underlying this targeting and intracellular trafficking of transient receptor potential (TRP) channels remain poorly understood, and
identifying proteins involved in these processes will provide insight into underlying mechanisms. Vision is dependent on the normal
Being in the right place at the right time is fundamental for sig-
naling proteins. Though the importance of regulating the spatial
distribution of signaling proteins is well recognized, the proteins
mediating this coordinated process remain largely unknown.
This is especially true for the transient receptor potential (TRP)
superfamily of ion channels. Although a few TRP channels have
been shown to interact with cellular components involved in
trafficking, such as cytoskeletal or vesicular proteins, specific ac-
cessory proteins required for TRP channel cellular localization
have not been identified.
TRP melastatin 1 (TRPM1) is the initial member of the
melastatin TRP subfamily and was identified in a screen analyz-
ing mRNA levels in malignant versus benign melanocytes (Dun-
largely intracellular role, possibly regulating melanin produc-
tion (Oancea et al., 2009; Patel and Docampo, 2009). Recently,
driven depolarizing bipolar cell (DBC) responses (Koike et al.,
The question of whether TRPs have intrinsic targeting infor-
mation encoded in their sequence or whether their residence in
the plasma membrane is mediated by extrinsic proteins remains
open. The dichotomy of TRPM1 channel localization in the in-
tracellular membranes of melanocytes versus dendritic tips of
likely influencing TRPM1 localization and, ultimately, function.
With this in mind, a protein expressed exclusively on the DBC
dendritic tips and critical for generating a TRPM1 response is a
strong candidate for regulating TRPM1 targeting to the plasma
Retinal DBCs detect changes in photoreceptor glutamate re-
lease via metabotropic glutamate receptor 6 (mGluR6). Gluta-
mate activates mGluR6 in the dendrites, which results in closure
of TRPM1. Mutations in either mGluR6 or TRPM1 in humans
result in congenital stationary night blindness (Audo et al., 2009;
Li et al., 2009; van Genderen et al., 2009; Nakamura et al., 2010).
In mice, primates, and humans, TRPM1 is localized in the den-
dritic tips of DBCs (Morgans et al., 2009; van Genderen et al.,
2009; Koike et al., 2010), and some mutations prevent TRPM1
gene are linked to night blindness in humans and mice (Bech-
J. N. Pearring’s present address: Duke University Eye Center, AERI, Room 5000, 2351 Erwin Road, Box 3802,
10060 • TheJournalofNeuroscience,July6,2011 • 31(27):10060–10066
Hansen et al., 2000; Gregg et al., 2003). In a nyctalopin mutant
mouse, Nyxnob, the DBCs do not have a glutamate response,
which is believed to be due to either malfunction of the TRPM1
channel or its absence from the dendrites (Gregg et al., 2007).
In this study, we used genetic, biochemical, and immunohis-
tochemical approaches to show that nyctalopin directly interacts
TRPM1 from the dendrites of DBCs. These data indicate that
tips of DBCs.
Animals. All experiments were performed using protocols approved by
the Animal Care and Use Committee at each institution, and the guide-
lines of the National Institutes of Health and the Society for Neurosci-
ence. Mice of either sex were used in experiments. The Nyxnobmice,
originally discovered on the BALBc/ByJ background (Pardue et al.,
1998), were backcrossed onto the C57BL/6J background for more than
seven generations. Controls were either littermates or age-matched
C57BL/6J mice (The Jackson Laboratory). Trpm1?/?mice were gener-
Mouse Mutant Archive. Molecular details of the targeted allele are avail-
able at http://www.emmanet.org/. Transgenic mice expressing a yellow
fluorescence protein (YFP)-nyctalopin in retinal bipolar cells were gen-
erated as previously described (Gregg et al., 2007) and will be referred to
as TgEYFP-NYX mice. Mice were housed in a 12 h light/dark cycle and
killed by anesthetic overdose or carbon dioxide exposure.
Plasmid construction. All cloning used the infusion cloning system
C57BL/6J retinas. A flag-tag (5?-GACTACAAGG ACGACGAC-3?) fol-
cDNA. For expression of a tagged nyctalopin, Strep-tag II (5?-
TGGAGCCACCCGCAGTTCGAAAAG-3?) was fused to enhanced YFP
(EYFP) and both were inserted after the codon for amino acid 19 of the
nyctalopin cDNA. Both fusion constructs, Flag-GFP-TRPM1 (FG-
the EcoR1 site of pcDNA3.1? (Invitrogen).
Membrane yeast two-hybrid. Full-length Nyx cDNA was cloned into
the pCCW-SUC bait vector (Dualsystems Biotech), such that the
fused to either the N or C terminus to create Cub-NYX and NYX-Cub,
respectively. Trpm1 cDNA was cloned into the pDLS-Nx prey vector.
Interactions were tested by cotransformation of bait and prey vectors
into NYM32 yeast. Incorporation of both plasmids was tested by growth
ble dropout), and interaction by growth on
plates lacking leucine, tryptophan, histidine,
and adenine (quadruple dropout). Interac-
tions were confirmed by using a colony lift as-
say that tests for expression of ?-galactosidase
and was performed as previously described
(Iyer et al., 2005).
down. HEK293T (human embryonic kidney
cells; ATCC) cells were grown in high-glucose
DMEM supplemented with 10% fetal bovine
serum, L-glutamine (2 mM), penicillin (50 IU/
ml), and streptomycin (Strep) (50 ?g/ml).
Two 10 cm plates were transiently transfected
using the CalPhos Mammalian Transfection
Kit (Clontech). Cells were harvested by soni-
cating in 400 ?l of NP-T lysis buffer (50 mM
to 8.0 with 1 M NaOH). Homogenate was
at 4°C. Forty microliters of total lysate was
saved, while the remainder was incubated with
buffer before proteins were eluted with 10 mM D-Biotin (Sigma-Aldrich)
in NP-T Lysis buffer. SDS sample buffer (62 mM Tris, 10% glycerol, 2%
SDS, and 5% ?-mercaptoethanol) was added, and samples were incu-
bated for 10 min at 70°C before Western blotting.
Western blotting. Protein fractions were analyzed on 4–12% NuPAGE
gels (Invitrogen), and proteins were transferred onto PVDF membranes
(GE Healthcare). Immunoblot analysis was performed using primary
antibodies; anti-GFP (1:2000, Cell Signaling Technology), anti-Flag (1:
2000, Sigma-Aldrich), and anti-TRPM1 (1:500) (Koike et al., 2010).
Anti-mouse (1:100,000, Sigma-Aldrich) and anti-rabbit (1:200,000,
with the advanced ECL detection system (Pierce).
mounts, and immunohistochemistry were performed as described pre-
anti-GFP conjugated to Alexa-488 (1:1000, Invitrogen), anti-ctbp2/rib-
eye (1:1000, BD Biosciences Pharmingen), anti-Na?/K?-ATPase (1:
300, Santa Cruz Biotechnology), and anti-TRPM1 (1:100) (Koike et al.,
2010). Secondary antibodies were Alexa-488 goat anti-rabbit, Alexa-555
goat anti-mouse, and Alexa-643-conjugated PNA (Invitrogen), all used
at the 1:1000 dilution. Images were collected using the Olympus FV300
confocal microscope with 60? oil-objective (1.45 numerical aperture).
Images shown are maximum projections of confocal stacks, adjusted for
contrast and brightness with Fluoview software.
?M), strychnine (10 ?M), and (1,2,5,6-tetrahydropyridin-4-yl) methyl-
itory conductances. The metabotropic receptor antagonist LY341495
(Tocris Bioscience) or TRP channel agonist capsaicin (Sigma-Aldrich)
were delivered to the retina from a pipette using positive pressure (2–4
psi) with a computer-controlled solenoid valve (Picospritzer, General
Valve Corp), and the mGluR6 agonist L-AP4 (4 ?M, Tocris Bioscience)
described (Snellman and Nawy, 2004; Shen et al., 2009). Data were ana-
lyzed off-line with Axograph X and Kaleidagraph (Synergy Software).
The holding potential for all cells was ?40 mV.
In mice lacking nyctalopin, Nyxnob, the DBCs do not respond to
glutamate (Gregg et al., 2007). We used whole-cell patch-
wild-type, Nyxnob, and TRPM1?/?mice. A, Wild-type rod bipolar cells (n ? 64) respond to a puff of the mGluR6 receptor
Rod bipolar cells in Nyxnobmice lack functional TRPM1 channels. Patch-clamp recordings of rod bipolar cells from
Pearringetal.•NyctalopinandSynapticLocalizationofTRPM1J.Neurosci.,July6,2011 • 31(27):10060–10066 • 10061
wild-type DBCs, the application of the mGluR6 antagonist,
LY341495, inactivates the mGluR6-mediated cascade, resulting
in closure of TRPM1 and generation of an outward current (Fig.
1A) (Shen et al., 2009). Consistent with our previous data,
mice (Fig. 1A) (Gregg et al., 2007). Application of capsaicin,
which has been shown to directly gate the DBC channel (Shen et
al., 2009), was then used to test whether any functional TRPM1
type cells, application of capsaicin produces a robust outward
absent from rod DBCs in Nyxnobmice (Fig. 1B). As a negative
The average peak response to LY341495 and capsaicin for each
mutant is summarized in Figure 1C. These data indicate that in
the Nyxnobmice TRPM1 channels responsive to capsaicin are
absent from DBC plasma membranes.
Given the patch-clamp data, we examined the relationship be-
tween nyctalopin and TRPM1 in the OPL to determine whether
the TRPM1 channel was correctly localized in the Nyxnobmice.
dritic tips of human, mouse, and monkey DBCs (Morgans et al.,
2009; van Genderen et al., 2009; Koike et al., 2010). To visualize
nyctalopin in DBCs, we used a line of mice, TgEYFP-NYX, that ex-
presses a functional EYFP-nyctalopin fusion protein in all DBCs
(Gregg et al., 2007). Immunohistochemical staining of retinal cross
sections of TgEYFP-NYX mice show that EYFP- nyctalopin expres-
image shows that TRPM1 and nyctalopin colocalize at the charac-
teristic synaptic puncta of DBCs (Fig. 2). The same colocalization
pattern of EYFP-NYX and TRPM1 was observed on the small-rod
To determine whether the expression and localization of nyc-
talopin and TRPM1 to the tips of DBC dendrites were mutually
dependent, we examined expression of TRPM1 in retinas from
Nyxnobmice and nyctalopin in retinas from Trpm1?/?/TgEYFP-
first analyzed the expression pattern of TRPM1in Nyxnobmice.
(green) and peanut agglutinin (PNA, red), a marker for the cone
synapses. In wild-type mice, TRPM1 colocalizes with PNA in a
(Fig. 3Ai, arrows). The small TRPM1 puncta observed above the
3Ai). As previously shown, there was no TRPM1 staining in the
of TRPM1 in the OPL of wild-type mice is absent in the Nyxnob
retinas. The extent of colocalization of TRPM1 and PNA on cone
data for the images; namely, that TRPM1 expression in the OPL is
Interestingly, in the absence of nyctalopin TRPM1 staining
remains in the DBC bodies. To determine the localization of this
staining, either in the plasma or biosynthetic membranes, we
10062 • J.Neurosci.,July6,2011 • 31(27):10060–10066Pearringetal.•NyctalopinandSynapticLocalizationofTRPM1
colabeled for TRPM1 and Na?/K?-ATPase, the latter of which
is a marker for the plasma membrane. The merged image of
TRPM1 and Na?/K?-ATPase shows robust staining of TRPM1
(green), indicating there is little colocalization (Fig. 3C). The
staining for Na?/K?-ATPase in wild-type mice was indistin-
guishable from that in Nyxnobmice (data not shown). These re-
sults indicate that the TRPM1 remaining
in the NyxnobDBCs is located in biosyn-
thetic membranes. This location also is
consistent with the patch-clamp data
showing a lack of functional TRPM1
branes (Fig. 1B). To determine whether
the decrease in TRPM1 expression in the
used quantitative Western blotting and
quantitative RT-PCR. Western blot
analyses of TRPM1 in whole retina ly-
Trpm1?/?mice show that the level of
TRPM1 is decreased in Nyxnobanimals
(Fig. 4). The level of TRPM1 mRNA in
the Nyxnobretinas was not different
from controls (data not shown), arguing
To examine whether nyctalopin ex-
pression is dependent on the presence of
tern of nyctalopin in Trpm1?/?/TgEYFP-
NYX mice. Figure 5 shows that in the
absence of Trpm1?/?nyctalopin staining
in the DBC dendrites is indistinguishable
from that in wild-type retinas. Just as in
wild-type retinas, EYFP-nyctalopin colo-
calizes with mGluR6 and is closely associ-
ated with ribeye, which is present as part
of the photoreceptor ribbon synapse
(Schmitz et al., 2000). Further, these data
show that mGluR6 also is localized and
expressed in a normal pattern in the
Trpm1?/?mice (Koike et al., 2010). In
summary, the data presented support the
hypothesis that nyctalopin is required for
the localization of TRPM1 at the tips of
DBC dendrites in the mouse retina.
Given the dependence of TRPM1 expres-
lopin, we examined whether the two
proteins interacted directly, using a genetic
approach was used to determine whether
nyctalopin and TRPM1 interacted (Johns-
son and Varshavsky, 1994; Stagljar et al.,
1998). This system uses a split ubiquitin as
the interaction sensor (Fig. 6A). Bait pro-
teins are fused to the C terminus of ubiqui-
tin that is, in turn, fused to a LexA-VP16
transcription factor. Prey proteins are
fused to the N terminus of ubiquitin
containing an I13G mutation, which prevents the two ubiqui-
tin fragments from interacting. Only if bait and prey proteins
interact is a functional ubiquitin formed, allowing recruit-
ment of cellular ubiquitinases, which cleave the fusion bait
protein freeing LexA-VP16 to enter the nucleus and activate
reporter genes (Fig. 6A).
Hoechst (Ci). In the absence of nyctalopin, TRPM1 staining remains in the soma of the DBCs surrounding the nucleus (Civ).
Pearringetal.•NyctalopinandSynapticLocalizationofTRPM1J.Neurosci.,July6,2011 • 31(27):10060–10066 • 10063
As a positive control we tested interaction between mGluR6
and G?O(Tian and Kammermeier, 2006). We also tested inter-
action between mGluR6 and TRPM1, nyctalopin and TRPM1,
and nyctalopin and Fur4 (Fig. 6B). The incorporation of both
on quadruple dropout plates (Fig. 6B, column 2) and by a
?-galactosidase assay (Fig. 6B, column 3). As expected,
mGluR6 and G?Oshow interaction by growth on quadruple
dropout plates and a positive ?-galactosidase assay (Fig. 6B,
row 1). There was no indication that mGluR6 and TRPM1 inter-
acted in this system (Fig. 6B, row 2). In contrast, nyctalopin and
TRPM1 showed interactions based on both growth and the
nonspecific interaction, we tested nyctalopin with Fur4, a yeast
plasma membrane protein, and there was
no indication of growth on the quadruple
was real, we used immunoprecipitation
assays. Nyctalopin was tagged with YFP
and a Strep tag. The Strep tag was used to
pull down complexes with Strep-tactin
magnetic beads (Schmidt and Skerra,
2007). A FLAG tag and GFP was added to
TRPM1 (FG-TRPM1). Both tagged con-
structs were cloned into pcDNA3.1 and
cotransfected either alone or in combina-
tion into HEK293T cells. Samples from
whole-cell lysates (Fig. 6, L) and proteins
bound and eluted from the Strep-tactin
FG-TRPM1 was detected by antibodies to
GFP and FLAG, respectively. In the mock
transfected cells, there were no bands
when using either anti-GFP or FLAG in-
dicating that the antibodies were specific
(Fig. 6C, lanes 1 and 2). Expression of SY-
NYX alone resulted in the expression of
the predicted 90 kDa SY-NYX protein
(Fig. 6C, lane 3), which was greatly en-
riched after pull down with Strep-tactin
beads, as seen by the increase in band in-
tensity (Fig. 6B, lane 4). When FG-
TRPM1 was transfected alone, a band corresponding to the 200
kDa FG-TRPM1 was present in the lysate (Fig. 6B, lane 5) and
FG-TRPM1 did not bind to the Strep-tactin beads (Fig. 6B, lane
6). When SY-NYX and FG-TRPM1 were cotransfected, both fu-
sion proteins were present in the lysate (Fig. 6B, lane 7). Fol-
lowing Strep-tactin purification of SY-NYX, a band for FG-
TRPM1 was present in the eluted fraction (Fig. 6B, lane 8),
indicating that the two proteins interact. In the eluted frac-
tion, the SY-NYX band is more intense than FG-TRPM1, sug-
gesting that only a fraction of the two proteins are interacting.
This situation also is consistent with what we see in the retina;
namely, that nyctalopin is only required for the expression of
TRPM1 at the tips of the DBCs and not in the intracellular
compartments. Combined with the yeast two-hybrid data, these
results demonstrate that nyctalopin and TRPM1 bind to one
the retinal DBC light response (Koike et al., 2010). How TRPM1
is localized to the DBC membrane in close proximity to other
members of the signal transduction cascade, such as mGluR6, is
an important question. In this study, we establish that the inter-
action between TRPM1 and nyctalopin is essential for TRPM1
localization to the tips of the DBC dendrites. Interestingly, nyc-
talopin is an extracellular protein that is attached to the mem-
or a single transmembrane domain in mice (O’Connor et al.,
nyctalopin contains only three intracellular amino acids, which
suggests that an entirely extracellular protein anchored to the
membrane is required for TRPM1 subcellular localization. The
localization of membrane proteins to the synapses is generally
present in the OPL of Trpm1?/?mice. B, EYFP-nyctalopin (green) and mGluR6 (red) colocalize in Trpm1?/?mice. C, EYFP-
10064 • J.Neurosci.,July6,2011 • 31(27):10060–10066Pearringetal.•NyctalopinandSynapticLocalizationofTRPM1
thought to involve cytoskeletal scaffolding proteins, many of
nyctalopin is extracellular; another ancillary transmembrane
protein would be needed to interact with intracellular scaffolding
complexes to hold the TRPM1 channel in the DBC synapse. Cur-
rently, no such protein has been identified in the DBC dendrite. In
addition to mGluR6 and TRPM1, a number of secondary proteins,
including G?5, R9AP, RGS7, RGS11, and nyctalopin, have been
cellular protein in this group and therefore is unlikely to be a direct
accessory protein to regulate localization of the TRPM1 channel to
the synapse. Exactly how nyctalopin positions the TRPM1 channel
extensively for ionotropic AMPA and NMDA channels (for re-
view, see Díaz, 2010). For example, transmembrane AMPA re-
ceptor regulatory proteins (TARPs) are accessory proteins that
mainly alter channel properties, though some also promote sur-
This targeting function appears restricted to TARPs containing
proteins to bind the PDZ domains or proteins already bound to
the scaffold. This provides for a dynamic association of channels
as well as a large number of signaling molecules localized to the
synapse. Many channels bind directly to PDZ domains, and this
has been shown to be the case for mGluR6 (Hirbec et al., 2002).
This interaction may be the reason mGluR6 is correctly localized
in the absence of nyctalopin (Ball et al., 2003). TRPM1 has not
been shown to bind to PDZ domains, and our data argue that its
localization to the PSD is dependent on nyctalopin.
Nyctalopin’s main extracellular domain is a curved structure,
et al., 2008). This structure is critical to nyctalopin’s function
because mutations that are predicted to disrupt it cause CSNB1
(Matsushima et al., 2005). Nyctalopin also contains a number of
potential glycosylation sites, which could be functionally impor-
tant. The exact nyctalopin motif mediating the TRPM1 interac-
tion is currently not known. Several LRR-containing proteins,
densin-180, Erbin, LGI (leucine-rich glioma inactivated), NGL
(netrin-G ligand), and SALM (synaptic adhesion-like molecule)
Pearringetal.•NyctalopinandSynapticLocalizationofTRPM1J.Neurosci.,July6,2011 • 31(27):10060–10066 • 10065
tant in synapse function (de Wit et al., 2009; Ko and Kim, 2007), Download full-text
although none are exclusively extracellular and required for tar-
geting of binding partners to the synapse.
One model for TRPM1 targeting to the DBC dendrites by
membrane. Nyctalopin expression is restricted to the DBC den-
is postulated to interact with integrins or other cell matrix pro-
interacts, thereby establishing its location. This would argue for
separate trafficking mechanisms for TRPM1 and nyctalopin,
nyctalopin is not dependent on interactions with TRPM1 since
nyctalopin is localized correctly in TRPM1-null mice (Fig. 5).
This postprocessing model of interaction also is consistent with
our heterologous expression data, where only a small fraction of
TRPM1 is coprecipitated with nyctalopin (Fig. 6).
In conclusion, this is the first report showing that an auxiliary
extracellular protein is required for localizing a TRP channel to a
there are a large number of leucine-rich repeat proteins and TRP
family members that could have a similar relationship. Elucidating
the detailed mechanism of TRPM1 dependence on nyctalopin will
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