scholarly journals Identification of PKCα-dependent phosphoproteins in mouse retina

2019 ◽  
Author(s):  
Colin M. Wakeham ◽  
Phillip A. Wilmarth ◽  
Jennifer M. Cunliffe ◽  
John E. Klimek ◽  
Gaoying Ren ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Functional Groups ◽  
Phorbol Ester ◽  
Bipolar Cells ◽  
Mouse Retina ◽  
Wild Type ◽  
Light Intensities ◽  
Retinal Rod ◽  
Wide Range ◽  
Rod Bipolar Cells

AbstractAdjusting to a wide range of light intensities is an essential feature of retinal rod bipolar cell (RBC) function. While persuasive evidence suggests this modulation involves phosphorylation by protein kinase C-alpha (PKCα), the targets of PKCα phosphorylation in the retina have not been identified. PKCα activity and phosphorylation in RBCs was examined by immunofluorescence confocal microscopy using a conformation-specific PKCα antibody and antibodies to phosphorylated PKC motifs. PKCα activity was dependent on light and expression of TRPM1, and RBC dendrites were the primary sites of light-dependent phosphorylation. PKCα-dependent retinal phosphoproteins were identified using a phosphoproteomics approach to compare total protein and phosphopeptide abundance between phorbol ester-treated wild type and PKCα knockout (PKCα-KO) mouse retinas. Phosphopeptide mass spectrometry identified over 1100 phosphopeptides in mouse retina, with 12 displaying significantly greater phosphorylation in WT compared to PKCα-KO samples. The differentially phosphorylated proteins fall into the following functional groups: cytoskeleton/trafficking (4 proteins), ECM/adhesion (2 proteins), signaling (2 proteins), transcriptional regulation (3 proteins), and homeostasis/metabolism (1 protein). Two strongly differentially expressed phosphoproteins, BORG4 and TPBG, were localized to the synaptic layers of the retina, and may play a role in PKCα-dependent modulation of RBC physiology. Data are available via ProteomeXchange with identifier PXD012906.SignificanceRetinal rod bipolar cells (RBCs), the second-order neurons of the mammalian rod visual pathway, are able to modulate their sensitivity to remain functional across a wide range of light intensities, from starlight to daylight. Evidence suggests that this modulation requires the serine/threonine kinase, PKCα, though the specific mechanism by which PKCα modulates RBC physiology is unknown. This study examined PKCα phosophorylation patterns in mouse rod bipolar cells and then used a phosphoproteomics approach to identify PKCα-dependent phosphoproteins in the mouse retina. A small number of retinal proteins showed significant PKCα-dependent phosphorylation, including BORG4 and TPBG, suggesting a potential contribution to PKCα-dependent modulation of RBC physiology.HighlightsPKCα is a major source of phosphorylation in retinal RBC dendrites and its activity in RBCs is light dependent.Proteins showing differential phosphorylation between phorbol ester-treated wild type and PKCα-KO retinas belong to the following major functional groups: cytoskeleton/trafficking (4 proteins), ECM/adhesion (2 proteins), signaling (2 proteins), transcriptional regulation (3 proteins), and homeostasis/metabolism (1 protein).The PKCα-dependent phosphoproteins, BORG4 and TPBG, are present in the synaptic layers of the retina and may be involved in PKCα-dependent modulation of RBC physiology.

Protein kinase modulation of GABAA currents in rabbit retinal rod bipolar cells

1996 ◽  
Vol 76 (5) ◽  
pp. 3070-3086 ◽  
Author(s):  
M. A. Gillette ◽  
R. F. Dacheux
Keyword(s):  
Protein Kinase ◽  
Phorbol Ester ◽  
Bipolar Cell ◽  
Kinase Inhibitor ◽  
Bipolar Cells ◽  
Calphostin C ◽  
Retinal Rod ◽  
Low Concentrations ◽  
Current Reduction ◽  
Rod Bipolar Cells

1. Protein kinase modulation of gamma-aminobutyric acid-A (GABAA)- and glycine-activated Cl- currents in freshly dissociated, morphologically identified rabbit retinal rod bipolar cells was studied under voltage clamp with the use of the whole cell patch-clamp technique. Responses to pulses of GABA and glycine were monitored before, during, and after application of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) and protein kinase C (PKC) activators, inactive analogues, and inhibitors. 2. Bath perfusion with either forskolin, an adenylate cyclase activator, or its inactive analogue, 1,9 dideoxyforskolin, reduced the GABA-activated Cl- currents by 30–50%; coapplication of N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide hydrochloride (H-8), a PKA inhibitor, did not prevent the forskolin effects. The membrane-permeable cAMP analogues, 8-bromo-cAMP and 8-(4-Chlorophenylthio)-cAMP, and intracellularly dialyzed cAMP, did not modulate either the GABA- or glycine-activated Cl- current. Perfusion of the phosphodiesterase inhibitor 3-isobutyl-1-methylxantine (IBMX) had no direct effect on the GABA-activated current and did not alter the results with cAMP or its membrane-permeable analogues. Collectively, these results make it very unlikely that PKA represents an important mechanism of either GABAA or glycine channel modulation in the rabbit rod bipolar cell. 3. Although the isoquinoline sulfonamide protein kinase inhibitor H-8 was without discernible effect, the related compounds 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochlorine (H-7) and N-(2-Aminoethyl)-5-isoquinolinesulfonamide dihydrochloride (H-9) both dramatically reduced the GABA response. H-7 also strongly reduced the response to glycine, whereas H-8 had no effect and H-9 had an intermediate effect. Because only certain members of this inhibitor class of agents proved effective, and their effectiveness appeared unrelated to the established activity profiles, these agents probably inhibit the Cl- currents in a phosphorylation-independent manner. Direct interaction of these inhibitors with binding sites in the GABAA receptor-channel complex has been previously reported in some other preparations. 4. The phorbol ester and PKC activator phorbol 12,13 dibutyrate (PDB) led to a 35-55% reduction in the GABA-activated Cl- current of the rod bipolar cell. The broad-spectrum kinase inhibitor staurosporine, and the more PKC-specific inhibitor calphostin C, had no direct effect on GABA responses, but prevented Cl- current reduction when coapplied with PDB. Phorbol 12-myristate 13-acetate (PMA) reduced the GABA-activated current in a fashion very similar to PDB. Staurosporine and calphostin C blocked the PMA effect. No reduction of Cl- current was seen with the inactive analogue, 4-alpha-PMA, used as a control for PKC-independent phorbol ester effects. 5. PDB effectively reduced the GABA-activated Cl- current of the rod bipolar cell at low concentrations, whereas PMA had a diminished effect at low concentrations. This is consistent with the reported concentration-dependent abilities of these agents to promote translocation of PKC-alpha immunoreactivity from the membrane to the cytosolic compartment in the rabbit retinal rod bipolar cell. Collectively, the data from phorbol esters, inactive analogues, and kinase inhibitors support the existence of a PKC-mediated mechanism for GABA-activated Cl- current reduction in these cells. 6. The naphthalenesulfonamide PKC activator N-(n-Heptyl)-5-chloro-1-naphthalenesulfonamide (SC-10) also potently and reversibly reduced the GABA-activated current. Staurosporine and calphostin C eliminated this effect. When the nonhydrolyzable guanosine 5'-triphosphate (GTP) analogue guanosine 5'-O-(3-thiotriphosphate) tetralithium salt (GTP-gamma-S) replaced GTP in the recording pipette, the SC-10-mediated GABA current reduction became irreversible.(ABSTRACT TRUNCATED)

scholarly journals The Effect of PKCα on the Light Response of Rod Bipolar Cells in the Mouse Retina

2015 ◽  
Vol 56 (8) ◽  
pp. 4961 ◽  
Author(s):  
Wei-Hong Xiong ◽  
Ji-Jie Pang ◽  
Mark E. Pennesi ◽  
Robert M. Duvoisin ◽  
Samuel M. Wu ◽  
...  
Keyword(s):  
Light Response ◽  
Bipolar Cells ◽  
Mouse Retina ◽  
Rod Bipolar Cells

Unique functional properties of the APB sensitive and insensitive rod pathways signaling light decrements in mouse retinal ganglion cells

2006 ◽  
Vol 23 (1) ◽  
pp. 127-135 ◽  
Author(s):  
GUO-YONG WANG
Keyword(s):  
Functional Properties ◽  
Ganglion Cells ◽  
Amacrine Cells ◽  
Bipolar Cells ◽  
Mouse Retina ◽  
Clear Cut ◽  
Types Of Information ◽  
Rod Bipolar Cells ◽  
Rod Pathway ◽  
Cone Bipolar Cells

Light decrements are mediated by two distinct groups of rod pathways in the dark-adapted retina that can be differentiated on the basis of their sensitivity to the glutamate agonist DL-2-amino-phosphonobutyric (APB). By means of the APB sensitive pathway, rods transmit light decrementsviarod bipolar cells to AII amacrine cells, then to Off cone bipolar cells, which in turn innervate the dendrites of Off ganglion cells. APB hyperpolarizes rod bipolar cells, thus blocking this rod pathway. With APB insensitive pathways, rods either directly synapse onto Off cone bipolar cells, or rods pass light decrement signal to cones by gap junctions. In the present study, whole-cell patch-clamp recordings were made from ganglion cells in the dark-adapted mouse retina to investigate the functional properties of APB sensitive and insensitive rod pathways. The results revealed several clear-cut differences between the APB sensitive and APB insensitive rod pathways. The latency of Off responses to a flashing spot of light was significantly shorter for the APB insensitive pathways than those for the APB sensitive pathway. Moreover, Off responses of the APB insensitive pathways were found to be capable of following substantially higher stimulus frequencies. Nitric oxide was found to selectively block Off responses in the APB sensitive rod pathway. Collectively, these results provide evidence that the APB sensitive and insensitive rod pathways can convey different types of information signaling light decrements in the dark-adapted retina.

scholarly journals Cross-synaptic synchrony and transmission of signal and noise across the mouse retina

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
William N Grimes ◽  
Mrinalini Hoon ◽  
Kevin L Briggman ◽  
Rachel O Wong ◽  
Fred Rieke
Keyword(s):  
Single Neuron ◽  
Amacrine Cells ◽  
Light Level ◽  
Bipolar Cells ◽  
Mouse Retina ◽  
Anatomical Connectivity ◽  
Physiological Conditions ◽  
Synaptic Noise ◽  
Mammalian Retina ◽  
Rod Bipolar Cells

Cross-synaptic synchrony—correlations in transmitter release across output synapses of a single neuron—is a key determinant of how signal and noise traverse neural circuits. The anatomical connectivity between rod bipolar and A17 amacrine cells in the mammalian retina, specifically that neighboring A17s often receive input from many of the same rod bipolar cells, provides a rare technical opportunity to measure cross-synaptic synchrony under physiological conditions. This approach reveals that synchronization of rod bipolar cell synapses is near perfect in the dark and decreases with increasing light level. Strong synaptic synchronization in the dark minimizes intrinsic synaptic noise and allows rod bipolar cells to faithfully transmit upstream signal and noise to downstream neurons. Desynchronization in steady light lowers the sensitivity of the rod bipolar output to upstream voltage fluctuations. This work reveals how cross-synaptic synchrony shapes retinal responses to physiological light inputs and, more generally, signaling in complex neural networks.

scholarly journals Expression and distribution of Trophoblast Glycoprotein in the mouse retina

2019 ◽  
Author(s):  
Colin M Wakeham ◽  
Gaoying Ren ◽  
Catherine W Morgans
Keyword(s):  
Time Course ◽  
Expression Patterns ◽  
Pdz Domain ◽  
Bipolar Cells ◽  
Hek293 Cells ◽  
Mouse Retina ◽  
Leucine Rich Repeat ◽  
Wild Type ◽  
Rich Domain ◽  
Activity State

AbstractWe recently identified the leucine-rich repeat adhesion protein, trophoblast glycoprotein (TPBG), as a novel PKCα-dependent phosphoprotein in retinal rod bipolar cells (RBCs). Since TPBG has not been thoroughly examined in the retina, this study characterizes the localization and expression patterns of TPBG in the developing and adult mouse retina using two antibodies, one against the N-terminal, leucine-rich domain and the other against the C-terminal PDZ-interacting motif. Both antibodies labeled dendrites and synaptic terminals of RBCs, as well as the cell bodies and dendrites of an uncharacterized class of amacrine cell. In transfected HEK293 cells, TPBG was localized to the plasma membrane and intracellular membranes and was associated with the tips of thin filopodia-like membrane projections. TPBG immunofluorescence in RBCs detected with the C-terminal antibody was strongly dependent on the activity state of the adult retina, with less labeling in dark-adapted compared to light-adapted retina, and less labeling in light-adapted PKCα knockout and TRPM1 knockout retinas compared to wild type, despite no change in total TPBG detected by immunoblotting. These results suggest that the C-terminal epitope is blocked in the dark-adapted and knockout retinas compared to light-adapted wild type retinas, possibly through interaction with a PDZ domain protein. During development, TPBG expression increases dramatically just prior to eye opening with a time course closely correlated with that of TRPM1 expression. In the retina, leucine-rich repeat proteins like TPBG have been implicated in the development and maintenance of functional bipolar cell synapses, and TPBG may play a similar role in RBCs.

scholarly journals Glycine Receptors and Glycinergic Synaptic Input at the Axon Terminals of Mammalian Retinal Rod Bipolar Cells

2003 ◽  
Vol 553 (3) ◽  
pp. 895-909 ◽  
Author(s):  
Jinjuan Cui ◽  
Yu‐Ping Ma ◽  
Stuart A. Lipton ◽  
Zhuo‐Hua Pan
Keyword(s):  
Synaptic Input ◽  
Bipolar Cells ◽  
Glycine Receptors ◽  
Axon Terminals ◽  
Retinal Rod ◽  
Rod Bipolar Cells
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