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SPECIFIC AIMS
The sour taste sensing ability of mammals depends on taste receptors cells (TRC) that
express PKD2L1, an ion channel in the polycystic kidney disease (PKD) family and transient
receptor potential (TRP) superfamily of ion channels. Interestingly, PKD2L1-expressing cells
lining the spinal cord central canal are capable of detecting changes in pH. It has not been
established whether PKD2L1 is, in fact, the mammalian sour taste receptor, yet it is thought that
additional proteins, such as PKD family member PKD1L3, are part of the molecular unit that senses
acids. The goal of this research is to determine how PKD1L3 and novel binding partners of
PKD2L1 that I seek to identify influence sour taste detection in TRCs and pH detection of the
cerebrospinal fluid by central canal cells (CC cells). I hypothesize that the ability of PKD2L1expressing cells to detect sour taste in a subset of TRCs and cerebrospinal pH in CC cells with
varying sensitivity depends on the binding partners of PKD2L1.
Specific Aim 1 To examine expression of PKD1L3 in spinal cord central canal cells and to
identify novel candidate binding partners associated with PKD2L1-mediated acid detection.
The expression of PKD1L3 and other candidate binding proteins of PKD2L1 in spinal cord CC cells
has not been examined. I will determine if PKD1L3 is expressed in PKD2L1-expressing cells
lining the spinal cord central canal by in situ hybridization and immunostaining. If PKD1L3 is not
expressed, this would suggest other proteins may associate with PKD2L1 in cells able to detect
cerebrospinal fluid pH with particular sensitivity. If PKD1L3 is expressed, it is not likely to be
responsible for the pH sensitivity differences between TRC and central canal cells. To isolate
additional candidate binding partners of PKD2L1, I will determine if any PKD family proteins are
expressed in PK2L1-expressing cells lining the central canal by in situ hybridization and
immunostaining. These results will provide preliminary evidence suggesting whether PKD1L3 or
other PKD family members may interact with PKD2L1 in CC cells to form the pH detector.
Specific Aim 2 To determine the ability of novel binding proteins identified in Specific Aim 1
to interact with and confer acid sensitivity to the putative PKD2L1 sour taste receptor and to
test the sufficiency of PKD2L1 and PKD1L3 expression for sour taste detection in TRCs.
First, I will overexpress PKD2L1 and any identified candidate binding partners in 293T cells and
perform co-immunoprecipitation to determine if these binding partners are able to interact with
PKD2L1. Second, I will investigate the ability of co-expressed PKD2L1 and putative binding
partners to form functional pH detectors by recording from transfected 293T cells stimulated with
solutions over a range of pH. Finally, I will test the sufficiency of PKD2L1 and PKD1L3
overexpression in mouse taste receptor cells that are normally unresponsive to acidic stimuli to
respond to sour compounds. Together, these experiments will inform whether PKD2L1 can
function with additional co-receptors and if specific binding partners of PKD2L1 are likely to be
responsible for pH sensitivity differences seen in the TRC and CC cell acid detectors.
Specific Aim 3 To determine the importance of PKD1L3 on sour taste and acid detection in
taste receptors and spinal cord central canal cells, respectively. I will generate mice lacking
Pkd1l3 using standard homologous recombination knock-out techniques to test the necessity of
PKD1L3 for sour taste/acid detection. The effect of PKD1L3 deficiency and any functional
redundancy of PKD1L3 on sour taste/acid detection will be tested with mouse behavioral taste
detection assays and electrophysiological recordings of the chorda tympani and central canal cells
during exposure to acidic solutions.
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BACKGROUND AND SIGNIFICANCE
Organization of mammalian taste reception
Precise taste detection enables animals to preferentially consume energy-containing matter
and avoid harmful substances, thus enhancing survival chances. Relatively little is known about the
gustatory system despite the fact that human beings derive pleasure (or disgust) from it on a daily
basis. While combinations of taste stimuli are limitless, there are five fundamental taste modalities
for the mammalian nervous system: sweet, bitter, salty, umami (monosodium glutamate flavor), and
souri. Specific receptors for three taste modalities (sweet, bitter, umami) have been identified 2-7.
This is consistent with the hypothesis of a labeled-line of gustatory coding in which taste receptor
cells (TRCs) sense and transmit information about a single taste modality that remains segregated
during relay to the brain8. While TRCs are located in all four taste tissue regions, the circumvallate,
foliate, fungiform, and palate papillae, individual taste receptors have variable distribution among
TRCs9. To understand how the nervous system integrates and behaviorally responds to taste stimuli
requires knowledge of the mechanisms directing specificity of the initial step of gustatory signal
transduction, the activation of the TRC by tastants. The identity of the sour taste receptor has
remained elusive, but new candidates are currently being investigated.
Receptors proposed to mediate sour taste detection
A variety of proteins have been proposed to act as the sour taste receptor. These proteins are
all ion channels. Whether these putative receptors are gated by the protons released in acidic
solution or by the anions themselves is not clear, but it is likely to be combination of the two.
Nearly two decades ago, it was suggested that the H-gated calcium channel10 or the inhibition of a
potassium channel11 mediated sour taste detection. Other possibilities include an amiloridesensitive sodium channel12, a hyperpolarization-activated cyclic nucleotide-gated channel13, and a
chloride channel14. Knockout animals are essential to determining whether these channels act as
sour taste receptors endogenously. Several years ago, the putative sour taste receptor, acid-sensing
degenerin 2 (ASIC2) was determined to have no role in sour taste detection showing thatmice
lacking ASIC2 had no defect in sour taste detection15. The most recent advancement in this field is
the identification of PKD2L1 as a candidate sour taste/acid receptor16.
Characteristics of the candidate sour taste/acid receptor PKD2L1
PKD2L1 (or TRPP3) belongs to a family of polycystic kidney disease (PKD) channels,
which is part of the transient receptor potential (TRP) ion channel superfamily17. Murine and
human PKD2L1 contains six transmembrane domains, 2 ER retention signal-like sequences, an EF
hand, and a coiled-coil domain18. PKD2L1 can form a non-selective cation channel when
heterologously expressed with either PKD1 or PKD1L318-20. Mutational analysis has shown that
interactions between PKD2L1 and PKD1 necessary for cell surface expression of these proteins in
293T cells depend on the coiled-coil domain of PKD2L118.
PKD2L1 is widely expressed in mouse tissues, including kidney, heart, brain, muscle, testes,
as determined in an early study21, and in portions of the gustatory system and spinal cord central
canal16. Specifically, PKD2L1 is expressed in a subset of TRCs that do not express known
receptors for sweet, umami, and bitter compounds16. These PKD2L1-expressing TRCs are
necessary for sour taste detection in mice16. This finding and the ability of PKD2L1 to be
stimulated by acidic tastants when expressed in 293T cells suggest that PKD2L1 mediates the acid
detection in these cells. To date, viable Pkd2l1 knockout animals have not been created.
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Analogous to its localization in a subset of TRCS necessary for sour taste detection,
PKD2L1 is also present in cells of the mammalian spinal cord central canal that detect pH in a
narrow range around pH 6.5-6.9 when stimulated in vitro16. CC neurons send processes into the
central canal, supporting the hypothesis that these neurons detect cerebrospinal fluid pH changes, a
role crucial for homeostasis. The PKD2L1-expressing cells in two different sensory contexts
diverge in terms of range and sensitivity to detecting pH. The pH of cerebrospinal fluid must be
controlled precisely in a small range around pH 7.4 to avoid physiological crisis, but the pH
sensitivity by sour taste TRCs is closer to pH 3.0. The sensitivity of the sour taste TRC is likely to
depend on the biochemical properties of the receptor PKD2L1 and its co-receptors, which are
largely unresolved in relevant model systems.
Proteins thought to associate with PKD2L1 to mediate sour taste reception
It is known that some PKD family proteins require associated proteins for proper cell
surface expression and channel formation. Founding members of this family, PKD1 and PKD2,
form functional channels exclusively as a heteromer in CHO cells22. The PKD1 subfamily contrasts
with PKD2 proteins in terms of their large size, long N-terminal extracellular domain, and 11
transmembrane domains containing the 6 transmembrane TRP-like channel domains. PKD1 family
proteins, which include PKD1, PKD1L1, PKD1L2, PKDREJ, and PKD1L3, contain a GPS
cleavage site, as well as C-type lectin and PLAT/H2 domains that are potentially responsible for
interactions with other proteins23. PKD2 proteins are currently thought to act as the ion channel
proper while PKD1 proteins are suggested to have a supporting, yet crucial, role in channel activity.
PKD2L1 and PKD1L3 require coexpression for cell surface localization and function as a sour
taste/acid receptor in 293T cells24. The experiments supporting this have only been performed in
non-taste tissue-derived cell lines and so would benefit from functional assays in cultured taste
receptor cells to confirm relevance to the gustatory systems. This preliminary characterization of
PKD2L1 and PKD1L3 raises intriguing and unanswered questions regarding the role of PKD1L3 in
sour taste detection by PKD2L1-expressing cells.
In addition to in vitro studies of PKD1L3 and PKD2L1 interactions, PKD1L3 has been
localized by in situ hybridization to regions of circumvallate and foliate taste tissue that express
PKD2L116,23,24. Interestingly, the fungiform and palate taste tissue express PKD2L1, but do not
express PKD1L3, suggesting there may be other proteins interacting with PKD2L1 in these regions.
Assessing expression of PKD1L3 and other binding proteins potentially associated with PKD2L1 is
a promising entry into investigating mechanisms of sour taste/acid detection.
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PRELIMINARY STUDIES
In support of my proposed research, I have investigated PKD2L1 as a candidate sour
taste/acid receptor and developed genetic tools that will continue to be useful for future taste
receptor studies.
PKD2L1 and PKD1L3 localization in taste papillae
A bioinformatics-based screen was carried out to identify candidate taste receptors. To
isolate proteins with characteristics of a taste receptor, I searched murine open reading frames
(ORF) for transmembrane domain-containing proteins indicative of potential cell surface
expression. These results were narrowed down by investigating only those ORFs with
corresponding expressed sequence tags exhibiting restricted tissue expression. Ubiquitously
expressed proteins are less likely to serve as taste receptors than those expressed specifically in taste
tissue. To identify those genes with enriched expression in taste tissue, expression levels of the 880
target sequences were analyzed against control tongue epithelium using RT-PCR. In situ
hybridization against cDNAs with greater expression levels than control revealed PKD2L1 as a
candidate taste receptor because of its strong expression in a subset of TRCs.
I raised a specific antibody against PKD21 to determine that the protein is expressed at the
taste pore, which is the appropriate localization for a taste receptor. In situ hybridization against
PKD2L1, PKD1L3, T1R3 (a component of the umami and sweet receptors), T2R (the bitter
receptor), and TRPM5 (the transduction channel for sweet, umami, and bitter detection) showed
that PKD2L1 expression does not overlap with receptors for non-sour taste modalities. PKD1L3 is
co-expressed with PKD2L1 in circumvallate and foliate, but not fungiform and palate, taste tissue.
This indicates that PKD1L3 may function as the sour taste co-receptor for PKD2L1 in two of the
four taste papillae.
Ablation of PKD2L1-expressing TRCs results in loss of sour taste perception
To study the role of PKD2L1-expressing TRCs in sour taste perception, I generated
transgenic mice in which PKD2L1-expressing TRCs are ablated due to attenuated diphtheria toxin
(DTA) expression. Briefly, I placed Cre recombinase under the control of the Pkd2l1 promoter and
crossed these mice with another transgenic mouse in which a floxed inactivation cassette lies
upstream of the DTA coding sequence. Double-positive progeny exhibit DTA expression
exclusively in cells expressing PKD2L1. In control and DTA-expressing cells, the number and
distribution of cells expressing T1R, T2R, and TRPM5 was normal, confirming the specificity of
the PKD2L1-Cre recombinase and DTA constructs. Lack of PKD2L1-expressing cells was seen in
double-positive, but not single-positive, progeny. I tested the functional implications of PKD2L1expressing cell ablation by stimulating mouse taste tissue with compounds perceived as sour, sweet,
umami, bitter, or salty. Citric acid, acetic acid, and hydrochloric acid were used in sour taste assays.
During the 25 seconds following stimulation, averaged responses were recorded from the chorda
tympani, the nerve that innervates taste cells of the tongue. Action potentials from chorda tympani
reflect TRC responses to taste stimulants. This is an established technique in the lab4. Mice lacking
PKD2L1-expressing cells did not respond to sour compounds but had normal responses to the other
four taste modalities, which indicates that cells expressing PKD2L1 are necessary for detection of
sour taste specifically. To confirm the validity of the results seen with PKD2L1-expressing cell
ablation, mice were generated with Cre recombinase under the control of the sweet receptor subunit
T1R2 promoter and crossed with DTA transgenic mice. Action potentials were recorded from the
chorda tympani in response to the sweet stimulants saccharin and acesulfame K. Mice lacking
T1R2-expressing cells did not respond to sweet compounds, but had normal responses to the other
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four taste modalities. Thus, this technique is specific and effective at targeting TRCs with uniquely
expressed proteins.
Expression of PKD2L1 in spinal cord central canal neurons that can detect pH
I investigated PKD2L1 expression in other murine body tissues by in situ hybridization and
immunhistochemistry and found that the only additional location of expression was in neurons
surrounding the central canal along the length of the spinal cord. This result contrasts with an
earlier study showing broader PK2L1 tissue expression21. A possible reason for this discrepancy is
that non-specific probes designed against the consensus sequence of PKD2L1 may have also
recognized family member PKD2 mRNA by Northern blot in this previous study. To determine
whether these PKD2L1-expressing cells could respond to acidic stimulants, I generated a transgenic
mouse in which green fluorescent protein (GFP) expression is controlled by the Pkd2l1 promoter.
This tool enabled us to identify the PKD2L1-expressing cells during electrophysiological
recordings. Patch-clamp recordings of GFP+ and GFP- cells were made in a spinal cord slice
preparation26 at pH 7.4, 6.9, and 6.5. GFP+ cells showed a higher action potential frequency than
GFP- cells when stimulated at 6.5 and 6.9, but not at pH 7.4, the normal pH of cerebrospinal fluid.
Recordings from stimuli below pH 6.5 were not obtained because the spinal cord slices became
damaged in solutions pH < 6.5. The localization of PKD2L1 in central canal neurons and the ability
of these cells to respond to acids near physiological pH of cerebrospinal fluid suggest that PKD2L1
may function as the pH detector for these cells as well as the sour taste receptor in the gustatory
system
Preliminary studies performed by my laboratory on PKD2L1 provide intriguing evidence to
suggest that this protein is the sour taste receptor in TRCs and the pH detector of CC neurons.
Furthermore, colocalization of PKD1L3 with PKD2L1 in the circumvallate and foliate papillae
determined by in situ hybridization, along with binding studies23, indicate that PKD1L3 is likely to
play an important, if not indispensable, role in sour taste detection. The identities of additional
PKD2L binding partners are worth pursuing, given the lack of PKD1L3 expression in fungiform
and palate papillae that express PKD2L1. Finally, the exquisitely sensitive pH detecting ability of
PKD2L1-expressing CC cells indicate that PKD2L1 may be a conserved protein involved in two
separate acid-sensing systems. My laboratory has been a pioneer during the recent rapid advances
in gustatory research and is well equipped with state-of-the-art methods and reagents to continue
investigating crucial questions in taste biology such as the protein(s) responsible for sour taste/acid
detection.
Genetic tools to study sour taste/acid detection in vitro
The study of molecular mechanisms of sour taste/acid reception would be greatly advanced
by in vitro genetic manipulation of TRCs and central canal cells followed by electrophysiological
recordings of responses to acidic stimulants. Taste receptor cells are more challenging to maintain
and transfect in vitro in comparison to other mammalian primary cell types. Methods to culture
functional rat taste receptor cells from which recordings can be made have been recently
developed27,28. In addition, both DNA introduction by adenovirus in rat and by liposome-mediated
plasmid transfection into rat and mouse taste receptors cells has shown between 50-90%
efficacy28,29. These tools would allow overexpression of mutant forms of taste receptors and their
binding proteins in cells more appropriate for taste reception study. Cell lines, such as HEK 293T,
may not have the machinery necessary for physiologically-relevant protein expression and stimulus
responses.
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EXPERIMENTAL PLAN
Specific Aim 1 To examine expression of PKD1L3 in spinal cord central canal cells and to
identify novel candidate binding partners associated with PKD2L1-mediated acid detection.
Aim 1.1
Rationale The expression of PKD1L3 in circumvallate and fungiform papillae, but not
in foliate and palate papillae suggests the existence of additional PKD2L1-binding proteins in sour
taste/acid sensing cells that do not express PKD1L3. I hypothesize that the candidate sour taste
receptor PKD2L1 requires a co-receptor(s) to detect sour taste in the gustatory system and pH in
cerebrospinal fluid, and that the specific co-receptor(s) is responsible for the differences in
sensitivities between the two acid-sensing systems.
Experimental Design The expression of PKD1L3 in neurons lining the spinal cord
central canal will be assessed to establish the possibility that PKD1L3 acts as a pH detecting coreceptor with PKD2L1 in the spinal cord CC. PKD1L3 expression will be tested with three
complementary methods. First, in situ hybridization against PKD1L3 will be carried out in mouse
spinal cord coronal sections across the length of the spinal cord from brain stem to the cauda equina
terminus. Second, fluorescent immuohistochemistry with anti-PKD1L3 antibodies will be used to
confirm in situ results. Finally, co-expression with PKD2L1 will be assessed using fluorescent
immunhistochemistry. Negative control staining with sense RNA probes or no primary antibody
will be used to exclude non-specific labeling.
Potential Results Based on the hypothesis that unidentified PKD2L1-binding proteins
in the CC provide the unique sensitivity of the pH-sensing neurons compared with the
PKD1L3/PKD2L1 co-expressing TRCs, I expect that PKD1L3 will not be co-expressed with
PKD2L1 in CC cells. If no signal is detected, this result will be confirmed with positive control in
situ hybridization and immunostaining against PKD1L3 in circumvallate and fungiform papillae
already shown to express PKD1L316. If PKD1L3 expression is detected in CC neurons, but does
not co-localize with PKD2L1, then this suggests that either 1) PKD1L3 has no function to detect pH
in central canal cells if PKD2L1 is the pH detector 2) PKD1L3 could be a pH receptor by itself or in
combination with another protein or 3) PKD1L3 has a non-pH detecting function in the spinal cord.
Furthermore, this would support the hypothesis that other binding proteins exist for PKD2L1. If
PKD1L3 is co-expressed with PKD2L1, then this would suggest that PKD1L3 is not the source of
differences in acid-detecting sensitivity between TRCs and CC cells.
Limitations/Alternatives If immunostaining and in situ hybridization fail to show
presence or absence of PKD1L3 in PKD2L1-expressing regions, reverse-transcriptase PCR (RTPCR) can be used to determine the mRNA expression of PKD1L3. To isolate the CC cells that
express PKD2L1, I will use the transgenic Pkd2l1 promoter-driven GFP mouse16. Spinal cord from
these mice will be extracted and GFP+ and GFP- cells isolated by FACS will be tested for PKD1L3
expression with RT-PCR. This transgenic mouse can also be used for immunohistochemistry to
assess PKD1L3 signal in GFP+ (PKD2L1-expressing) cells.
Aim 1.2
Rationale The purpose of this experiment is to determine whether other PKD family
members are co-localized with PKD2L1 in CC neurons as a first step to identify potential pH
detection co-receptors of PKD2L1. The choice of these candidate proteins is based on studies
showing PKD1 and PKD2 heteromers are necessary for functional channel expression at the cell
surface22. Other PKD1 family proteins are likely to contain domains necessary for binding
interactions with PKD2L1. Furthermore, the finding that these proteins are not expressed in TRCs,
suggests that they could have a CC-specific acid-sensing role23. As discussed in Aim 1.1 Potential
Results, the utility of this assay is not dependent on the result of PKD1L3 expression studies.
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Experimental Design Expression of PKD1 family proteins PKD1, PKD1L1, PKD1L2,
and PKDREJ will be tested in CC cells using in situ hybridization and/or immunostaining in
PKD2L1-expressing CC cells with methods described in Aim 1.1. Any PKD1 proteins that are
found to co-localize with PKD2L1 will be further tested for expression with RT-PCR in GFP+ cells
from the CC of PKD2L1 promoter-GFP mice.
Potential Results All, a select few, or none of the candidates tested may co-localize
with PKD2L1 in CC cells. Any candidates that co-localize may be involved with a PKD2L1
complex that senses cerebrospinal fluid pH and will be investigated in Specific Aim 2. If none of
the candidates are detected, this result will be confirmed with positive control in situ hybridization
and immunostaining against tissues in which expression has already been determined, such as the
kidney for PKD1.
Limitations/Alternatives If no candidate binding proteins are expressed in the CC
cells, I will uncover novel candidates that are differentially expressed in PKD2L1-expressing TRCs
and PKD2L1-expressing CC cells using microarray analysis. GFP+ (PKD2L1-expressing) cells will
be FACS-purified from extracted taste tissue and central canal spinal cord tissue of Pkd2l1
promoter-GFP transgenic mice. These two PKD2L1-expressing cell populations will be used for
microarray studies. Gene expression data from GFP- taste tissue and GFP- central canal cells will
be compared to their respective GFP+ tissue counterparts as a method of subtracting gene
expression data that are due to properties not associated with acid sensing. Candidate genes for
PKD2L binding proteins will be selected based on 1) expression levels in GFP+ CC that are similar
to PKD2L1 in GFP+ CC cells 2) lack of or very low expression in GFP+ TRCs and 3) putative
transmembrane domains.
Specific Aim 2 To determine the ability of novel binding proteins identified in Specific Aim 1
to interact with and confer acid sensitivity to the putative PKD2L1 sour taste/acid receptor
and to test the sufficiency of PKD2L1 and PKD1L3 expression for sour taste detection in
TRCs
Aim 2.1
Rationale Candidate proteins that are co-expressed with PKD2L1 in CC cells may
interact with PKD2L1 to form an acid receptor. PKD1L3 has been shown to co-immunoprecipitate
with PKD2L1 and form a functional acid-responsive receptor when overexpressed in 293T cells.
This experiment will test the binding ability of candidate binding partners identified in Specific Aim
1 to interact with PKD2L1 in a similar system.
Experimental Design I will overexpress PKD2L1 and candidate partners in 293T cells
by co-transfecting plasmids containing the wild-type cDNA sequence of Pkd2l1 or candidate
partners. HEK 293T cells are the optimal cell type for this co-immunoprecipitation assay because
they are easily transfected and provide a substantial amount of protein. The candidate partner
cDNA will be tagged with HA at the N-terminal and the PKD2L1 cDNA will be Flag-tagged for
immunoprecipitation. Cell lysates will be collected for protein and will be precipitated with either
HA or FLAG antibodies, followed by western blot analysis with HA and FLAG antibodies to test if
overexpressed PKD2L1 and the putative binding partner interact with each other. Coimmunoprecipitation with HA-PKD1L3 and FLAG-PKD2L1 will serve as a positive control as this
has been published.
Potential Results If a candidate partner co-immunoprecipitates with PKD2L1 when cotransfected into 293T cells, this would provide evidence that these two proteins can bind in
eukaryotic cells and merit further investigation to build on this correlation. If no candidate partners
co-immunoprecipitate with PKD2L1, this would suggest that these two proteins do not interact
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when expressed at these levels without the presence of additional protein partners and may not act
as the acid co-receptor in CC cells.
Limitations/Alternatives One limitation of this assay is the use of 293T cells as a model
system instead of TRCs/CC neurons. While this system works effectively to test putative protein
interactions, it would also be possible to test potential interactions using a yeast two-hybrid system.
In this experiment, I would ligate cDNA of WT Pkd2l1 (the “bait”) to the GAL4 DNA-binding
domain sequence and ligate cDNA of a WT candidate partner(s) (the “prey”) to the transcriptional
activation domain sequence. These vectors would be introduced into yeast with a reporter gene
controlled by a UAS promoter to which GAL4 binds. Positive yeast colonies in which the reporter
gene is expressed would indicate binding interactions between PKD2L1 and the candidate partner.
Despite the limitations of these cell models, any interactions will be further investigated in Aim 2.3
using primary cultures of mouse TRCs.
Aim 2.2 Rationale This experiment will test the ability of binding partners identified in Aim 2.1
to provide responses to solutions over a pH range when co-expressed with PKD2L1 in 293T cells.
The purpose is to contrast the pH response range of these binding partners with the response range
of 293T cells transfected with wild-type Pkd2l1 and Pkd1l3, shown to be around pH 3.024. This is
fueled by the hypothesis that PKD2L1-expressing CC neurons are sensitive to pH changes around
6.5-7.4 because of specific binding partners that differ from those in TRCs. I hypothesize that
candidate binding proteins identified in CC cells will confer a pH sensitivity around pH 6.5 when
overexpressed with PKD2L1 in 293T cells.
Experimental Design As in the above-described experiments, I will co-transfect 293T
cells with wild-type Pkd2l1 and wild-type binding partner gene constructs, in order to specifically
test if different binding partners confer sensitivities to different pH values. The vector with Pkd2l1
cDNA will contain an IRES-GFP sequence while the vector with the candidate binding partner
cDNA will contain an IRES-DsRED sequence so cells expressing both gene constructs should emit
both green and red fluorescence. Electrophysiological recordings with voltage-clamp whole cell
methods (at -60 mV) will be conducted 36 hours after transfection. A total of 10-15 cells will be
stimulated over a range of pH (2.8 to 7.4) using different concentrations of citric acid or HCl. All
whole cell currents will be measured in the presence of 100 uM amiloride to eliminate any
depolarizing effect of amiloride-sensitive Na+ channels. To ensure that observed responses are due
exclusively to the two overexpressed genes, responses from stimulated 293T cells that are either
mock-transfected or transfected with a single gene construct will be recorded.
Potential Results If transfected 293T cells do not depolarize in response to a range of
acidic stimulants, this would suggest that PKD2L1 and the candidate partner do not form a sour
taste receptor. If depolarization is seen at low pH (3.0), this would suggest that this
PKD2L1/candidate partner complex are not likely to serve as the pH detector in CC cells, which
depolarize at pH 6.5 and 6.9. If depolarization is seen close to pH 6.5, this would provide evidence
that this PKD2L1/candidate partner complex may serve as the pH detector in CC cells.
Limitations/Alternatives An alternative experimental technique that provides data on
populations of cells is calcium dye-based imaging of cellular responses to acids. Transfected cells
would be subsequently loaded with Fluo-4 and Fura-red. Fluo-4/Fura-red ratios representing
depolarization would be calculated for a field of cells before and after stimulation with solutions of
different pH as described as above. Constructs would be made without DsRed or EGFP to prevent
interference with emissions from calcium dyes. A limitation of this experiment is its dependence on
the identification of novel binding partners of PKD2L1 in Specific Aim 1. If none are identified, I
will focus on an equally interesting question of what properties of PKD1L3 confer sensitivity to the
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PKD2L1-PKD1L3 receptor complex by performing identical experiments with PKD1L3 domain
mutants.
Aim 2.3 Rationale This experiment answers the question of whether PKD2L1 and PKD1L3 are
sufficient for sour taste reception in cultured mouse TRCs, a more relevant system than 293T
studies.
Experimental Design Taste tissue from previously generated transgenic mice
expressing GFP under the Pkd2l1-promoter will be harvested and grown in culture. The culture will
contain a mixture of GFP+ (PKD2L1-expressing) and GFP- (non-PKD2L1-expressing) cells. I will
confirm that these cultured GFP- cells express other taste receptors such as T1R3 or T2R by
immunocytochemistry. As shown in my preliminary studies, ablation of PKD2L1-expressing cells
eliminates all sour taste response in mice. Thus, all GFP- TRCs in the culture will not be
responsive to sour stimuli under normal conditions. Wild-type Pkd2l1 and wild-type Pkd1l3-IRESDsRed will be individually cloned into puromycin-resistance gene vectors. Cultured mouse TRCs
will be transfected with these two constructs using liposomes29, followed by application of
puromycin for selection purposes. The cells that survive puromycin and are GFP- DsRed+ will
express exogenous PKD2L1 and PKD1L3. During stimulation with acids (as described in Aim 2),
electrophysiological recordings will be taken from these cells, as well as negative control GFP- and
positive control GFP+ cells from a separate, untransfected culture. To assess the validity of this
technique in detecting taste-specific stimulation of TRCs, I will also record from untransfected
GFP- and GFP+ cells that are exposed to either acidic (citric acid, acetic acid, hydrochloric acid),
sweet (saccharin, acesulfame K), umami (glutamate), bitter (quinine), and salty (sodium choloride)
compounds.
Potential Results If no action potentials are recorded from transfected mouse TRCs, this
would suggest that the PKD2L1and PKD1L3 do not act as sour taste receptor. If action potentials
are recorded from GFP- DsRed+ mouse TRCs when stimulated with solutions of pH 2.0-3.0 (acetic
acid, citric acid, HCl), this would confirm that expression of PKD2L1 and PKD1L3 is sufficient to
confer sour taste detection in mouse TRCs not normally responsive to sour tastes. If GFP- DsRed(expressing exogenous PKD2L1 but not PKD1L3) is stimulated by sour stimuli, this would imply
that PKD1L3 is not a necessary component of the sour taste receptor. It is expected that GFPDsRed+ mouse TRCs will respond to non-sour taste modalities because GFP- TRCs should not
express PKD2L1 endogenously and so are likely to have cellular machinery to respond to at least
one non-sour taste stimulus.
Limitations/Alternatives A limitation of this study is the inability to rule out whether a
failure to observe sour taste responses in GFP- DsREd+ cells is because PKD2L1/PKD1L3 is not
the sour taste receptor or because of restrictions of the GFP- cell characteristics. GFP- TRCs do not
express PKD2L1 and are likely to detect one of the other four taste modalities under normal
circumstances. Thus, these cells may not possess the additional molecular components required for
sour taste detection or may possess inhibiting molecules that prevent proper function of
PKD2L1/PKD1L3. In the event of such a negative result, these experiments may be attempted in
cultured rat TRCs, which are thought to express a non-functional PKD2L1 protein and do not
express PKD1L3.
Aim 3 To determine the importance of PKD1L3 on sour taste and acid detection in taste
receptors and spinal cord central canal cells, respectively.
Rationale The hypothesis that PKD1L3 is necessary for sour taste detection, but
dispensable for CC cell-mediated acid detection, can best be addressed by creating a Pkd1l3
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knockout mouse and determining sour taste/acid detection phenotypes of this transgenic mouse.
Since PKD1L3 is expressed in TRCs, the result of testing its expression in CC cells (Specific Aim
1) does not affect the necessity of creating a Pkd1l3 knockout mouse to investigate effects of
Pkd1l3 deficiency and any functional redundancy of PKD1L3 on sour taste detection.
Experimental Design I will use standard homologous recombination knockout techniques
to create a PKD1L3-deficient mouse. Figure 1 illustrates the components of the targeting vector.
Of the 60.3 Kb of mouse Pkd1l3 genomic sequence, Exon 1 (307 bp beginning with ATG start
codon) will be replaced with the neomycin-resistance gene to disrupt the Pkd1l3 gene. This vector
will be introduced into 129SV agouti embryonic stem cells (ES) and positive clones will be selected
for with neomycin. Gancyclovir will be applied to the ES cells to kill those cells containing any
randomly inserted vector that has not recombined homologously with the endogenous gene to
eliminate thymidine kinase. Clones surviving both neo/gancyclovir treatment will be implanted into
a C57Bl/6 black pseudo-pregnant mother. When chimeric offspring are produced, they will be
assessed for germ-line transmission by crossing the chimeras with wild-type mice. After
heterozygotes are backcrossed five generations into 129SV background, I will cross PKD1L3+/animals produce knockout and wild-type littermates for phenotypic comparison. The lack of
PKD1L3 expression will be tested by immunohistochemistry and in situ hybridization against
PKD1L3 in tissues of post-natal and adult mice. I will also perform co-staining against PKD2L1 on
taste tissue and spinal cord CC tissue to determine if PKD1L3 deficiency affects PKD2L1 cellular
localization as suggested by previous studies24.
Exon 1 (307 bp)
Pkd1l3 gene on Chromosome 8
Neo-resistance
gene
thymidine kinase
Gene disruption targeting construct
3 Kb of homology
3 Kb of homology
Figure 1: PKD1L3 knock-out targeting vector
construct (Not drawn to scale)
To test defects in sour taste detection, two assays will be performed once mice have matured
to 8 weeks of age. First, a taste preference test will be performed on wild-type and knock-out mice
to determine whether loss of PKD1L3 affects the detection of acidic solutions. Individual mice will
be exposed to a bottle of water pH 7.4 and a bottle of 50 mM citric acid solution (pH 2.3) and the
volume of liquid consumed from each bottle over 48 hrs will be an indicator of taste preference. I
will switch the bottle position after 24 hrs to control for position preference. Knockout mouse taste
preferences will be compared with taste preferences of wild-type mice, which prefer neutral water
to acidic water. Because defects in odor detection could confound this taste preference assay, mice
will be examined for impairments in odor detection by observing responses to odors that are
naturally aversive or attractive. A failure to avoid an aversive odor, such as toluquinone, would
indicate Pkd1l3 knockout mice may have odor detection defects. A more quantitative method of
testing sour taste abilities will be to measure action potentials from the chorda tympani when taste
tissue is stimulated with acidic (citric acid, acetic acid, hydrochloric acid), sweet (saccharin,
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acesulfame K), umami (glutamate), bitter (quinine), and salty compounds (sodium chloride).
Anesthetized adult knockout mice will be placed in a head-holder for stabilization. I will dissect out
the intact chorda tympani and attach a recording electrode. Solutions would be presented to taste
papillae over a period of 20 s at a constant flow rate of 4 ml/min, with two-minute rinses of water
following each taste trial. Responses will be integrated over 25 seconds following the stimulus to
allow for potential delayed responses and to ensure sufficient data collection.
If PKD1L3 is expressed in CC cells (Specific Aim 1), I will measure responses to
application of solutions from three different pH values (6.5, 6.9, 7.4) in CC cells spinal cord slices
from 8 week old wild-type and knock-out mouse using a cell-attached patch-clamp technique. I will
record from all cells surrounding the central canal (approximately 30 cells) from 5 different slices
along spinal cord. This should provide a sufficiently high probability of recording from PKD2L1expressing cells that were previously shown to detect pH changes in order to determine with
confidence whether loss of PKD1L3 affects pH sensing in the central canal.
Potential Results If knockout mice drink equivalent amounts of citric acid and water, and
WT mice drink lower amounts of citric acid than water, this would suggest that PKD1L3 deficiency
impairs sour taste detection. Alternatively, if knockout mice drink less citric acid solution than
water as is expected for the wild-type, this would suggest that PKD1L3 deficiency does not impair
sour taste detection. If genetic compensation occurs in the knockout animal, it may not be clear
whether PKD1L3 is involved in with the sour taste receptor complex.
For chorda tympani recordings, if no action potentials are recorded when taste tissue is
stimulated with acidic solutions, this would be evidence that PKD1L3 is necessary to detect the
presence of sour compounds. Conversely, normal responses to acid stimulation in comparison to
WT would indicate that PKD1L3 deficiency has not effect on sour taste detection. The validity of
these results will be confirmed with control stimulation of wild-type mouse to ensure that
recordings can be made from the chorda tympani in response to sour taste stimulation. Even if
PKD1L3 acts with PKD2L1 to sense acids, it is possible that a response may be detected if cells that
express PKD2L1 but not PKD1L3 are capable of sensing acids. There is some evidence for this,
including the calcium imaging-based finding that 23-25% of TRCs in slices respond to citric acid15
while only 20% of TRCs express both PKD2L1 and PKD1L324. Thus, by applying the tastants to
specific regions of taste tissue known to express (circumvallate and foliate) or not express
(fungiform and palate) PKD1L3, the requirement of PKD1L3 for sour taste detection can be
differentiated based on papillae.
If the hypothesis that PKD1L3 does not mediate pH detection in PKD2L1-expressing CC
cells is correct, the loss of PKD1L3 should not eliminate the wild-type responses to pH 6.5-6.9. If
PKD1L3 must be expressed for CC cells to respond to pH changes, it is likely that the knockout will
be lethal due to inability to maintain cerebrospinal fluid at pH 7.4.
Limitations/Alternatives This experiment is dependent on the survival of the Pkd1l3
knock-out mouse. If PKD1L3 is expressed in CC cells and is essential for cerebrospinal fluid pH
detection, the knockout may be lethal. Alternatively, lethality may result because PKD1L3 is
indispensable in an undetermined cell type. The lethality would point to an intriguing role for
PKD1L3 necessary for organism survival, but it would preclude any study of PKD1L3 as a putative
component of the sour taste receptor. To address this compelling question, an RNA interferencebased in vitro technique could be employed to knock down Pkd1l3 mRNA transcripts. First, I will
create a transgenic mouse in which GFP is driven by the Pkd1l3 promoter (as described in previous
studies) to aid the identification of PKD1L3+ cells in subsequent experiments. PKD1L3-expressing
circumvallate and fungiform papillae from these Pkd1l3 promoter-GFP mice will be extracted and
cultivated. Lentivirus that contains short hairpin RNA (shRNA) against mouse Pkd1l3 mRNA will
be generated in 293T cells and used to infect cultured mouse TRCs. The benefit of using lentiviral
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shRNA delivery is that transduction of genetic material can occur in post-mitotic cells that include
Type II elongate epithelial TRCs that expressing taste receptors. The lentiviral construct would
contain an IRES sequence between the shRNA coding sequence and the DsRED sequence in order
to determine which cultured cells were infected and stably expressing shRNA. Expression of both
GFP and DsREd would indicate a cell with levels of Pkd1l3 mRNA significantly lower than
transcript levels of GFP+ DsRED- cells. Electrophysiological recordings of infected cells would be
taken in the presence of sour taste stimulation as described in Specific Aim 2 to determine how
significantly decreased levels of PKD1L3 affect responses to sour tastes.
CONCLUSION
The perception of five distinct taste modalities allows mammals to distinguish food from
toxin. The interest in gustation has fueled research to identify how sweet, bitter, umami, salty, and
sour compounds stimulate taste cells and how these signals are transduced to the brain. Despite
intense investigation, elements of the sour taste detector remain to be characterized. In light of the
great interest in PKD2L1 as a candidate sour taste receptor and cerebrospinal pH detector, an
indispensable component of sour taste receptor studies would be the investigation of PKD1L3 and
other proteins associated with the PKD2L1-receptor complex that are likely to be involved in sour
taste/acid detection. The different pH sensitivity of PKD2L1-expressing cells in taste tissue and CC
cells motivates this examination of PKD1L3 as a putative sour taste co-receptor of PKD2L1 and to
identify additional candidate binding proteins that may interact with PKD2L1 in CC cells.
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