag-glr-3(cDNA,P121L) This paper pSX2869
Plasmid: PcDNA3.1-Flag-glr-3(cDNA,P130L) This paper pSX2870
Plasmid: Pmyo-3::glr-3(cDNA,M582R)::sl2::mcherry This paper pSX2795
Plasmid: Pmyo-3::glr-3(cDNA,Q584R)::sl2::mcherry This paper pSX2796
Plasmid: Pgcy-5::glr-3(cDNA,M582R):sl2::CFP This paper pSX2793
Plasmid: Pgcy-5::glr-3(cDNA,Q584R):sl2::CFP This paper pSX2794
Plasmid: PcDNA3.1-Flag-mGluK2(M620R) This paper pSX2797
Plasmid: PcDNA3.1-Flag-mGluK2(Q622R) This paper pSX2798
Plasmid: PcDNA3.1-Flag-mGluK2(P151L) This paper pSX2868
Plasmid: Pmyo-3::mGluK2(M620R)::sl2::CFP This paper pSX2802
Plasmid: Pmyo-3::mGluK2(Q622R)::sl2::CFP This paper pSX2803
Plasmid: Pgcy-5::mGluK2(M620R)::sl2::CFP This paper pSX2800
Plasmid: Pgcy-5::mGluK2(Q622R)::sl2::CFP This paper pSX2801
Plasmid: Pgcy-5::goa-1(cDNA)::sl2::CFP This paper pSX2822
Software and Algorithms
MetaFluor Molecular Devices Inc. N/A
GraphPad GraphPad Software, Inc N/A
Cell 178, 1375–1386.e1–e5, September 5, 2019 e3
otide variants and mapped the mutation in xu261 to the gene glr-3, which mutated P121 residue to L. By testing the deletion null
mutant glr-3(tm6403), we found that it exhibited the same cold-sensing phenotype as glr-3(xu261) in the intestine, which was rescued
by transgenic expression of wild-type glr-3 gene in the intestine.
C. elegans calcium imaging and behavioral assays
Calcium imaging was performed on an Olympus IX73 inverted microscope under a 40X objective. Images were acquired using an
ORCA-Flash 4.0 sCMOS camera (Hammatsu Inc.) with MetaFluor software (Molecular Devices Inc.). To image the intestine, ASER
neuron and body-wall muscle cells in response to temperature stimuli, we glued worms on a cover glass covered with a thin layer
of agarose pad. A Bipolar In-line Cooler/Heater (SC-20 from Warner Instruments) was used to control the temperature of the
recording solution, which was perfused toward the worm. Recording buffer: 10 mM HEPES at pH 7.4, 5 mM KCl, 145 mM NaCl,
1.2 mM MgCl2, 2.5 mM CaCl2, 10 mM glucose. The temperature was initially set at 23
C; after achieving a basal line, the temperature
was cooled to 10
C over 120 s, and heated back to 23
C. Faster cooling can be achieved by perfusing pre-cooled (4
C) solution
toward the subject, and similar results were obtained (Figures S2A–S2F); but since this protocol does not permit precise control
of the end temperature, we focused on the use of the former. To image ASER neuron in response to salt gradient, we used a microfluidic
system as described previously (Wang et al., 2016). Briefly, worms were loaded into the chip mounted on the microscope, and
50 to 0 mM salt concentration steps were performed by switching solutions administered to the nose tip (25 mM potassium phosphate
[pH 6.0], 1 mM CaCl2, 1 mM MgSO4, 0.02% gelatin, and either 50 or 0 mM NaCl with glycerol to adjust osmolarity to 350
mOsm). For intestine calcium imaging, we scored the peak percentage change in the ratio of GCaMP/DsRed fluorescence. For
ASER and muscle imaging, we scored the peak percentage change in in the intensity of GCaMP fluorescence.
Cooling-evoked swimming assay was performed in a recording chamber (RC-26 from Warner Instruments) filled with M9 buffer
using day 1 adult hermaphrodites. The temperature was initially set at 21
C, gradually cooled to 18
C over 40 s, and then heated
back to 21
C by slowly perfusing the chamber with M9 buffer of varying temperatures with a Bipolar In-line Cooler/Heater (Warner
Instruments). The number of turns that worms displayed every 10 s was scored.
Cooling-evoked probe assay was performed in an environmentally controlled room set at 21
C and 30% humidity. Day 1 adult
hermaphrodites were tested. Animals were tested on standard NGM plates dried for one hour without lids to remove any excess surface
moisture. Prior to testing, plates were seeded with a thin lawn of fresh OP50 in the center of the plate to prevent animals from
leaving, which was allowed to dry for 10 minutes prior to placing animals on the plate. To deliver localized cooling stimuli to a region of
the worm, we used a custom-built thermoelectric cooling probe with a tip size of 50 mm. The design and fabrication of the cooling
device will be described elsewhere. The probe tip was pre-cooled to 17
C and placed $100 mm above the head of the animal (without
touching the animal) for 5 s using a micromanipulator. An avoidance response was scored if the worm stopped forward movement
and initiated a reversal with at least half a head swing within the 5 s period. Five animals were tested on a single plate. Each animal
was tested only once as we observed C. elegans could quickly adapt to acute cooling stimuli, resulting in a blunted response upon
multiple trials. The response rate for five animals on one plate was averaged and counted as a single trial, and repeated for a total of
10 trials per genotype.
Salt chemotaxis assay was carried out as previously described (Tomioka et al., 2006). Briefly, young adult animals were collected,
washed, and transferred with chemotaxis buffer (25 mM potassium phosphate (pH 6.0), 1 mM CaCl2, 1 mM MgSO4) followed by a
final wash in ddH2O. Test plates were made using 10 cm diameter assay plates (5 mM KPO4, pH 6.0, 1 mM CaCl2, 1mM MgSO4, 2%
agar), on which a salt gradient was formed by placing a 2% agar plug (10 mm diameter) made in chemotaxis buffer containing
100 mM of NaCl. Fifty to two hundred animals were tested for each plate. The chemotaxis index (C.I.) was calculated as described
previously (Tomioka et al., 2006).
Cell culture and calcium imaging
CHO cells were cultured in DMEM/F12 media with 10% FBS (heat-inactivated) at 37
C under 5% CO2. DMEM media with 10% FBS
(heat-inactivated) were used to culture COS-7 and HeLa cells. Cells grown on cover glasses coated with poly-lysine were transfected
in 35 mm dishes using Lipofectamine 2000 (ThermoFisher) for 4 hours. The lipid/DNA ratio was 3:1, and the total DNA was 1.5 mg
except for fish GluK2. For fish GluK2, to maximize its expression level, we used 12 mL lipid and 3 mg DNA for transfection.
18 hours after the transfection, cells were loaded with 4 mM Fura-2 AM and 0.2% Pluronic F127 in HBSS buffer at 37
C for 30 min,
washed for 3 times with HBSS, and incubated in the recording buffer for 30 min before calcium imaging. It is important to use a ratiometric
dye such as Fura-2 to image cold responses, as single wavelength dyes may show intrinsic responses to cold. Recording
buffer: 10 mM HEPES at pH 7.4, 5 mM KCl, 145 mM NaCl, 1.2 mM MgCl2, 2.5 mM CaCl2, 10 mM glucose. Calcium imaging was
performed as described above for C. elegans imaging with the following modification: to obtain better Fura-2 images under 340/
380 nm, we used a 40x water-immersion objective with high transmission efficiency of UV light. The same temperature control protocol
was used as described above for C. elegans imaging. For those experiments involving inhibitors, cells were pretreated with
inhibitors (mSIRK: 50 mM; YM-254890: 10 mM) right after Fura-2 AM loading. For PTX, we pretreated cells with 100 ng/ml of PTX
for 10 hours before the imaging experiment.
e4 Cell 178, 1375–1386.e1–e5, September 5, 2019
Mouse DRG neuron culture, transfection, qPCR, and calcium imaging
DRG culture and siRNA transfection were carried out as described previously (Lou et al., 2013). Briefly, mice were euthanized at the
age of P14-15 by CO2 inhalation using a protocol approved by the Institutional Animal Care and Use Committee at University of Michigan.
DRGs were isolated from T10-L6 and collected in Ca2+
- and Mg2+
-free Hank’s buffered salt solution (HBSS). Following isolation,
DRGs were digested with papain (1.5 mg/ml, MilliporeSigma) for 10 minutes and then collagenase/dispase for 12 minutes (1 mg/ml,
MilliporeSigma) at 37
C. After digestion, the culture was then triturated and dissociated using fire-polished Pasteur pipettes for
further siRNA transfection. To knockdown GluK2 mRNA transcripts, a pool of 4 different siRNA (250 nM, QIAGEN. Target sequences:
ACGCAGATTGGTGGCCTTATA, AAGGTACAATCTTCGACTTAA, TCGCTTCATGAGCCTAATTAA, TACAGGCAGAATTACATTTAA)
was electroporated into the freshly dissociated DRG neurons using P3 Primary Cell 4D-NucleofectorTM
X Kit (V4XP-3024, Lonza).
A scrambled siRNA (250 nM, QIAGEN) was transfected as a control. A MaxGFP construct (1 ml, Lonza) was co-transfected as a
marker.
Two days post-transfection, DRG neurons were harvested, and the total RNA was extracted with TRIzol (Life Technologies) for
quantitative real-time PCR (qPCR) analysis. qPCR reactions were performed in a 384-well format using Power SYBR Green (Thermo
Fisher Scientific). Relative mRNA levels were calculated using the DDCT method and normalized to Tbp, 36B4. Primer sequences:
mGluK2 fwd: GCGCAACCATGACGTTTTTCAAG, mGluK2 rev: CCATGGGAGTGCCAACACCATAG.
Calcium imaging of cultured DRG neurons was carried out two days post-transfection. The imaging protocol was similar to that
described above for CHO cell imaging, except that a 20x rather than 40x water-immersion objective with high transmission efficiency
of UV light was used for imaging. DRG neurons showing R 25% increase in fura-2 F340/F380 fluorescence ratio (DR/R) in response to
temperature stimulation were scored positive.
Mouse DRG in situ hybridization (RNAscope)
PFA-fixed DRG samples from P30 mice was frozen in optimal cutting temperature (OCT) freezing medium and then the DRGs were
used to prepare six adjacent sections at 12 mm thickness. GluK2 mRNA transcript in the DRG sections was detected using RNAscope
assay (Wang et al., 2012a). The probes were designed and provided by Advanced Cell Diagnostics, Inc (Advanced Cell Diagnostics,
Newark, CA). Staining was performed using the RNAscope multiplexed fluorescent in situ hybridization kit, according to manufacturer’s
instructions. For quantification of GluK2-positive population, neurons that had at least four positive signals (observed as
puncta) within the periphery of the neurons, as defined by the phase contrast image, were considered as GluK2-positive. Around
300-450 neurons from each animal were analyzed for GluK2 mRNA transcripts. For categorization of GluK2-positive neuronal population,
the type of the neurons was assigned by measuring their size. Only the neurons containing nuclei were counted. As described
previously (Fang et al., 2005), neurons with a surface area < 400 mm2
, 400-800 mm2
, and > 800 mm2
were categorized as small-diameter,
medium-diameter and large-diameter cells, respectively.
Electrophysiology
Whole-cell patch-clamp recording was performed on an Olympus IX73 inverted microscope under a 40x objective with a Multiclamp
700B amplifier. Transfected cells were identified with a co-transfection RFP marker. Bath solution (in mM): 145 NaCl, 2.5 KCl, 1
CaCl2, 1 MgCl2, 10 glucose, and 5 HEPES (pH adjusted to 7.2). Pipette solution: 115 K-gluconate, 15 KCl, 1 MgCl2, 10 HEPES,
0.25 CaCl2, 20 sucrose, 5 BAPTA, and 5 Na2ATP. Glutamate (10 mM) was diluted in bath solution and perfused toward the cell using
a rapid perfusion system (Bio-Logic) to evoke glutamate-gated currents. Pipette resistance: 2-4 MU. Series resistance and capacitance
were compensated during recording. Voltage was clamped at À70 mV.
QUANTIFICATION AND STATISTICAL ANALYSIS
Quantification and statistical parameters were indicated in the legends of each figure, including the statistical method, error bars
(SEM), n numbers, and p values. We applied ANOVA, t test, and c2
test to determine statistical significance. Specifically, for those
analyses involving multiple group comparisons, we applied one-way ANOVA followed by a post hoc test (Bonferroni test). For those
only involving two groups, we applied t test (Figure 7C) and c2
test (Figure 7F). We considered p values of < 0.05 significant. All analyses
were performed using SPSS Statistics software (IBM, Inc).
DATA AND CODE AVAILABILITY
This study did not generate/analyze datasets or code.
Cell 178, 1375–1386.e1–e5, September 5, 2019 e5
Supplemental Figures
-1
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100 s
WT
Intestine
GCaMP∆R/R(fold)
WT
Intestine
GCaMP∆R/R(fold)
***
glr-3(tm6403) glr-3(xu261)
***
23℃
10℃
glr-3(tm6403)
glr-3(xu261)
A B
Figure S1. Additional Data Regarding Cooling-Evoked Calcium Response in the Intestine, Related to Figure 1
(A and B) Both glr-3(tm6403) and glr-3(xu261) mutant worms showed a severe defect in cooling-evoked calcium response in the intestine. (A) Sample traces. (B)
Bar graph. Error bars: SEM n R 10. ***p < 0.0001 (ANOVA with Bonferroni test).
-0.4
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ASER
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***
***Fura-2∆R/R(fold)
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WT
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GCaMP∆F/F(fold)
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Turningrate(Hz)
21oC
18oC
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glr-3(tm6403); trpa-1(ok999)
trpa-1(ok999) H
***
Probe assay
***
Swimming assay
GCaMP∆F/F(fold)
Figure S2. Rapid Cooling Evokes GLR-3/GluK2-Dependent Calcium Response in C. elegans (ASER Neuron and Muscles) and CHO Cells and
Additional Data on Cold-Avoidance Behavior, Related to Figures 2 and 3
(A–F) Rapid cooling was achieved by perfusing pre-cooled solution toward the worm or CHO cells that express GLR-3/GluK2. We observed cooling-evoked
calcium response similar to that described in the main figures. But since this protocol did not permit precise control of the end temperature from experiment to
experiment (varying from 5-8
C), we focused on the use of the conventional cooling protocol. Error bars: SEM n R 12. ***p < 0.0001 (ANOVA with Bonferroni test).
(G) TRPA-1 is not involved in cold-avoidance behavior in a swimming assay.
(H) glr-3(tm6403) mutant worms are defective in cold-avoidance behavior in a probe assay, which was rescued by an ASER-specific glr-3 transgene. A pre-cooled
probe, which cooled the air temperature near the head of the worm crawling on an agar plate, triggered backward movement (reversals). Error bars: SEM n R 10.
***p < 0.0001 (ANOVA with Bonferroni test).
0
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Fura-2∆R/R(fold)
23℃
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mGluK2
GLR-3
10℃ 100 s
C Hela
HelaD
Fura-2∆R/R(fold)
***
***
***
***
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GLR-3
Fura-2∆R/R(fold)
CHO
23℃
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-Ca2+ +Ca2+
E F
-0.2
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1.6Fura-2∆R/R(fold)
CHO -Ca2+ +Ca2+
***
***
ns ns
Figure S3. Heterologous Expression of GLR-3 and Mouse GluK2 in COS-7 and HeLa Cells Confers Cold Sensitivity, and Cooling-Evoked
Calcium Increase Mediated by GLR-3/GluK2 Primarily Results from Calcium Influx, Related to Figure 3
(A–D) COS-7 cells and HeLa cells were transfected with GLR-3 and mGluK2 or vector control. Cooling evoked calcium response in GLR-3 and mGluK2
transfected cells but not control cells. (A-B) COS-7 cells. Error bars: SEM n R 15. ***p < 0.0001 (ANOVA with Bonferroni test). (C-D) HeLa cells. Error bars: SEM
n R 20. ***p < 0.0001 (ANOVA with Bonferroni test).
(E and F) Cooling-evoked calcium increase mediated by GLR-3/GluK2 primarily results from calcium influx. Cooling evoked no or little calcium response in GLR-3/
GluK2-expressing CHO cells in the absence of extracellular calcium, but evoked robust calcium increase when calcium was present in the bath solution. (E)
Sample traces. (F) Bar graph. Error bars: SEM n R 18. p = 0.601, p = 0.773, ***p < 0.0001 (ANOVA with Bonferroni test).
Figure S4. Cooling to 18
C Evokes Calcium Response in ASER Neuron, Related to Figure 4
(A–C) Sample traces showing that the activation threshold of ASER neuron is around 18
C. Cooling to 23
C (A) and 20
C (B) failed to evoke calcium response in
ASER neuron, while cooling to 18
C did (C). The traces in blue represent calcium traces, while those in black denote temperature changes.
glutamate glutamateMock mGluk2
mGluk2(Q622R)
GLR-3
50ms
500pA
mGluk2(M620R) glutamate
glutamate
glutamate
C D
E F
HG
glutamatemGluk2(P151L)
A B
-SPPTRPSTHLHPHLNA-
-DNKDSFYVSLYPDFSS-
L
121
L
130
GLR-3
141
L
151
mGluK2
119
620 622
-FWFGVGALM(Q/R)QGSE-
-FWFTVCSLM(Q/R)QGSE-GLR-3
mGluK2
R R
R R
582 584
574
612
I
0
500
1000
1500
Currentamplitude(pA)
Figure S5. Glutamate-Gated Current Is Only Detected in Mouse GluK2 but Not the Channel-Dead Mouse GluK2 Variants or GLR-3, Related to
Figures 4 and 5
(A) Sequence alignment of mouse GluK2 and GLR-3 in the M2 pore region. The Q/R RNA editing site is denoted in blue. The residues mutated in this study are
marked in red.
(B) Sequence alignment mouse GluK2 and GLR-3 in the N-terminal ATD domain where point mutations were generated in this study. The residues mutated are
marked in red.
(C–I) Whole-cell recording of glutamate-gated currents of wild-type and various variants of mouse GluK2 as well as GLR-3 expressed in CHO cells. Glutamate:
10 mM. (C-H) Sample traces. (I) Bar graph. n R 4. Error bars: SEM.
23℃
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A B
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Bradykinin
CHO
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Intestine
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Figure S6. Additional Data on G Protein Signaling in CHO Cells and Cooling-Evoked Calcium Response in C. elegans Intestine, Related to
Figure 6
(A and B) Bradykinin-evoked calcium response in CHO cells is sensitive to the Gq/11 inhibitor YM-254890 but not the Gi/o inhibitor PTX. Bradykinin (10 mM)
evoked robust calcium response in CHO cells, which was blocked by pre-incubation with YM-254890 (10 mM) but not by PTX (100 ng/ml). (A) Sample traces. (B)
Bar graph. Error bars: SEM n R 20. ***p < 0.0001 (ANOVA with Bonferroni test).
(C andD) goa-1(n1134) and glr-3(tm6403) mutant worms show a severe defect in cooling-evoked calcium response in the intestine. (C) Sample traces. (D) Bar
graph. Error bars: SEM n R 10. ***p < 0.0001 (ANOVA with Bonferroni test). The WT and glr-3(tm6403) sample traces and data are identical to those in Figure S1
and are included for the purpose of comparison. These experiments were carried out along with glr-3(xu261) mutant worms shown in Figure S1 at the same time.