A Role for Nyctalopin, a Small Leucine-Rich Repeat Protein, in Localizing the TRP Melastatin 1 Channel to Retinal Depolarizing Bipolar Cell Dendrites

Article (PDF Available)inThe Journal of Neuroscience : The Official Journal of the Society for Neuroscience 31(27):10060-6 · July 2011with26 Reads
DOI: 10.1523/JNEUROSCI.1014-11.2011 · Source: PubMed
Expression of channels to specific neuronal sites can critically impact their function and regulation. Currently, the molecular mechanisms 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 function of retinal depolarizing bipolar cells (DBCs), which couple a metabotropic glutamate receptor 6 to the TRP melastatin 1 (TRPM1) channel to transmit signals from photoreceptors. We report that the extracellular membrane-attached protein nyctalopin is required for the normal expression of TRPM1 on the dendrites of DBCs in mus musculus. Biochemical and genetic data indicate that nyctalopin and TRPM1 interact directly, suggesting that nyctalopin is acting as an accessory TRP channel subunit critical for proper channel localization to the synapse.
A Role for Nyctalopin, a Small Leucine-Rich Repeat Protein,
in Localizing the TRP Melastatin 1 Channel to Retinal
Depolarizing Bipolar Cell Dendrites
Jillian N. Pearring,
* Pasano Bojang Jr,
* Yin Shen,
Chieko Koike,
Takahisa Furukawa,
Scott Nawy,
and Ronald G. Gregg
Departments of
Biochemistry and Molecular Biology and
Ophthalmology and Visual Sciences, University of Louisville, Louisville, Kentucky 40202,
Departments of Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, Bronx, New York 10461,
Department of Developmental Biology,
Osaka Bioscience Institute, Suita, Osaka 565-0874, Japan, and
PRESTO, Japanese Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan
Expression of channels to specific neuronal sites can critically impact their function and regulation. Currently, the molecular mechanisms
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
function of retinal depolarizing bipolar cells (DBCs), which couple a metabotropic glutamate receptor 6 to the TRP melastatin 1 (TRPM1)
channel to transmit signals from photoreceptors. We report that the extracellular membrane-attached protein nyctalopin is required for
the normal expression of TRPM1 on the dendrites of DBCs in mus musculus. Biochemical and genetic data indicate that nyctalopin and
TRPM1 interact directly, suggesting that nyctalopin is acting as an accessory TRP channel subunit critical for proper channel localization
to the synapse.
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-
can et al., 1998). TRPM1 in melanocytes is now believed to play a
largely intracellular role, possibly regulating melanin produc-
tion (Oancea et al., 2009; Patel and Docampo, 2009). Recently,
TRPM1 was identified in the retina, where it is required for light-
driven depolarizing bipolar cell (DBC) responses (Koike et al.,
2010). These results established that TRPM1 also functions in the
plasma membrane.
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
DBCs suggests that extrinsic factors, specific to each cell type, are
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
delivery to the DBC dendrites (Nakamura et al., 2010). The small
leucine-rich repeat (LRR) protein nyctalopin is critical for gener-
ating a DBC response because spontaneous mutations in the Nyx
gene are linked to night blindness in humans and mice (Bech-
Received Feb. 25, 2011; revised May 11, 2011; accepted May 19, 2011.
Author contributions: J.N.P., P.B., S.N., and R.G.G. designed research; J.N.P., P.B., and Y.S. performed research;
Y.S., C.K., and T.F. contributed unpublished reagents/analytic tools; J.N.P., P.B., Y.S., and S.N. analyzed data; J.N.P.
and R.G.G. wrote the paper.
This work was supported byNationalEyeInstituteGrantEY12354 (R.G.G.) and National Natural Science Founda-
tion of China Grant 81000395 (Y.S.). We thank Dr. Vadim Arshavsky for many valuable discussions.
*J.N.P. and P.B. contributed equally to this work.
J. N. Pearring’s present address: Duke University Eye Center, AERI, Room 5000, 2351 Erwin Road, Box 3802,
Durham, NC 27710.
Y. Shen’s present address: Department of Ophthalmology, Renmin Hospital of Wuhan University, Wuhan, China,
C. Koike’s present address: College of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1Noji-Higashi, Ku-
satsu, Shiga 525-8577, Japan.
Correspondence should be sent to Ronald G. Gregg, 319 Abraham Flexner Way, A Building, Room 616, Louisville,
Kentucky 40202. E-mail: ron.gregg@louisville.edu.
Copyright © 2011 the authors 0270-6474/11/3110060-07$15.00/0
10060 The Journal of Neuroscience, July 6, 2011 31(27):10060 –10066
Hansen et al., 2000; Gregg et al., 2003). In a nyctalopin mutant
mouse, Nyx
, 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
with TRPM1 and that the lack of nyctalopin leads to an absence of
TRPM1 from the dendrites of DBCs. These data indicate that
nyctalopin is required for localization of TRPM1 to the dendritic
tips of DBCs.
Materials and Methods
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 Nyx
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
ated by Lexicon Genetics (Trpm1
) and obtained from the European
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
(Clontech). PCR of full-length Trpm1 was performed using retinal cDNA
generated using Superscript III (Invitrogen) and total RNA isolated from
C57BL/6J retinas. A flag-tag (5-GACTACAAGG ACGACGAC-3) fol-
lowed by GFP cDNA that was fused to the 5 end of a full-length TRPM1
cDNA. For expression of a tagged nyctalopin, Strep-tag II (5-
(EYFP) and both were inserted after the codon for amino acid 19 of the
nyctalopin cDNA. Both fusion constructs, Flag-GFP-TRPM1 (FG-
TRPM1) and Strep-tagII-EYFP-nyctalopin (SY-NYX) were inserted into
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
C-terminal ubiquitin moiety and a LexA-VP16 transcription factor were
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
on plates lacking leucine and tryptophan (dou-
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
and was performed as previously described
(Iyer et al., 2005).
Streptomycin-tactin magnetic bead pull
down. HEK293T (human embryonic kidney
cells; ATCC) cells were grown in high-glucose
DMEM supplemented with 10% fetal bovine
L-glutamine (2 mM), penicillin (50 IU/
ml), and streptomycin (Strep) (50
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
, 300 mM NaCl, 0.05% Tween-20, pH
to 8.0 with 1
M NaOH). Homogenate was
cleared by centrifuging at 15,000 g for 10 min
at 4°C. Forty microliters of total lysate was
saved, while the remainder was incubated with
l of Strep-tactin magnetic beads (Qiagen)
on a rocker at 4°C for 2–3 h. Beads were washed three times in NP-T lysis
buffer before proteins were eluted with 10 m
MD-Biotin (Sigma-Aldrich)
in NP-T Lysis buffer. SDS sample buffer (62 m
M 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,
Pierce) HRP-conjugated secondary antibodies were used to detect bands
with the advanced ECL detection system (Pierce).
Retinal preparation and immunohistochemistry. Retinal sections, whole
mounts, and immunohistochemistry were performed as described pre-
viously (Gregg et al., 2007). Antibodies and their dilutions are as follows:
anti-GFP conjugated to Alexa-488 (1:1000, Invitrogen), anti-ctbp2/rib-
eye (1:1000, BD Biosciences Pharmingen), anti-Na
-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.
Patch-clamp recordings. Retinal slices from 4- to 6-week-old C57BL/6J
and Nyx
mice were collected for patch-clamp recordings as previously
described (Snellman and Nawy, 2004; Shen et al., 2009). Picrotoxin (100
M), strychnine (10
M), and (1,2,5,6-tetrahydropyridin-4-yl) methyl-
phosphinic acid (50
M) were included in all experiments to block inhib-
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)
was added to the bath. Whole-cell recordings were obtained as previously
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.
TRPM1 currents are absent from DBCs lacking nyctalopin
In mice lacking nyctalopin, Nyx
, the DBCs do not respond to
glutamate (Gregg et al., 2007). We used whole-cell patch-
clamping to further examine TRPM1 currents in Nyx
DBCs. In
Figure 1. Rod bipolar cells in Nyx
mice lack functional TRPM1 channels. Patch-clamp recordings of rod bipolar cells from
wild-type, Nyx
, and TRPM1
mice. A, Wild-type rod bipolar cells (n 64) respond to a puff of the mGluR6 receptor
antagonist, LY341495, with an outward current. TRPM1
(n 6) and Nyx
(n 5) rod bipolar cells do not respond to the
addition of LY341495. B, An outward response was elicited in wild-type rod bipolar cells (n 46) by a puff of the TRPM1 channel
agonist, capsaicin. Rod bipolar cell recordings in TRPM1
(n 6) and Nyx
(n 6) mutants have no response to the addition
of capsaicin. C, Summary of the peak response from LY341495 and capsaicin applications. **p 0.0001. Holding potential for all
cells was 40 mV.
Pearring et al. Nyctalopin and Synaptic Localization of TRPM1 J. Neurosci., July 6, 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,
LY341495 failed to induce a current in rod DBCs from the Nyx
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
remained in the membrane in the absence of nyctalopin. In wild-
type cells, application of capsaicin produces a robust outward
current similar to LY341495 (Fig. 1 B). However, this current was
absent from rod DBCs in Nyx
mice (Fig. 1B). As a negative
control, DBC recordings were performed in TRPM1
mice and
showed no response to either LY341495 or capsaicin (Fig. 1 A, B).
The average peak response to LY341495 and capsaicin for each
mutant is summarized in Figure 1C. These data indicate that in
the Nyx
mice TRPM1 channels responsive to capsaicin are
absent from DBC plasma membranes.
TRPM1 localization to DBC dendrites is dependent on
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 Nyx
TRPM1 is expressed on the DBC somas, and as puncta on the den-
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-
sion is restricted to DBC dendrites (Fig. 2A) (Gregg et al., 2007), and
that TRPM1 also is expressed as discrete puncta on the dendritic tips
of DBCs and additionally in the DBC somas (Fig. 2A). The merged
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
DBC dendrites (Fig. 2 B, arrows) and large-cone DBC dendrites (Fig.
2B, arrowheads) in retina flat-mounts.
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
mice and nyctalopin in retinas from Trpm1
NYX mice. Both these mouse lines have normal retinal morphol-
ogy (Ball et al., 2003; Morgans et al., 2009; Koike et al., 2010). We
first analyzed the expression pattern of TRPM1in Nyx
Figure 3 shows retinal cross sections immunostained for TRPM1
(green) and peanut agglutinin (PNA, red), a marker for the cone
synapses. In wild-type mice, TRPM1 colocalizes with PNA in a
pattern consistent with expression on the tips of cone DBC dendrites
(Fig. 3Ai, arrows). The small TRPM1 puncta observed above the
cone terminals represent staining at the tips of rod BC dendrites (Fig.
3Ai). As previously shown, there was no TRPM1 staining in the
retinas of Trpm1
mice (Fig. 3Aii) (Morgans et al., 2009; Koike et
al., 2010). Figure 3Aiii shows that the characteristic punctate staining
of TRPM1 in the OPL of wild-type mice is absent in the Nyx
retinas. The extent of colocalization of TRPM1 and PNA on cone
terminals was quantified by measuring fluorescence intensity in the
green and red channels (Fig. 3B). These data reinforce the qualitative
data for the images; namely, that TRPM1 expression in the OPL is
dependent on nyctalopin expression.
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
Figure2. The TRPM1 channel colocalizes with nyctalopin inDBCdendrites. A, Immunohistochemistry of EYFP-nyctalopin (green) and TRPM1 (red) in retinal cross sections from TgEYFP-NYX mice.
The merged images show that expression of TRPM1 in the OPL colocalizes with the EYFP-nyctalopin fusion protein. Scale bar, 10
m. The OPL at higher magnification is shown in the side panels.
Scale bar, 5
m. B, Whole-mount sections through the OPL of TgEYFP-NYX mice, 5
m z-stack. EYFP-nyctalopin and TRPM1 are localized to both rod DBC dendrites (arrows) and cone DBC dendrites
(arrowheads). OPL, Outer plexiform layer; IPL, inner plexiform layer. Scale bar, 5
10062 J. Neurosci., July 6, 2011 31(27):10060 –10066 Pearring et al. Nyctalopin and Synaptic Localization of TRPM1
colabeled for TRPM1 and Na
-ATPase, the latter of which
is a marker for the plasma membrane. The merged image of
TRPM1 and Na
-ATPase shows robust staining of TRPM1
(green), indicating there is little colocalization (Fig. 3C). The
staining for Na
-ATPase in wild-type mice was indistin
guishable from that in Nyx
mice (data not shown). These re
sults indicate that the TRPM1 remaining
in the Nyx
DBCs is located in biosyn
thetic membranes. This location also is
consistent with the patch-clamp data
showing a lack of functional TRPM1
channels in the Nyx
DBC plasma mem
branes (Fig. 1 B). To determine whether
the decrease in TRPM1 expression in the
OPL decreased overall levels of TRPM1, we
used quantitative Western blotting and
quantitative RT-PCR. Western blot
analyses of TRPM1 in whole retina ly-
sates from wild-type, Nyx
, and
mice show that the level of
TRPM1 is decreased in Nyx
(Fig. 4). The level of TRPM1 mRNA in
the Nyx
retinas was not different
from controls (data not shown), arguing
that the reduction in protein expression is a
post-transcriptional effect.
To examine whether nyctalopin ex-
pression is dependent on the presence of
TRPM1, we examined the expression pat-
tern of nyctalopin in Trpm1
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
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.
Nyctalopin interacts with TRPM1
Given the dependence of TRPM1 expres-
sion on the tips of DBC dendrites on nycta-
lopin, we examined whether the two
proteins interacted directly, using a genetic
and a biochemical approach.
A membrane yeast two-hybrid (MYTH)
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. 6 A). 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. 6 A).
Figure 3. The TRPM1 channel is absent from DBC dendrites in Nyx
mice. A, Immunohistochemical analysis of TRPM1 (green)
localization in wild-type (Ai), Trpm1
(Aii), and Nyx
mice (Aiii). PNA (red) served as a marker for cone terminals in the OPL.
TRPM1 is present in the OPL (arrows) as well as in the somas within the inner nuclear layer (INL) (arrowheads) of wild-type retinas
and is absent from Trpm1
retinas. TRPM1 is absent from the dendritic tips of both rod and cone bipolarcells but remains in the
somas of the bipolar cells (arrowheads) in Nyx
retina. Scale bar, 5
m. B, Analyses of TRPM1 and EYFP-nyctalopin immunoflu
orescence at cone terminals in wild-type and Nyx
retinas. A 2.1
m line was drawn through the center of PNA-positive puncta,
and the distribution of fluorescence intensity along this line was used to generate profiles in which the red trace corresponds to
PNA, and the green to TRPM1. The traces shown are an average of 10 intensity scans within a single section. C, TRPM1 staining in
the INL of wild-type and Nyx
mice. In wild-type mice, TRPM1 is in the soma of the DBCs surrounding the nucleus, stained with
Hoechst (Ci). In the absence of nyctalopin, TRPM1 staining remains in the soma of the DBCs surrounding the nucleus (Civ).
-ATPasestaining in the INL was used asa marker for the DBC plasma membrane(Cii,v). TRPM1 and Na
not colocalize in DBCs from wild-type or Nyx
mice, suggesting TRPM1 staining is in biosynthetic membranes of DBCs (Ciii,vi).
Scale bar, 5
Pearring et al. Nyctalopin and Synaptic Localization of TRPM1 J. Neurosci., July 6, 2011 31(27):10060 –10066 10063
As a positive control we tested interaction between mGluR6
and G
(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
bait and prey plasmids was confirmed by growth on double drop-
out plates (Fig. 6B, column 1). Interactions were tested by growth
on quadruple dropout plates (Fig. 6B, column 2) and by a
-galactosidase assay (Fig. 6 B, column 3). As expected,
mGluR6 and G
show 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
-galactosidase assay (Fig. 6 B, row 3). To ensure that this was not a
nonspecific interaction, we tested nyctalopin with Fur4, a yeast
plasma membrane protein, and there was
no indication of growth on the quadruple
dropout plates (Fig. 6B, row 4). Combined,
these data indicate that in the MYTH system
nyctalopin and TRPM1 interact.
To further validate that this interaction
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
beads (Fig. 6, E) were analyzed by Western
blot (Fig. 6). The presence of SY-NYX and
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. 6 B, lane 4). When FG-
TRPM1 was transfected alone, a band corresponding to the 200
kDa FG-TRPM1 was present in the lysate (Fig. 6 B, lane 5) and
FG-TRPM1 did not bind to the Strep-tactin beads (Fig. 6 B, lane
6). When SY-NYX and FG-TRPM1 were cotransfected, both fu-
sion proteins were present in the lysate (Fig. 6 B, lane 7). Fol-
lowing Strep-tactin purification of SY-NYX, a band for FG-
TRPM1 was present in the eluted fraction (Fig. 6 B, 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 TRPM1 channel is the nonselective cation channel mediating
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-
brane by either a glycosylphosphatidylinositol anchor in humans
or a single transmembrane domain in mice (O’Connor et al.,
2005). Human nyctalopin has no intracellular region, and mouse
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
Figure 4. TRPM1 protein levels are reduced in Nyx
mice. Western blot analysis of TRPM1
expression in retinas from wild-type, Nyx
, and Trpm1
mice. Total retinal lysate (80
was loaded in each lane. Blots were probed with anti-TRPM1 or anti-
-tubulin. Band intensi-
ties of the indicated proteins were determined by NIH ImageJ software and were normalized to
levels of
-tubulin present in the same sample. Error bars are SEM values. *p 0.01. Data are
averages from three separate groups of animals.
Figure 5. Trpm1
mice have normal expression of nyctalopin at the dendrites of the DBCs. A, Immunohistochemistry for
EYFP-nyctalopin (green) and TRPM1 (red) in retinal cross sections from Trpm1
/TgEYFP-NYX mice. EYFP-nyctalopin puncta are
present in the OPL of Trpm1
mice. B, EYFP-nyctalopin (green) and mGluR6 (red) colocalize in Trpm1
mice. C, EYFP-
nyctalopin (green) and ribeye (red) do not colocalize, rather they are in close apposition in Trpm1
mice, as has been reported
for wild-type mice (Gregg et al., 2007). Scale bars, 5
10064 J. Neurosci., July 6, 2011 31(27):10060 –10066 Pearring et al. Nyctalopin and Synaptic Localization of TRPM1
thought to involve cytoskeletal scaffolding proteins, many of
which contain PDZ domains (Feng and Zhang, 2009). Given that
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
localized to the DBC dendritic tip (Gregg et al., 2007; Morgans et al.,
2007; Rao et al., 2007; Cao et al., 2009). Nyctalopin is the only extra-
cellular protein in this group and therefore is unlikely to be a direct
component of the transduction cascade, suggesting it is acting as an
accessory protein to regulate localization of the TRPM1 channel to
the synapse. Exactly how nyctalopin positions the TRPM1 channel
to the region of the postsynaptic density (PSD) is unknown.
The role of accessory targeting proteins has been studied most
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-
face expression and targeting and/or stability of AMPA receptors.
This targeting function appears restricted to TARPs containing
intracellular domains that interact with PDZ domains of proteins
that are part of the PSD. The PSD serves as a scaffold for synaptic
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,
formed by the LRRs, which is a known interaction domain (Bella
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)
families of proteins and LRRTM2, have been shown to be impor-
Figure6. A, TRPM1 interacts with nyctalopin. Schematic representation of the yeast-based splitubiquitinsystem.Cub,C-terminalhalfofubiquitin;Nub,N-terminal half of ubiquitin (carrying the
I13G mutation); LV, LexA-VP16. Positive interactions between two membrane-bound proteins, such as with TRPM1 and nyctalopin, reconstitute a functional ubiquitin protein that cleaves the LV
transcription factor. LV then translocates to the nucleus and induces the expression of His3, Ade, and LacZ reporter genes. B, Incorporation of plasmids into yeast is verified by growth on double
dropout plates lacking leucine and tryptophan (first column). Specific interactions are detected by growth on quadruple dropout plates lacking leucine, tryptophan, histidine, and adenine (second
column), and are confirmed by
-galactosidase assay (third column). mGluR6 and G
were used as a positive control and show interaction. TRPM1 did not interact with mGluR6, but did interact
with nyctalopin, as shown by robust growth on quadruple dropout plates and positive
-galactosidase assay. NYX did not show interactions with the yeast plasma membrane protein, Fur4. C,
Strep-tactin magnetic bead pull downs were performed on total lysate (L) collected from HEK293 cells transiently transfected with SY-NYX and/or FG-TRPM1. Anti-GFP antibody detects enrichment
from SY-NYX in the elution (E; lane 4). Anti-flag antibody does not detect FG-TRPM1 in the E fraction when expressed alone (lane 6); however, a small fraction of FG-TRPM1 is found in the E fraction
when cotransfected with SY-NYX (lane 8). D, Schematic representing recombinant SY-NYX and FG-TRPM1 proteins.
Pearring et al. Nyctalopin and Synaptic Localization of TRPM1 J. Neurosci., July 6, 2011 31(27):10060 –10066 10065
tant in synapse function (de Wit et al., 2009; Ko and Kim, 2007),
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
nyctalopin would be that positioning only occurs in the dendritic
tips after trans-Golgi trafficking of TRPM1 and insertion into the
membrane. Nyctalopin expression is restricted to the DBC den-
dritic tips (Gregg et al., 2007), and its leucine-rich repeat domain
is postulated to interact with integrins or other cell matrix pro-
teins (Heinegård, 2009). Therefore, a plausible model is that nyc-
talopin is localized to the DBC dendritic tips, after which TRPM1
interacts, thereby establishing its location. This would argue for
separate trafficking mechanisms for TRPM1 and nyctalopin,
which is consistent with our results that the dendritic targeting of
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
specific neuronal compartment or synapse. While the expression of
nyctalopin and TRPM1 is somewhat restricted in tissue distribution,
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
lead to new findings with respect to the mechanisms controlling the
targeting of TRP channels to specific neuronal compartments.
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    • "Therefore, our study showed a reduction in the expression of major components of the mGluR6 signaling cascade in APLP2-KO. Interestingly, GRM6-and NYXand LRIT3 deficient mouse, three models for cCSNB, showed a nearly absent mGluR6 at the dendritic tips of cone ON-bipolar cells [71,[89][90][91] , indicating a reduction in ON-bipolar cells of major components of the mGluR6 signaling cascade in mouse models of cCSNB. By RT-qPCR, we demonstrated that APLP2 deletion alters the expression of TRPM1, GRM6 and CACNA1F, three major genes involved in CSNB. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Amyloid precursor protein knockout mice (APP-KO) have impaired differentiation of amacrine and horizontal cells. APP is part of a gene family and its paralogue amyloid precursor-like protein 2 (APLP2) has both shared as well as distinct expression patterns to APP, including in the retina. Given the impact of APP in the retina we investigated how APLP2 expression affected the retina using APLP2 knockout mice (APLP2-KO) Results: Using histology, morphometric analysis with noninvasive imaging technique and electron microscopy, we showed that APLP2-KO retina displayed abnormal formation of the outer synaptic layer, accompanied with greatly impaired photoreceptor ribbon synapses in adults. Moreover, APLP2-KO displayed a significant decease in ON-bipolar, rod bipolar and type 2 OFF-cone bipolar cells (36, 21 and 63 %, respectively). Reduction of the number of bipolar cells was accompanied with disrupted dendrites, reduced expression of metabotropic glutamate receptor 6 at the dendritic tips and alteration of axon terminals in the OFF laminae of the inner plexiform layer. In contrast, the APP-KO photoreceptor ribbon synapses and bipolar cells were intact. The APLP2-KO retina displayed numerous phenotypic similarities with the congenital stationary night blindness, a non-progressive retinal degeneration disease characterized by the loss of night vision. The pathological phenotypes in the APLP2-KO mouse correlated to altered transcription of genes involved in pre- and postsynatic structure/function, including CACNA1F, GRM6, TRMP1 and Gα0, and a normal scotopic a-wave electroretinogram amplitude, markedly reduced scotopic electroretinogram b-wave and modestly reduced photopic cone response. This confirmed the impaired function of the photoreceptor ribbon synapses and retinal bipolar cells, as is also observed in congenital stationary night blindness. Since congenital stationary night blindness present at birth, we extended our analysis to retinal differentiation and showed impaired differentiation of different bipolar cell subtypes and an altered temporal sequence of development from OFF to ON laminae in the inner plexiform layer. This was associated with the altered expression patterns of bipolar cell generation and differentiation factors, including MATH3, CHX10, VSX1 and OTX2. Conclusions: These findings demonstrate that APLP2 couples retina development and synaptic genes and present the first evidence that APLP2 expression may be linked to synaptic disease.
    Full-text · Article · Jun 2016
    • "In Nyx nob mice, the a-wave and the flicker responses in range C were comparable in size to those in control mice 1.5 log cd s/m 2 ) was not attenuated in Nyx nob mice (Fig. 1a, b), indicating normal functionality of rod photoreceptor outer segments. Thus, the results obtained herein (Fig. 1a, b) were entirely consistent with such a postsynaptic abnormality [9, 20, 21]. In the flicker ERG, the responses in range A were remarkably different in configuration between Nyx nob and control mice, i.e., no clear positive-going response and no oscillations in Nyx nob mice (Fig. 1c). "
    [Show abstract] [Hide abstract] ABSTRACT: Purpose: Marked attenuation of the single-flash electroretinographic (ERG) b-wave in the presence of a normal-amplitude or less-attenuated a-wave is commonly referred to as the "negative ERG." The purpose of this study was to investigate whether the disparate origins of the negative ERG in three murine models can be discriminated using flickering stimuli. Methods: Three models were selected: (1) the Nyx (nob) mouse model of complete congenital stationary night blindness, (2) the oxygen-induced retinopathy (OIR) rat model of retinopathy of prematurity (ROP), and (3) the Rs1 knockout (KO) mouse model of X-linked juvenile retinoschisis. Directly after a dark-adapted, single-flash ERG luminance series, a flicker ERG frequency series (0.5-30 Hz) was performed at a fixed luminance of 0.5 log cd s/m(2). This series includes frequency ranges that are dominated by activity in (A) the rod pathways (below 5 Hz), (B) the cone ON-pathway (5-15 Hz), and (C) the cone OFF-pathway (above 15 Hz). Results: All three models produced markedly attenuated single-flash ERG b-waves. In the Nyx (nob) mouse, which features postsynaptic deficits in the ON-pathways, the a-wave was normal and flicker responses were attenuated in ranges A and B, but not C. The ROP rat is characterized by inner-retinal ischemia which putatively affects both ON- and OFF-bipolar cell activity; flicker responses were reduced in all ranges (A-C). Notably, the choroid supplies the photoreceptors and is thought to be relatively intact in OIR, an idea supported by the nearly normal a-wave. Finally, in the Rs1 KO mouse, which has documented abnormality of the photoreceptor-bipolar synapse affecting both ON- and OFF-pathways, the flicker responses were attenuated in all ranges (A-C). The a-wave was also attenuated, likely as a consequence to schisms in the photoreceptor layer. Conclusion: Consideration of both single-flash and flickering ERG responses can discriminate the functional pathology of the negative ERG in these animal models of human disease.
    Article · Mar 2016
    • "It is interesting to note that mutations in non-ion channel proteins, required for the correct localization of TRPM1, have also been reported to cause CSNB1. One of these proteins, a small leucine-rich repeat protein, Nyctalopin (Nyx), interacts directly with TRPM1 and is required to target the channel to the dendritic tips of the rod ON-bipolar cells (Hansen et al., 2000; Pearring et al., 2011). In a recent study, rod ON-bipolar cell function in a Nyx-deficient mouse was restored by intravitreal injection of AAVs encoding wild-type Nyx (Scalabrino et al., 2015). "
    [Show abstract] [Hide abstract] ABSTRACT: The eye is the sensory organ of vision. There, the retina transforms photons into electrical signals that are sent to higher brain areas to produce visual sensations. In the light path to the retina, different types of cells and tissues are involved in maintaining the transparency of avascular structures like the cornea or lens, while others, like the retinal pigment epithelium, have a critical role in the maintenance of photoreceptor function by regenerating the visual pigment. Here, we have reviewed the roles of different ion channels expressed in ocular tissues (cornea, conjunctiva and neurons innervating the ocular surface, lens, retina, retinal pigment epithelium, and the inflow and outflow systems of the aqueous humor) that are involved in ocular disease pathophysiologies and those whose deletion or pharmacological modulation leads to specific diseases of the eye. These include pathologies such as retinitis pigmentosa, macular degeneration, achromatopsia, glaucoma, cataracts, dry eye, or keratoconjunctivitis among others. Several disease-associated ion channels are potential targets for pharmacological intervention or other therapeutic approaches, thus highlighting the importance of these channels in ocular physiology and pathophysiology.
    Full-text · Article · Dec 2015
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