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Synaptic transmission between light-sensory photoreceptor cells and downstream ON-bipolar neurons plays an important role for vertebrate vision. This process is mediated by the G protein coupled receptor pathway involving glutamate receptor mGluR6 and effector channel TRPM1. The signal transmission occurs on a rapid timescale, however the molecular organization that ensures timely signaling in this cascade is unknown. Genetic studies in human patients and animal models reveal that ON-bipolar cell signaling depends on a synaptic protein nyctalopin. We have conducted a proteomic search for proteins associated with nyctalopin in the mouse retina and identified TRPM1 as the binding partner. We further demonstrate that nyctalopin additionally interacts with mGluR6 receptor. Disruption of mGluR6 prevented targeting of TRPM1 to the postsynaptic compartment of ON-bipolar neurons. These results reveal a unique macromolecular organization of the mGluR6 cascade, where principal signaling components are scaffolded by nyctalopin, creating an organization essential for the correct localization of the signaling ensemble and ultimately intact transmission of the signal at the first visual synapse.
Synaptic transmission between neurons of the retina is critical for vertebrate vision. Absorption of light by photoreceptors hyperpolarizes the cells and leads to the suppression of neurotransmitter glutamate release from their axonal terminals. Across the synapse, changes in glutamate concentration are sensed by the second order neurons, bipolar cells (BC). Rod photoreceptor neurons are active under low illumination conditions and signal predominantly to the ON type of bipolar cells (ON-BC) that depolarize in response to light-evoked reduction in synaptic glutamate concentration (Sharpe and Stockman, 1999; Taylor and Smith, 2004). The ON-BC play essential role for dim vision and their dysfunction is one of the leading causes of the congenital types of night blindness (Berger et al., 2010).
It has been established that glutamate reception of ON-BC is mediated by the mGluR6 receptor (Nakajima et al., 1993; Masu et al., 1995) and involves the activation of G protein, Go (Nawy, 1999; Dhingra et al., 2000). Inactivation of either mGluR6 (Masu et al., 1995; Maddox et al., 2008) or Go (Dhingra et al., 2000) in mice leads to a complete absence of ON-BC responses to light. Genetic studies revealed that synaptic transmission to ON-BC is also abolished in mice carrying inactivating mutation in nyctalopin gene (Gregg et al., 2003). Nyctalopin is a membrane anchored protein that contains several leucine-rich repeat domains, ubiquitous structural motifs important for protein-protein interaction (Poopalasundaram et al., 2005). In the retina, nyctalopin is expressed at the dendritic tips of ON-BC, where it closely co-localizes with mGluR6 forming prominent synaptic puncta (Morgans et al., 2006; Gregg et al., 2007). Single cell electrophysiological studies demonstrated that ON-BC lacking nyctalopin are insensitive to direct application of glutamate suggesting the involvement of this protein in modulating the components of the signal transduction pathway (Gregg et al., 2007).
One of the most significant recent breakthroughs in understanding ON-BC signaling has been the identification of the TRPM1 as the main effector channel that mediates ON-BC responses to mGluR6 activation (Morgans et al., 2009; Shen et al., 2009; Koike et al., 2010). Like mGluR6 and nyctalopin, TRPM1 is specifically enriched at dendritic tips and its elimination in mice completely abolishes ON-BC responses. Genetic association studies indicate that mutations in TRPM1 (Audo et al., 2009; van Genderen et al., 2009; Nakamura et al., 2010), mGluR6 (Zeitz et al., 2005; O’Connor et al., 2006) and nyctalopin (Bech-Hansen et al., 2000; Leroy et al., 2009) account for the majority of congenital night blindness cases in human patients. However, beyond general knowledge regarding the identity of the main players involved in ON-BC responses to light, our understanding of the mechanistic details pertaining to the organization of the signaling pathway is very limited.
In this study we report identification of TRPM1 and mGluR6 as nyctalopin binding partners. We further establish that the post-synaptic accumulation of TRPM1 complex is dependent on mGluR6. These studies indicate that the three main components of the ON-BC signaling pathway are linked together in a single macromolecular complex providing an insight into the compartmentalization of the postsynaptic signaling machinery of retina ON-BC neurons.
Antibodies against mouse recombinant nyctalopin (aa19-450), TRPM1 (aa1423-1622) and mGluR6 (aa853-871) were generated in sheep. Antibodies were affinity purified on the antigen-containing column and used throughout the study. Specificity of all antibodies have been verified by their ability to recognize cognate proteins transiently expressed in transfected HEK293T cells and to precipitate them from native retinas as determined by mass-spectrometry. For double immunolabeling experiments guinea pig anti-mGluR6 antibodies were used (gift from Dr. Furukawa). Commercial antibodies were: mouse anti-β-actin (Sigma), mouse anti-CtBP2 (BD Biosciences), mouse anti-HA (Millipore) and mouse anti-c-myc (Roche).
Cloning of rat mGluR6 was described previously (Cao et al., 2009). The C-terminal HA tagged mouse nyctalopin was amplified from mouse cDNA clone (Clone ID: 5362608, Open Biosystems) and cloned into a pcDNA3.1/V5-His-TOPO (Invitrogen) vector. C-terminal HA or tandem affinity purification (TAP) (Burckstummer et al., 2006) tags were appended before the stop codon by PCR. To obtain N-terminal c-myc tagged full length mouse TRPM1 expression construct, TRPM1(349–4869) was amplified from mouse cDNA clone (Clone ID 100068120, Open Biosystems), supplemented with TRPM1(1–348) amplified from mouse TRPM1 cDNA clone (Clone ID A930013B11, imaGenes) and cloned into a pcDNA3.1/V5-His-TOPO (Invitrogen) vector.
nob3 and nob4 mouse strains were obtained from The Jackson Laboratory and the NIH Neurogenomics Project, respectively. Both male and female mice (1–3 months old) were used in the study. Mice were maintained on a diurnal 12 h light/dark cycle. Procedures involving mice strictly followed NIH guidelines and were approved by the IACUC at University of Minnesota.
Whole retinas were removed from mice, lysed by sonication in 1% Triton X-100 containing buffer and subjected to immunoprecipitation as previously described (Martemyanov et al., 2005; Cao et al., 2009). The beads were washed three times with ice-cold IP buffer and proteins were eluted with 50 μL SDS sample buffer (62 mM Tris, 10% glycerol, 2% SDS, and 5% β-mercaptoethanol). Resulting samples were allowed to enter the SDS-PAGE gel by applying ~150mV for 15–20 min. Gels were fixed with using 5% acetic acid in 50% methanol, stained by NOVEX colloidal blue (Invitrogen). Stained areas were cut out, digested with trypsin (Promega) and alkylated as described (Shevchenko et al., 2006). The resulting peptide mixtures were desalted, resolved by high pressure liquid chromatography and analyzed using LTQ-Orbitrap XL mass spectrometer as previously described (Posokhova et al., 2011).
HEK293T cells were cultured at 37°C and 5% CO2 in DMEM supplemented with 10% FBS, and 4 mM L-glutamine. Cells were transfected at ~70% confluency using Lipofectamine LTX (Invitrogen) and harvested 24 hours later. Cells were lysed in ice-cold PBS IP buffer by sonication and lysates were cleared by centrifuged at 14,000 rpm for 15 minutes. For immunoprecipitation experiments, the supernatant was incubated with 10μl of protein G beads (GE Healthcare) and 5 μg antibodies on a rocker at 4°C for 1 hour. After three washes with PBS IP buffer, proteins were eluted with 50 μl of SDS sample buffer and analyzed by SDS-PAGE, followed by Western blotting using HRP conjugated secondary antibodies and an ECL West Pico (Thermo Scientific) detection system. Pull-down experiments with TAP-tagged constructs were performed using Streptavidin Sepharose (GE Healthcare). Procedures replicated ones used for the IP experiments except Triton X-100 in cell lysis buffer was substituted for 0.5% E10C12 and proteins were eluted from the beads with 25 μl of 3 mM biotin. For quantitative analysis, Western blotting was performed with IRDye 680- and IRDye 800-labeled secondary antibodies (Li-Cor Biosciences) and the signal was detected and quantified by an Odyssey Infrared Imaging System (Li-Cor Biosciences). The integrated intensity of each band of interest was measured in a corresponding channel with a top-bottom background setting. Integrated intensity of β-actin was used for data normalization.
Immunohistochemical detection of proteins in retinal sections was performed as described previously (Cao et al., 2008). Dissected eyecups were fixed for 15 minutes with 4% paraformaldehyde, blocked with 10% donkey serum for 1 hour and incubated with primary antibody in for 1 hour. After four-washes sections were incubated with fluorophore-conjugated secondary antibodies for 1 hour before mounting in Fluromount (Sigma). Images were taken by the Olympus Fluoview 1000 confocal microscope.
In order to gain an insight into the functional role of nyctalopin at the molecular level we have conducted a proteomic search for its binding partners in the retina. We began by developing antibodies to nyctalopin that would recognize native protein. Antibodies directed against globular portion of the nyctalopin efficiently and specifically recognized nyctalopin expressed transiently in transfected HEK293T cell (Fig.1A). Furthermore, when used for immunoprecipitation, these antibodies could substantially deplete nyctalopin from the cellular lysates and dramatically enrich the protein in the eluate fractions demonstrating their utility for affinity purification of nyctalopin (Fig.1A).
When tested in native retinas, nyctalopin antibodies failed to recognize specific bands in the total retina lysate. However, when used for the immunoprecipitation the antibodies were able to pull-down a 52 kDa protein that corresponds to the predicted molecular weight of nyctalopin and matches the migration position of nyctalopin expressed in HEK293T cells (Fig.1B). This immunoprecipitation was specific as non-immune IgG failed to pull down the band. We have next conducted a preparative immunoprecipitation reaction with 14 mouse retinas and subjected eluates to tandem mass-spectrometric analysis. MS/MS sequencing of precursor peptides identified 6 unique peptides with at least 95% confidence that precisely matched the sequence of Mus musculus nyctalopin derived from the database (Fig.1C). Importantly, none of the nyctalopin peptides were detected in the control immunoprecipitation reaction where non-immune IgG beads were used instead. These results allow us to conclude with confidence that our antibodies can specifically immunoprecipitate nyctalopin from native mouse retinas.
Examination of proteins pulled down with anti-nyctalopin antibodies identified the presence of TRPM1. A sequence of single unique peptide matching to amino acids 541–548 of mouse TRPM1 protein was deduced with high confidence by MS/MS fragmentation (Fig.1D). Western blot analysis of samples from immunoprecipitation confirmed the presence of full-length TRPM1 protein in eluates where anti-nyctalopin antibodies were used for the pull-down (Fig.1E). Importantly, no TRPM1 protein was detected by either mass-spectrometry or Western blotting in control experiments where non-immune IgGs were used instead. These data indicate that nyctalopin specifically interacts with TRPM1 in native retinas.
We next examined whether binding between nyctalopin and TRPM1 could be reconstituted in heterologous expression system. First, epitope tagged constructs encoding c-myc-TRPM1 and HA-nyctalopin were co-transfected into mammalian HEK293T cells. We were able to effectively precipitate nyctalopin from the cellular lysates using anti-HA antibodies (Fig.2A). Examination of the eluate fraction revealed robust presence of c-myc-TRPM1 only in samples where nyctalopin was present, indicating specific co-precipitation of TRPM1. Conversely, in reciprocal immunoprecipitation experiment, nyctalopin was specifically present in the eluates when TRPM1 was pulled down with anti c-myc antibodies (Fig.2A). Similar results were obtained when specific antibodies were used to precipitate untagged proteins in both forward and reverse directions (Fig.2B). Furthermore, when attached to the TAP protein module, nyctalopin, but not TAP alone, was able to effectively pull-down TRPM1 from cellular lysates (Fig.2C)
mGluR6 is the principle receptor of the ON-BC that controls closing of TRPM1 channel through the activation of the intracellular signaling cascade. We therefore, next examined a possibility that nyctalopin could be additionally involved in the interactions with mGluR6. As evident from the Fig. 2D, in cells co-transfected with both nyctalopin and mGluR6, but not in the cells transfected with only one protein, mGluR6 was effectively co-precipitated with nyctalopin in both forward and reverse immunoprecipitation reactions (Fig.2D). These data indicate that nyctalopin can form protein-protein interactions with two principle components of the ON-BC signal transduction pathway: the receptor mGluR6 and the effector channel TRPM1.
Binding of nyctalopin to both mGluR6 and TRPM1 suggests that these proteins exist in ternary complex. Because mGluR6 has been previously shown to target associated proteins, e.g. R7 RGS proteins (Cao et al., 2009), to the dendritic tips of ON-BC we have tested a possibility that mGluR6 also drives post-synaptic accumulation of TRPM1 channels. Therefore, we have next performed immunohistological studies to examine the role of mGluR6 in subcellular localization of TRPM1. Consistent with recent reports (Morgans et al., 2009; Koike et al., 2010) we found that TRPM1 staining was located on the plasma membrane and specifically enriched at the tips of the ON-BC dendrites forming characteristic punctate staining pattern completely overlapping with mGluR6 immunofluorescence (Fig3A). The TRPM1-positive puncta immediately opposed pre-synaptic photoreceptor terminals revealed by labeling with synaptic ribbon marker, CtBP2 (Fig.3B). Strikingly, we found complete lack of this punctate post-synaptic accumulation of TRPM1 in retinas of nob3 and nob4 mice that do not express mGluR6 protein (Fig.3B). At the same time, the plasma membrane localization of TRPM1 in the mutant retinas was unchanged and largely resembled that observed in the wild type samples. We have further noticed that mutant retinas consistently yielded lower fluorescence intensities when stained for TRPM1. This observation was confirmed by Western blotting that revealed a 30% reduction in the levels of TRPM1 protein in nob3 and nob4 mutant retinas lacking mGluR6 (Fig.4). These observations indicate that macromolecular complex formation with mGluR6 is essential for the synaptic targeting and expression of TRPM1 in ON-BC.
The sign-inverting synapse between photoreceptors and ON-BC neurons provides a conversion of the light-induced hyperpolarizing response of photoreceptors into the excitatory depolarization of the downstream ON-BC. The main players, as well as the key steps of this process at the postsynaptic site have now been established (Vardi et al., 2002; Okawa and Sampath, 2007; Morgans et al., 2010). In the hypothetical sequence of events, decrease in the glutamate release by the photoreceptors during light response leads to a reduction in mGluR6 activation, subsequent Go deactivation and opening of the TRPM1 channels, that are kept closed in the dark by tonic mGluR6-Gαo signaling (Vardi et al., 2002; Snellman et al., 2008). This makes TRPM1 the central effector channel ultimately responsible for providing ON-BC responses to light (Morgans et al., 2010).
Currently, the mechanisms governing gating and cellular regulation of TRPM1 in ON-BC neurons are completely unknown. What is known, however, is the remarkable speed with which ON-BC mount the response. The TRPM1 channels fully open in less than 100 ms from the moment mGluR6 receptors detect changes in glutamate concentration (Field and Rieke, 2002). In comparison, corresponding step in G protein cascade of rod photoreceptors, deactivation of rhodopsin-transducin signaling and opening of the cGMP-gated channels, takes approximately 5 times longer (Pugh, 2006; Fain et al., 2010). These considerations suggest that postsynaptic G protein cascade of ON-BC is organized in a way that allows streamlining signal transmission from mGluR6 to TRPM1 channel. Indeed, mGluR6 cascade displays a remarkable compartmentalization with all key components being enriched at the tips of the ON-BC dendrites in immediate apposition from the photoreceptor axonal terminals.
One of the main results of our study is a demonstration that mGluR6 is required for targeting of TRPM1 channel to the dendritic tips, where it functions. In the absence of mGluR6, transmembrane channel TRPM1 is still able to reach plasma membrane but completely looses its accumulation at postsynaptic sites. This observation parallels our earlier report showing that targeting of RGS proteins to the dendritic tips is also mediated by their association with mGluR6. Together, these findings indicate an important role that mGluR6 receptor plays in subcellular targeting of pathway components in addition to its role in signaling.
The second key finding of this study is the demonstration of the macromolecular complex formation between principle components of the ON-BC signaling cascade: TRPM1 and mGluR6 which both physically associate with synaptic protein nyctalopin. We propose that assembly of this complex plays an essential role in enabling fast transmission of signal from mGluR6 to TRPM1, ensuring millisecond timing kinetics of ON-BC responses to light. Because neither mGluR6 (Beqollari et al., 2009; Masuho et al., 2010) nor TRPM1 (Oancea et al., 2009; Koike et al., 2010; Lambert et al., 2011) appear to require nyctalopin for their functional activity in cells per se, we envision that the role of nyctalopin is to coordinate the assembly of mGluR6-TRPM1 complex in the optimal configuration for signaling, which ultimately makes the fast signal transmission possible. Loss of nyctalopin in this scheme would disrupt the coupling of mGluR6 receptor to TRPM1 to result in inefficient response that would not reach the threshold level necessary for the generation of the light response. This situation is reminiscent of invertebrate phototransduction cascade where the coupling of the G protein coupled receptor to the TRP effector channel requires participation of auxiliary scaffolding protein (INAD) that brings the components in the complex together and plays a critical role in visual signaling (Tsunoda and Zuker, 1999; Wang and Montell, 2007). Interestingly, several G protein coupled receptors carry extracellular nyctalopin-like leucine-rich repeat (LRR) domains (Hsu et al., 2000), and there is also a precedent for the involvement of LRR-containing proteins in allosteric modulation of the ion channel properties (Yan and Aldrich, 2010). These observations suggest a possible general mechanism of synaptic signaling where numerous LRR proteins (Ko and Kim, 2007) could be involved in modulating signal transmission events from G protein coupled receptors to TRP channels.
We thank Dr. Mathew D. Stone for the help with the protein identification experiments. This work was supported by NIH grants EY018139 and DA026405. The authors recognize the Center for Mass Spectrometry and Proteomics at the University of Minnesota and various supporting agencies, including the National Science Foundation for Major Research Instrumentation grants 9871237 and NSF-DBI-0215759 used to purchase the instruments described in this study.