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, 7 (10), 1079-87

Essential Role of Ca2+-binding Protein 4, a Cav1.4 Channel Regulator, in Photoreceptor Synaptic Function

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Essential Role of Ca2+-binding Protein 4, a Cav1.4 Channel Regulator, in Photoreceptor Synaptic Function

Françoise Haeseleer et al. Nat Neurosci.

Abstract

CaBP1-8 are neuronal Ca(2+)-binding proteins with similarity to calmodulin (CaM). Here we show that CaBP4 is specifically expressed in photoreceptors, where it is localized to synaptic terminals. The outer plexiform layer, which contains the photoreceptor synapses with secondary neurons, was thinner in the Cabp4(-/-) mice than in control mice. Cabp4(-/-) retinas also had ectopic synapses originating from rod bipolar and horizontal cells tha HJt extended into the outer nuclear layer. Responses of Cabp4(-/-) rod bipolars were reduced in sensitivity about 100-fold. Electroretinograms (ERGs) indicated a reduction in cone and rod synaptic function. The phenotype of Cabp4(-/-) mice shares similarities with that of incomplete congenital stationary night blindness (CSNB2) patients. CaBP4 directly associated with the C-terminal domain of the Ca(v)1.4 alpha(1)-subunit and shifted the activation of Ca(v)1.4 to hyperpolarized voltages in transfected cells. These observations indicate that CaBP4 is important for normal synaptic function, probably through regulation of Ca(2+) influx and neurotransmitter release in photoreceptor synaptic terminals.

Conflict of interest statement

COMPETING INTERESTS STATEMENT

The authors declare that they have no competing financial interests.

Figures

Figure 1
Figure 1
CaBP4 protein sequence, tissue distribution and immunolocalization. (a) Primary structure of mouse (m), rat (r), bovine (b) and human (h) CaBP4 (accession numbers AY039218, XM_344981, AY048883 and AY039271). The identical residues in all sequences are in white on a black background. Conservative substitutions are in white on a gray background. Functional EF-hand motifs are shown as shaded boxes and nonfunctional EF-hand motif as an open box. Arrows indicate the intronexon junction of the CABP4 gene. (b) Northern blot analysis of CaBP4 mRNA from various human (left) or rat (right) tissues. Control hybridization with a P-labeled β-actin mRNA is also shown. (c) PCR analysis of CaBP4 transcript in various mouse tissues. A positive control was carried out with primers specific for glyceraldehyde-3-phosphate dehydrogenase (G3PDH). (d) In situ hybridization of CaBP4 transcripts in mouse retina using antisense (left) and sense (right) RNA. (e) In situ hybridization of CaBP4 transcripts in monkey retina using antisense (left) and sense (right) RNA. Inset, immunoreactivity with antibodies to red and green opsin (left) and blue opsin (right) was covisualized with CaBP4 transcripts (blue signal). (f) Presynaptic localization of mouse CaBP4 (red) demonstrated by covisualization with a presynaptic protein (PSD95; green). Arrows indicate rod spherules and cone pedicles. (g) Colocalization of synaptic vesicle proteins SV2 and synaptophysin (green) with mouse CaBP4 (red). In f and g, yellow indicates overlap of two immunoreactivities and blue indicates cell nuclei (Hoechst 33342 staining). INL, inner nuclear layer; ONL, outer nuclear layer; OPL, outer plexiform layer. (df) Scale bars, 20 μm.
Figure 2
Figure 2
Characterization of CaBP4 knockout mice. (a) Immunoblotting of retinal extracts from Cabp4+/+, Cabp4+/− and Cabp4−/− mice probed with anti-CaBP4 or anti-synaptophysin (control). (b) Immunolocalization of CaBP4 in the retina of Cabp4+/+ mouse (left). Lack of CaBP4 immunoreactivity in the retina of Cabp4−/− mouse (right). Scale bars, 20 μm. Nuclei are visualized by staining with Hoechst 33342 dye (blue). (c) The thickness of individual retinal layers from 8-to 10-week-old Cabp4+/+ (black bars) and Cabp4−/− mice (gray bars) measured at 1.25 mm inferior from the optic nerve head. *P < 0.01. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; PR, photoreceptor outer and inner segments; WR, whole retina. (d,e) Rod outer segment (ROS; d) and outer plexiform layer (ORPL; e) thickness (in micrometers) plotted as a function of the retinal location (in millimeters) from the optic nerve head. (f) Montage of cross-sections through the retinas of 2-month-old mice analyzed by transmission electron microscopy. The outer plexiform layer is thinner in Cabp4−/− mice than it is in Cabp4+/+ mice. IS, inner segment; OS, outer segment. (g) Higher magnification of a cross-section through the outer plexiform layer. Photoreceptor terminals are present in the outer plexiform layer of Cabp4+/+ mice (arrow), but fewer and altered terminals are observed in Cabp4−/− mice. A synaptic ribbon is shown at higher magnification (inset; scale bars, 0.2 μm).
Figure 3
Figure 3
Synaptic connection between photoreceptors and bipolar cells. (a) Immunolocalization of GluR1 (red) and PSD95 (green). In Cabp4+/+ (left) and Cabp4−/− (right) mice, GluR1 is expressed in off-cone bipolar cell dendrites proximal to cone pedicles. PSD95 is localized proximal to the plasma membrane of the photoreceptor synaptic terminals. (b) Immunolocalization of mGluR6 (red) and PSD95 (green). A photoreceptor presynapse in a Cabp4+/+ mouse shows morphologically distinguishable rod spherules and cone pedicles (arrows indicate cone pedicles). Cabp4−/− mice have disorganized synaptic processes.(c) Immunolocalization of CaBP4 (red) and the presynaptic marker bassoon (green). Insets show higher-magnification images. (d) Immunolocalization of calbindin, a marker for H1 horizontal cells. Each picture is a three-dimensional projection made from multiple stacks of confocal images. (e,f) Three-dimensional projections of PKCα (red, a marker for rod bipolar cells) and PSD95 (green) immunolocalization in (e) Cabp4+/+ and (f) Cabp4−/− mice. Arrow indicates ectopic synapse in the outer nuclear layer between rod photoreceptor and bipolar cells. In af, nuclei were visualized by staining with Hoechst 33342 dye. Scale bars, 20 μm (in inset, 5 μm).
Figure 4
Figure 4
Current responses of Cabp4+/+ and Cabp4−/− rod outer segments. (a) Flash family measured from a Cabp4+/+ rod. Average responses (5–100 trials at each flash strength) are superimposed for flashes producing 0.65, 1.3, 7, 14, 28, 56, 112 and 224 Rh*. (b) Flash family measured from a Cabp4−/− rod as in a. Flash strengths are 1.9, 3.8, 7.6, 14, 26, 56, 112 and 224 Rh*. (c) Stimulus-response relationship for the Cabp4+/+ (•) and Cabp4−/− (○) rod in a and b. Response amplitudes were normalized to the maximal response and were plotted against flash strength. Saturating exponential fits to the data were used to estimate the half-saturating flash strength.
Figure 5
Figure 5
Voltage-clamp responses of Cabp4+/+ and Cabp4−/− rod bipolars. (a) Flash family measured from a Cabp4+/+ rod bipolar held at −60 mV. Average responses are superimposed for flashes producing 0.28, 0.55, 1.1, 2.2, 4.5, 9 and 18 Rh*/rod. (b) Flash family measured from a Cabp4−/− rod bipolar as in a. Flash strengths are 2.8, 5.6, 11, 22, 44, 90 and 180 Rh*/rod. (c) Stimulus-response relationship for the Cabp4+/+ (•) and Cabp4−/− (○) rod bipolar cells in a and b. (d) Stimulus-response relationship as in c with the response amplitudes normalized to the maximal response. Hill curves that were fit to the data were used to estimate the half-saturating flash strength. (e) Effect of APB on the dark current and saturating response of a Cabp4+/+ rod bipolar. Saturating flashes producing 36 Rh*/rod were delivered every 3 s. The two traces shown are responses measured before and after adding 8 μM APB to the superfusion solution. (f) Effect of APB on the dark current and saturating response of a Cabp4−/− rod bipolar, as in e. Flash strength was 180 Rh*/rod.
Figure 6
Figure 6
Single-flash ERG responses of increasing intensity for Cabp4−/− and Cabp4+/+ mice. (a,c) Serial ERG responses to increasing flash stimuli obtained from Cabp4−/− and Cabp4+/+ mice under (a) dark-adapted and (c) light-adapted conditions. Single-flash timing is indicated by the filled triangle. (b) Plotted ERG a-wave and b-wave amplitudes in response to increasing stimuli in Cabp4−/− mice showed significantly lower responses as compared with those in Cabp4+/+ mice in dark-adapted conditions (***P < 0.0001; n = 8). (d) The b-wave amplitudes in Cabp4−/− mice were also lower as compared with those in Cabp4+/+ mice in light-adapted conditions (*P < 0.01; n = 8) but the a-wave amplitudes showed smaller differences. Light-adapted responses were examined after bleaching at 1.4 log cd·m−2 for 15 min.
Figure 7
Figure 7
CaBP4 interacts with and modulates Cav1.4. (a) Affinity chromatography of purified recombinant mCaBP4 on Ca1.4vα1 column. The eluted fractions were probed with anti-CaBP4. Lanes 1–4, elution with 3 mM EGTA; lane 5, final elution with 3 mM EGTA; lanes 6–8: further elution with 0.1 M glycine buffer, pH 2.1. (b) Colocalization of CaBP4-DsRed2 with Cav1.4-GFP in HEK293 cells. Confocal images of HEK293 cells transfected with mCaBP4-DsRed2 and Cav1.4–GFP (left three panels) or transfected with mCaBP4-DsRed2 alone (right panel). The yellow color indicates colocalization of the overexpressed fusion proteins. Scale bars, 12.5 μ) m. (c) Modulation of Cav1.4 activation by CaBP4 in transfected HEK293T cells. Whole-cell Ca2+ currents recorded in cells transfected with Cav1.4 subunits (α1 1.4, β2A, α2δ) alone (left) or cotransfected with CaBP4 (right). Shown are representative traces of Ca2+ currents evoked by 50-ms steps from a holding voltage of −80 mV to various test voltages. Extracellular recording solution contained 20 mM Ca2+ and intracellular solution contained 5 mM EGTA. CaBP4 enhanced activation of Ca2+ currents at negative voltages as indicated. (d) Current-voltage relationship from cells transfected with Cav1.4 alone (○) or cotransfected with CaBP4 (•). For each test voltage (Vm), I/Imax represents the current amplitude measured at 45 ms normalized to the maximal current amplitude obtained in the series (mean ± s.e.m.; n = 7–10).

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