A dual role for Cav1.4 Ca2+ channels in the molecular and structural organization of the rod photoreceptor synapse

Abstract

Synapses are fundamental information processing units that rely on voltage-gated Ca²⁺ (Ca_v) channels to trigger Ca²⁺-dependent neurotransmitter release. Ca_v channels also play Ca²⁺-independent roles in other biological contexts, but whether they do so in axon terminals is unknown. Here, we addressed this unknown with respect to the requirement for Ca_v1.4 L-type channels for the formation of rod photoreceptor synapses in the retina. Using a mouse strain expressing a non-conducting mutant form of Ca_v1.4, we report that the Ca_v1.4 protein, but not its Ca²⁺ conductance, is required for the molecular assembly of rod synapses; however, Ca_v1.4 Ca²⁺ signals are needed for the appropriate recruitment of postsynaptic partners. Our results support a model in which presynaptic Ca_v channels serve both as organizers of synaptic building blocks and as sources of Ca²⁺ ions in building the first synapse of the visual pathway and perhaps more broadly in the nervous system.

Keywords: calcium; mouse; neuroscience; photoreceptor; retina; synapse.

Figure 1.. Characterization of G369i KI mouse strain to study the requirement of Ca_v1.4 Ca²⁺ signals in rod synaptogenesis.

(A), potential roles of Ca_v1.4 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 the rod spherule; Ca_v1.4 clusters beneath the ribbon that tethers glutamate (Glu)-filled synaptic vesicles (SVs). In Ca_v1.4 KO retina, ribbons do not form, and HC and RBC neurites sprout into the outer nuclear layer (ONL). The efficacy of a non-conducting Ca_v1.4 mutant channel to support rod synaptogenesis will depend on whether Ca²⁺ influx through Ca_v1.4 is required. (B), schematic of Ca_v1.4 pore-forming subunit with four homologous repeats (I–IV) each with six transmembrane domains; location of the glycine insertion in domain I (G369i) is indicated. G369i mutation (red). (C), left, representative Ba²⁺ currents (I_Ba) evoked by 20 ms voltage steps from −90 mV to the indicated voltages in transfected HEK293T cells. Boxed regions indicate gating currents evident in cells co-transfected with β₂ + α₂δ−1 and WT or G369i mutant channels but not in cells transfected with β₂ and α₂δ−1 alone. Upper right, representative gating currents from boxed regions (left) elicited by a voltage step to +70 mV. Lower right, graph shows I–V relationship for cells co-transfected with β₂ + α₂δ−1 alone (n = 5), or co-transfected with WT (n = 9) or G369i mutant channels (n = 6). Points represent mean ± SEM. (D), representative traces (left) and average amplitudes of peak rod Ca²⁺ currents (I_Ca, right) recorded in acute slices from WT, G369i KI, and Ca_v1.4 KO mouse retinas. Stimulus protocol was a 1 s voltage ramp from −90 to +50 mV. Points represent individual recorded rods with the mean and SEM indicated with bars. P values were determined by one-way ANOVA with Uncorrected Fisher’s Least Significant Difference post-hoc test. (E), confocal micrographs of the OPL of wild-type (WT) and G369i KI retina double-labeled with antibodies against Ca_v1.4 and CtBP2 (CtBP2 labeling removed here for clarity). (F), expanded view of the boxed regions in E showing both CtBP2 and Ca_v1.4 labeling. Arrows depict arc-shaped ribbons apposed to Ca_v1.4 labeling. Scale bars, 5 μm. (G) representative voltage responses from flash ERGs recorded in dark- adapted WT (n = 4) and G369i KI (n = 4) mice. Arrowheads indicate time of flash. Numbers indicate flash intensities (cd•s /m²). (H, I), a-wave amplitudes (H) and b-wave amplitudes (I) measured from recordings obtained as in (G). Points represent mean ± SEM.

Figure 1—figure supplement 1.. Characterization of G369i mutation in transfected HEK293T cells.

(A) Representative Ba²⁺ currents (I_Ba) evoked by 50 ms voltage step from −80 to 0 mV in HEK293T cells co-transfected with wild-type (WT) (black) or G369i (red) Ca_v1.4 channels and auxiliary subunits β₂ and α₂δ−4. (B) Graph showing I–V relationship for cells co-transfected with WT or G369i mutant channels. I_Ba was evoked by 50 ms voltage steps from −70 to +70 mV in +10 mV increments. I_Ba is plotted against each test voltage (V_m). Points represent mean ± SEM. Parentheses indicate number of cells. (C) HEK293T cells co-transfected as in A and immunofluorescently labeled using FLAG-tag antibodies. Scale bar = 10 μm.

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 genotyping. PAM, protospacer adjacent motif. cRNA, CRISPR RNA. (B) Gel image of PCR products that were amplified from genomic DNA followed by Fnu4HI digestion. Following digestion, two DNA fragments of ~330 base pairs (bp) and 60 bp (not shown) are present if the G369i mutation and Fnu4HI restriction site are within the CACNA1F gene. Note: the CACNA1F gene lies on the X chromosome, which precludes male mice from being heterozygous for the G369i mutation. (C) Western blot analysis of whole retina lysates collected from WT, G369i KI and Ca_v1.4 KO mice. Blots were probed with antibodies against Ca_v1.4 and β-actin. Bands corresponding to Ca_v1.4 were present in WT and G369i KI retina lysates, but not lysates from Ca_v1.4 KO mice.

Figure 2.. Rod ribbons still form in the absence of Ca_v1.4 Ca²⁺ signals.

(A) Confocal micrographs of the OPL of WT, G369i KI and Ca_v1.4 KO mice labeled with antibodies against CtBP2 to mark ribbons (arrows). Arrowheads depict spheres resembling immature ribbons. (B) Cumulative frequency of rod ribbon lengths in WT, G369i KI, and Ca_v1.4 KO. Ribbon states less than and greater than 0.95 µm were classified as spheres and ribbons, respectively. (C, D) Violin plots of rod ribbon lengths (C) and sphericity (D) obtained from dataset in B. **** p<0.001 by one-way ANOVA with Tukey’s multiple comparison post-hoc test. WT, n = 1264 ribbons; KO, n = 348 ribbons; G369i KI, n = 1322 ribbons. Data were collected from the retinas of at least six individual mice per genotype. (E) Schematic of a rod active zone and associated proteins. SV, synaptic vesicle. (F–I) Confocal micrographs (maximum z-projections) of the OPL of WT, G369i KI and Ca_v1.4 KO mice double-labeled with antibodies against CtBP2 and PSD95 (F), Ribeye and β-dystroglycan (β-Dys), (G) CtBP2 and Bassoon (H) and CtBP2 and RIM2 (I). Arrows and arrowheads depict ribbons and spheres, respectively. Scale bars, 5 μm in A, 2 μm in F-I.

Figure 3.. Postsynaptic signaling at rod synapses is partially intact in the absence of Ca_v1.4 Ca²⁺ signals.

(A), schematic illustrating synaptic transmission at rod-RBC synapses. In the dark, rods are depolarized, which activates Ca_v1.4 and Ca²⁺-triggered glutamate (E) release. Activation of the mGluR6/G-protein pathway by glutamate or L-AP4 inhibits TRPM1-dependent channels. Light signals or the mGluR6 antagonist CPPG activate the ON pathway by disinhibiting the TRPM1-dependent conductance. (B–D), confocal micrographs of the OPL of WT, G369i KI and Ca_v1.4 KO mice triple-labeled with antibodies against Ribeye, mGluR6, and TRPM1. In B,C, only signals for Ribeye and mGluR6 (B) or TRPM1 (C) are shown for clarity. (C) Higher magnification views of boxed regions are shown in the merged image (D). Arrows and arrowheads depict ribbons and spheres, respectively. (E–F), whole-cell patch clamp electrophysiology of RBCs recorded in WT, G369i KI, and Ca_v1.4 KO mouse retinal slices. E, left, representative image of Lucifer Yellow-filled RBC showing typical lobular-shaped terminal (arrowhead). Right, representative current responses to CPPG puffs in RBCs held at −60 mV and +60 mV. Lines within each current trace are low-pass FFT filtered data. F, peak amplitudes of CPPG-evoked EPSCs. Each shape represents individual cells recorded at −60 and +60 mV. Bars represent mean ± SEM. ****p<0.0001, **p<0.01, *p<0.05, by one-way ANOVA with Fishers least significant difference post-hoc test. Scale bars, 2 μm in B,C, 1 μm in D, 15 μm in E.

Figure 4.. Rod synapses lack invaginating HC and RBC neurites in the absence of Ca_v1.4 Ca²⁺ signals.

(A), confocal micrographs of the ONL and OPL of WT, G369i KI and Ca_v1.4 KO retinas immunolabeled for calbindin and PKCα. The schematic below illustrates neurite sprouting in each genotype. (B), quantification of the area in the ONL occupied by HCs (calbindin) or RBCs (PKCα). One-way ANOVA with Fishers least significant difference post-hoc test. ****p<0.0001, **p<0.01, *p<0.05. n = 6 mice for each genotype. (C), confocal micrographs of the OPL of WT and the ONL and OPL of G369i KI retinas immunolabeled for Ribeye and Calbindin (upper panels) or PKCα (lower panels). (D), confocal micrographs of the ONL of G369i KI retinas that were immunolabeled for CtBP2 and Ca_v1.4 (left), PSD95 (middle) or Bassoon (right). (E), TEM image of a rod terminal in a WT retina. (F,G), serial block-face scanning electron microscopy images (F’,G’) and 3D reconstructions (F’’–G’’’) of rod terminals in a G369i KI retina. Rod terminals located within the OPL (F) and ONL (G). The yellow star F’’ indicates a neurite from HC2 sprouting into the ONL. Insets in F’’ are a rotated view. G’’’ is a side view of G’’. Arrows depict anchored ribbons. Arrowheads in G’’’ depict additional ribbon. Asterisks in G’ indicate rod somas. Scale bars, 10 μm in A, 2 μm in C-D, 1 μm in E,F’,G’. Rib, ribbon. RBC, rod bipolar cell. HC, horizontal cell.

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 terminals of G369i KI mice. Filled circles represent the means. Bars represent the SEM. (C) Additional SBFSEM reconstruction of a G369i KI spherule at the OPL/ONL border. Panels are labeled as in Figure 4F–G.

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 of Ribeye-mGluR6 distances. Solid lines represent the medians. Dashed lines represent the quartiles. p-value determined by unpaired t-test. WT, n = 864. G369i KI, n = 1390.