Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|B, Gp, H, M, R||WB||M||Purified||Monoclonal Antibody|
|Description||Anti-G Protein Goα Antibody, clone 2A|
|Presentation||Purified Immunoglobulin. Liquid in 0.02M PBS, 0.25M NaCl, pH 7.6, containing 0.1% sodium azide as a preservative.|
|Safety Information according to GHS|
|Material Size||100 µg|
Anti-G Protein Goα Antibody, clone 2A SDS
|MOUSE ANTI-Goa MONOCLONAL ANTIBODY||2942490|
|MOUSE ANTI-Goa MONOCLONAL ANTIBODY - 2064709||2064709|
|MOUSE ANTI-Goa MONOCLONAL ANTIBODY - 2181270||2181270|
|MOUSE ANTI-Goa MONOCLONAL ANTIBODY - 2326385||2326385|
|MOUSE ANTI-Goα - 2500634||2500634|
|MOUSE ANTI-Goα -2557660||2557660|
|MOUSE ANTI-Goα -2724457||2724457|
|MOUSE ANTI-Goα -2750317||2750317|
|MOUSE ANTI-Goα -2840466||2840466|
|MOUSE ANTI-Goα MONOCLONAL ANTIBODY||3043674|
|Reference overview||Application||Species||Pub Med ID|
|Restoring the ON Switch in Blind Retinas: Opto-mGluR6, a Next-Generation, Cell-Tailored Optogenetic Tool.|
van Wyk, M; Pielecka-Fortuna, J; Löwel, S; Kleinlogel, S
PLoS biology 13 e1002143 2015
Photoreceptor degeneration is one of the most prevalent causes of blindness. Despite photoreceptor loss, the inner retina and central visual pathways remain intact over an extended time period, which has led to creative optogenetic approaches to restore light sensitivity in the surviving inner retina. The major drawbacks of all optogenetic tools recently developed and tested in mouse models are their low light sensitivity and lack of physiological compatibility. Here we introduce a next-generation optogenetic tool, Opto-mGluR6, designed for retinal ON-bipolar cells, which overcomes these limitations. We show that Opto-mGluR6, a chimeric protein consisting of the intracellular domains of the ON-bipolar cell-specific metabotropic glutamate receptor mGluR6 and the light-sensing domains of melanopsin, reliably recovers vision at the retinal, cortical, and behavioral levels under moderate daylight illumination.
|Oligomeric state of purified transient receptor potential melastatin-1 (TRPM1), a protein essential for dim light vision.|
Agosto, MA; Zhang, Z; He, F; Anastassov, IA; Wright, SJ; McGehee, J; Wensel, TG
The Journal of biological chemistry 289 27019-33 2014
Transient receptor potential melastatin-1 (TRPM1) is essential for the light-induced depolarization of retinal ON bipolar cells. TRPM1 likely forms a multimeric channel complex, although almost nothing is known about the structure or subunit composition of channels formed by TRPM1 or any of its close relatives. Recombinant TRPM1 was robustly expressed in insect cells, but only a small fraction was localized to the plasma membrane. Similar intracellular localization was observed when TRPM1 was heterologously expressed in mammalian cells. TRPM1 was affinity-purified from Sf9 cells and complexed with amphipol, followed by detergent removal. In blue native gels and size exclusion chromatography, TRPM1 migrated with a mobility consistent with detergent- or amphipol-bound dimers. Cross-linking experiments were also consistent with a dimeric subunit stoichiometry, and cryoelectron microscopy and single particle analysis without symmetry imposition yielded a model with approximate 2-fold symmetrical features. Finally, electron microscopy of TRPM1-antibody complexes revealed a large particle that can accommodate TRPM1 and two antibody molecules. Taken together, these data indicate that purified TRPM1 is mostly dimeric. The three-dimensional structure of TRPM1 dimers is characterized by a small putative transmembrane domain and a larger domain with a hollow cavity. Blue native gels of solubilized mouse retina indicate that TRPM1 is present in two distinct complexes: one similar in size to the recombinant protein and one much larger. Because dimers are likely not functional ion channels, these results suggest that additional partner subunits participate in forming the transduction channel required for dim light vision and the ON pathway.
|Efficient transduction and optogenetic stimulation of retinal bipolar cells by a synthetic adeno-associated virus capsid and promoter.|
Cronin, T; Vandenberghe, LH; Hantz, P; Juttner, J; Reimann, A; Kacsó, AE; Huckfeldt, RM; Busskamp, V; Kohler, H; Lagali, PS; Roska, B; Bennett, J
EMBO molecular medicine 6 1175-90 2014
In this report, we describe the development of a modified adeno-associated virus (AAV) capsid and promoter for transduction of retinal ON-bipolar cells. The bipolar cells, which are post-synaptic to the photoreceptors, are important retinal targets for both basic and preclinical research. In particular, a therapeutic strategy under investigation for advanced forms of blindness involves using optogenetic molecules to render ON-bipolar cells light-sensitive. Currently, delivery of adequate levels of gene expression is a limiting step for this approach. The synthetic AAV capsid and promoter described here achieves high level of optogenetic transgene expression in ON-bipolar cells. This evokes high-frequency (~100 Hz) spiking responses in ganglion cells of previously blind, rd1, mice. Our vector is a promising vehicle for further development toward potential clinical use.
|p53 selectively regulates developmental apoptosis of rod photoreceptors.|
Vuong, L; Brobst, DE; Ivanovic, I; Sherry, DM; Al-Ubaidi, MR
PloS one 8 e67381 2013
Retinal cells become post-mitotic early during post-natal development. It is likely that p53, a well-known cell cycle regulator, is involved in regulating the genesis, differentiation and death of retinal cells. Furthermore, retinal cells are under constant oxidative stress that can result in DNA damage, due to the extremely high level of metabolic activity associated with phototransduction. If not repaired, this damage may result in p53-dependent cell death and ensuing vision loss. In this study, the role of p53 during retinal development and in the post-mitotic retina is investigated. A previously described super p53 transgenic mouse that expresses an extra copy of the mouse p53 gene driven by its endogenous promoter is utilized. Another transgenic mouse (HIP) that expresses the p53 gene in rod and cone photoreceptors driven by the human interphotoreceptor retinoid binding protein promoter was generated. The electroretinogram (ERG) of the super p53 mouse exhibited reduced rod-driven scotopic a and b wave and cone-driven photopic b wave responses. This deficit resulted from a reduced number of rod photoreceptors and inner nuclear layer cells. However, the reduced photopic signal arose only from lost inner retinal neurons, as cone numbers did not change. Furthermore, cell loss was non-progressive and resulted from increased apoptosis during retinal developmental as determined by TUNEL staining. In contrast, the continuous and specific expression of p53 in rod and cone photoreceptors in the mature retinas of HIP mice led to the selective loss of both rods and cones. These findings strongly support a role for p53 in regulating developmental apoptosis in the retina and suggest a potential role, either direct or indirect, for p53 in the degenerative photoreceptor loss associated with human blinding disorders.
|Expression of voltage-gated calcium channel α(2)δ(4) subunits in the mouse and rat retina.|
De Sevilla Müller, LP; Liu, J; Solomon, A; Rodriguez, A; Brecha, NC
The Journal of comparative neurology 521 2486-501 2013
High-voltage activated Ca channels participate in multiple cellular functions, including transmitter release, excitation, and gene transcription. Ca channels are heteromeric proteins consisting of a pore-forming α(1) subunit and auxiliary α(2)δ and β subunits. Although there are reports of α(2)δ(4) subunit mRNA in the mouse retina and localization of the α(2)δ(4) subunit immunoreactivity to salamander photoreceptor terminals, there is a limited overall understanding of its expression and localization in the retina. α(2)δ(4) subunit expression and distribution in the mouse and rat retina were evaluated by using reverse transcriptase polymerase chain reaction, western blot, and immunohistochemistry with specific primers and a well-characterized antibody to the α(2)δ(4) subunit. α(2)δ(4) subunit mRNA and protein are present in mouse and rat retina, brain, and liver homogenates. Immunostaining for the α(2)δ(4) subunit is mainly localized to Müller cell processes and endfeet, photoreceptor terminals, and photoreceptor outer segments. This subunit is also expressed in a few displaced ganglion cells and bipolar cell dendrites. These findings suggest that the α(2)δ(4) subunit participates in the modulation of L-type Ca(2+) current regulating neurotransmitter release from photoreceptor terminals and Ca(2+)-dependent signaling pathways in bipolar and Müller cells.
|Metabotropic glutamate receptor 6 signaling enhances TRPM1 calcium channel function and increases melanin content in human melanocytes.|
Devi, S; Markandeya, Y; Maddodi, N; Dhingra, A; Vardi, N; Balijepalli, RC; Setaluri, V
Pigment cell & melanoma research 26 348-56 2013
Mutations in TRPM1, a calcium channel expressed in retinal bipolar cells and epidermal melanocytes, cause complete congenital stationary night blindness with no discernible skin phenotype. In the retina, TRPM1 activity is negatively coupled to metabotropic glutamate receptor 6 (mGluR6) signaling through Gαo and TRPM1 mutations result in the loss of responsiveness of TRPM1 to mGluR6 signaling. Here, we show that human melanocytes express mGluR6, and treatment of melanocytes with L-AP4, a type III mGluR-selective agonist, enhances Ca(2+) uptake. Knockdown of TRPM1 or mGluR6 by shRNA abolished L-AP4-induced Ca(2+) influx and TRPM1 currents, showing that TRPM1 activity in melanocytes is positively coupled to mGluR6 signaling. Gαo protein is absent in melanocytes. However, forced expression of Gαo restored negative coupling of TRPM1 to mGluR6 signaling, but treatment with pertussis toxin, an inhibitor of Gi /Go proteins, did not affect basal or mGluR6-induced Ca(2+) uptake. Additionally, chronic stimulation of mGluR6 altered melanocyte morphology and increased melanin content. These data suggest differences in coupling of TRPM1 function to mGluR6 signaling explain different cellular responses to glutamate in the retina and the skin.
|Quantitative confocal imaging of the retinal microvasculature in the human retina.|
Tan, PE; Yu, PK; Balaratnasingam, C; Cringle, SJ; Morgan, WH; McAllister, IL; Yu, DY
Investigative ophthalmology & visual science 53 5728-36 2012
We investigated quantitatively the distribution of blood vessels in different neural layers of the human retina.A total of 16 human donor eyes was perfusion-fixed and labeled for endothelial f-actin. Retinal eccentricity located 3 mm superior to the optic disk was studied using confocal scanning laser microscopy. Immunohistochemical methods applied to whole-mount and transverse sections were used to colocalize capillary networks with neuronal elements. Capillary morphometry, diameter, and density measurements were compared among networks.Four different capillary networks were identified and quantified in the following regions: Nerve fiber layer (NFL), retinal ganglion cell (RGC) layer, border of the inner plexiform layer (IPL) and superficial boundary of the inner nuclear layer (INL), and boundary of the deep INL and outer plexiform layer. The innermost and outermost capillary networks demonstrated a laminar configuration, while IPL and deep INL networks displayed a complex three-dimensional configuration. Capillary diameter in RGC and IPL networks were significantly less than in other networks. Capillary density was greatest in the RGC network (26.74%), and was significantly greater than in the NFL (13.69%), IPL (11.28%), and deep INL (16.12%) networks.The unique metabolic demands of neuronal sub-compartments may influence the morphometric features of regional capillary networks. Differences in capillary diameter and density between networks may have important correlations with neuronal function in the human retina. These findings may be important for understanding pathogenic mechanisms in retinal vascular disease.
|Gβ3 is required for normal light on responses and synaptic maintenance.|
Anuradha Dhingra,Hariharasubramanian Ramakrishnan,Adam Neinstein,Marie E Fina,Ying Xu,Jian Li,Daniel C Chung,Arkady Lyubarsky,Noga Vardi
The Journal of neuroscience : the official journal of the Society for Neuroscience 32 2012
Heterotrimeric G-proteins, comprising Gα and Gβγ subunits, couple metabotropic receptors to various downstream effectors and contribute to assembling and trafficking receptor-based signaling complexes. A G-protein β subunit, Gβ(3), plays a critical role in several physiological processes, as a polymorphism in its gene is associated with a risk factor for several disorders. Retinal ON bipolar cells express Gβ(3), and they provide an excellent system to study its role. In the ON bipolar cells, mGluR6 inverts the photoreceptor's signal via a cascade in which glutamate released from photoreceptors closes the TRPM1 channel. This cascade is essential for vision since deficiencies in its proteins lead to complete congenital stationary night blindness. Here we report that Gβ(3) participates in the G-protein heterotrimer that couples mGluR6 to TRPM1. Gβ(3) deletion in mouse greatly reduces the light response under both scotopic and photopic conditions, but it does not eliminate it. In addition, Gβ(3) deletion causes mislocalization and downregulation of most cascade elements and modulators. Furthermore, Gβ(3) may play a role in synaptic maintenance since in its absence, the number of invaginating rod bipolar dendrites is greatly reduced, a deficit that was not observed at 3 weeks, the end of the developmental period.
|mGluR6 deletion renders the TRPM1 channel in retina inactive.|
Ying Xu,Anuradha Dhingra,Marie E Fina,Chieko Koike,Takahisa Furukawa,Noga Vardi
Journal of neurophysiology 107 2012
In darkness, glutamate released from photoreceptors activates the metabotropic glutamate receptor 6 (mGluR6) on retinal ON bipolar cells. This activates the G protein G(o), which then closes transient receptor potential melastatin 1 (TRPM1) channels, leading to cells' hyperpolarization. It has been generally assumed that deleting mGluR6 would render the cascade inactive and the ON bipolar cells constitutively depolarized. Here we show that the rod bipolar cells in mGluR6-null mice were hyperpolarized. The slope conductance of the current-voltage curves and the current noise were smaller than in wild type. Furthermore, while in wild-type rod bipolar cells, TRPM1 could be activated by local application of capsaicin; in null cells, it did not. These results suggest that the TRPM1 channel in mGluR6-null rod bipolar cells is inactive. To explore the reason for this lack of activity, we tested if mGluR6 deletion affected expression of cascade components. Immunostaining for G protein subunit candidates Gα(o), Gβ(3), and Gγ(13) showed no significant changes in their expression or distribution. Immunostaining for TRPM1 in the dendritic tips was greatly reduced, but the channel was still present in the soma and primary dendrites of mGluR6-null bipolar cells, where a certain fraction of TRPM1 appears to localize to the plasma membrane. Consequently, the lack of TRPM1 activity in the null retina is unlikely to be due to failure of the channels to localize to the plasma membrane. We speculate that, to be constitutively active, TRPM1 channels in ON bipolar cells have to be in a complex, or perhaps require an unidentified factor.
|Melatonin receptors are anatomically organized to modulate transmission specifically to cone pathways in the retina of Xenopus laevis.|
Allan F Wiechmann,David M Sherry
The Journal of comparative neurology 520 2012
Melatonin receptors have been identified in several retinal cell types, including photoreceptors, horizontal cells, amacrine cells, and ganglion cells. Recent reports suggest that melatonin potentiates signaling from rods to inner retinal neurons. However, the organization of the melatonin receptors mediating this action in the outer plexiform layer (OPL) is not clear. To assess melatonin receptor localization in the OPL, double-label confocal immunohistochemistry for Mel1a or Mel1b melatonin receptors was performed in combination with markers for cone photoreceptors (calbindin, XAP-1) and ON bipolar cells (guanine nucleotide binding protein alpha, Goα) on the retina of Xenopus laevis. Both Mel1a and Mel1b receptors were specifically associated with processes contacting the pedicles of cones, but localized to processes from different sets of second-order neurons. Mel1a receptors localized to the large axonal processes of horizontal cells, while Mel1b receptors localized to the dendrites of OFF bipolar cells. Both receptors also localized to third-order amacrine and ganglion cells and their processes in the inner plexiform layer. This study indicates that Mel1a and Mel1b melatonin receptors are expressed specifically in the Xenopus OPL to modulate transmission from cones to horizontal cells and OFF bipolar cells, respectively; they are second-order neurons that predominantly contact ribbon synapses and display OFF responses to light. When combined with results from recent physiological studies, the current results suggest a conserved function for melatonin in enhancing transmission from rods to second-order neurons across species, although the precise mechanisms by which melatonin enhances this transmission are likely to vary in a species-dependent manner.
|Anti-G Protein Goalpha, clone 2A - Data Sheet|