A dual role for Cav1.4 Ca2+ channels in the molecular and structural organization of the rod photoreceptor synapse
- Department of Molecular Physiology and Biophysics, University of Iowa, United States
- Iowa Neuroscience Institute, University of Iowa, United States
- Pappajohn Biomedical Institute, University of Iowa, United States
- Department of Neuroscience, University of Wisconsin-Madison, United States
- Department of Ophthalmology, University of California, San Francisco, United States
- Department of Biochemistry, University of Iowa, United States
- Department of Ophthalmology, United States
- Department of Ophthalmology and Visual Science, University of Wisconsin-Madison, United States
Figures
Figure 1 with 2 supplements
Characterization of G369i KI mouse strain to study the requirement of Cav1.4 Ca2+ signals in rod synaptogenesis.
(A), potential roles of Cav1.4 in the assembly of rod synapses. In the outer plexiform layer (OPL) of WT mice, neurites of two horizontal cells (HCs) and one rod bipolar cell (RBC) invaginate into …
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Figure 1—source data 1
Traces corresponding to ICa that were used for analyses of rod peak ICa in Figure 1D are shown in the ‘.PDF’ file.
Individual values obtained for peak ICa in different cells are listed in the ‘.xlsx’ file.
- https://cdn.elifesciences.org/articles/62184/elife-62184-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Characterization of G369i mutation in transfected HEK293T cells.
(A) Representative Ba2+ currents (IBa) evoked by 50 ms voltage step from −80 to 0 mV in HEK293T cells co-transfected with wild-type (WT) (black) or G369i (red) Cav1.4 channels and auxiliary subunits …
Figure 1—figure supplement 2
Characterization of the G369i KI mouse line.
(A) CRISPR-Cas9 genome editing was used to insert a glycine residue between G369 and V370. The restriction site recognized by the endonuclease Fnu4HI was included in the repair template for …
Figure 2
Rod ribbons still form in the absence of Cav1.4 Ca2+ signals.
(A) Confocal micrographs of the OPL of WT, G369i KI and Cav1.4 KO mice labeled with antibodies against CtBP2 to mark ribbons (arrows). Arrowheads depict spheres resembling immature ribbons. (B) …
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Figure 2—source data 1
The image archive contains all images used in quantitative analyses in Figure 2B–D.
The data were exported as 8-bit ‘tif’ files (1600 × 1600 pixels). Values obtained for individual data points (and outlier analysis) for summary graphs in Figure 2B–D are contained in ‘.xlsx’ files.
- https://cdn.elifesciences.org/articles/62184/elife-62184-fig2-data1-v2.zip
Figure 3
Postsynaptic signaling at rod synapses is partially intact in the absence of Cav1.4 Ca2+ signals.
(A), schematic illustrating synaptic transmission at rod-RBC synapses. In the dark, rods are depolarized, which activates Cav1.4 and Ca2+-triggered glutamate (E) release. Activation of the …
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Figure 3—source data 1
Traces corresponding to EPSCs that were used for analyses of EPSC amplitudes in Figure 3F are included in the ‘.PDF’ file; individual values obtained for EPSC amplitudes in different cells are listed in the ‘.xlsx’ file.
- https://cdn.elifesciences.org/articles/62184/elife-62184-fig3-data1-v2.zip
Figure 4 with 2 supplements
Rod synapses lack invaginating HC and RBC neurites in the absence of Cav1.4 Ca2+ signals.
(A), confocal micrographs of the ONL and OPL of WT, G369i KI and Cav1.4 KO retinas immunolabeled for calbindin and PKCα. The schematic below illustrates neurite sprouting in each genotype. (B), …
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Figure 4—source data 1
The image archive contains all images used in quantitative analyses in Figure 4B.
The data were exported as 8-bit ‘tif’ files (800 × 600 pixels). Values obtained for individual data points (and outlier analysis) for summary graphs in are contained in ‘.xlsx’ files.
- https://cdn.elifesciences.org/articles/62184/elife-62184-fig4-data1-v2.zip
Figure 4—figure supplement 1
Multiple ribbons are found in rod terminals of G369i KI mice.
(A) Electron micrographs of rod terminals with multiple ribbons (left) and a club-shaped ribbon (right). Arrows depict separate ribbons. (B) Number of total and floating ribbons in four rod …
Figure 4—figure supplement 2
Nearest-neighbor analysis of Ribeye- and mGluR6-labeled structures.
(A) Deconvolved confocal images from Figure 2B-D. Arrows indicate arc-shaped ribbons in apposition to mGluR6/TRPM1. (B) Cumulative frequency of distances between Ribeye and mGluR6. (C) Violin plot …
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Figure 4—figure supplement 2—source data 1
The image archive contains all images used in quantitative analyses in Figure 4—figure supplement 2B; Figure 4—figure supplement 2C.
The data were exported as 8-bit ‘tif’ files (1024 × 1024 pixels). Values for individual data points (and outlier analysis) for summary graphs in Figure 4—figure supplement 2B are Figure 4—figure supplement 2C contained in excel files.
- https://cdn.elifesciences.org/articles/62184/elife-62184-fig4-figsupp2-data1-v2.zip
Tables
Table 1
Antibodies used in this study.
| Antibody | Host/clonality | Manufacturer | Cat no. | RRID |
|---|---|---|---|---|
| Bassoon | ms-IgG2A | Thermo Fisher Scientific | MA1-20689 | AB_2066981 |
| Cone Arrestin | rabbit | Millipore | AB15282 | AB_1163387 |
| Calbindin (D-28K) | mouse-IgG1 | Sigma | C9848 | AB_476894 |
| PKCα | mouse-IgG2A | Invitrogen | MA1-157 | AB_2536865 |
| rabbit | UC Santa Cruz | SC-208 | AB_2168668 | |
| Ctbp2 | mouse-IgG1 | BD Biosciences | 612044 | AB_399431 |
| Ribeye | rabbit | Synaptic Systems | 192 103 | AB_2086775 |
| Psd95 | mouse-IgG2A | UC Davis/NIH NeuroMab | 75–028 | AB_2292909 |
| RIM2 | rabbit | Synaptic Systems | 140–103 | AB_887776 |
| mGluR6-366 | mouse | Dr. Theodore Wensel | N/A | N/A |
| TRPM1 | mouse-IgG1 | Dr. Theodore Wensel | N/A | N/A |
| Cav1.4 | rabbit | Lee Lab Ab167 | N/A | AB_2650487 |
