November 2008
Volume 49, Issue 11
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Retinal Cell Biology  |   November 2008
Characterization of Ca2+-Binding Protein 5 Knockout Mouse Retina
Author Affiliations
  • Fred Rieke
    From the Howard Hughes Medical Institute and the
    Departments of Physiology and Biophysics and
  • Amy Lee
    Department of Pharmacology and Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, Georgia.
  • Françoise Haeseleer
    Ophthalmology, University of Washington, Seattle, Washington; and the
Investigative Ophthalmology & Visual Science November 2008, Vol.49, 5126-5135. doi:10.1167/iovs.08-2236
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      Fred Rieke, Amy Lee, Françoise Haeseleer; Characterization of Ca2+-Binding Protein 5 Knockout Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2008;49(11):5126-5135. doi: 10.1167/iovs.08-2236.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. The goal of this study was to investigate, with the use of CaBP5 knockout mice, whether Ca2+-binding protein 5 (CaBP5) is required for vision. The authors also tested whether CaBP5 can modulate expressed Cav1.2 voltage-activated calcium channels.

methods. CaBP5 knockout (Cabp5 −/−) mice were generated. The retinal morphology and visual function of 6-week-old Cabp5 −/− mice were analyzed by confocal and electron microscopy, single-flash electroretinography, and whole-cell patch-clamp recordings of retinal ganglion cells. The interaction and modulation of Cav1.2 channels by CaBP5 were analyzed using affinity chromatography, gel overlay assays, and patch-clamp recordings of transfected HEK293 cells.

results. No evidence of morphologic changes and no significant difference in the amplitude of the ERG responses were observed in CaBP5 knockout mice compared with wild-type mice. However, the sensitivity of retinal ganglion cell light responses was reduced by approximately 50% in Cabp5 −/− mice. CaBP5 directly interacted with the CaM-binding domain of Cav1.2 and colocalized with Cav1.2 in rod bipolar cells. In transfected HEK293T cells, CaBP5 suppressed calcium-dependent inactivation of Cav1.2 and shifted the voltage dependence of activation to more depolarized membrane potentials.

conclusions. This study provides evidence that lack of CaBP5 results in reduced sensitivity of rod-mediated light responses of retinal ganglion cells, suggestive of a role for CaBP5 in the normal transmission of light signals throughout the retinal circuitry. The interaction, colocalization, and modulation of Cav1.2 by CaBP5 suggest that CaBP5 can alter retinal sensitivity through the modulation of voltage-gated calcium channels.

CaBP5 is a member of a subfamily of neuronal Ca2+-binding proteins that are highly similar to calmodulin (CaM). 1 2 3 4 CaBPs can modulate various targets also regulated by CaM, including voltage-gated calcium channels, 5 6 7 TRP channels, and inositol 1,4,5-trisphosphate (IP3) receptors. 8 9 10 11 CaBP4, thus far the most characterized member of the CaBP subfamily, is localized at the photoreceptor synaptic terminals. CaBP4 is essential for normal development and maintenance of the photoreceptor output synapse, likely through the modulation of photoreceptor Ca2+ channels and transmitter release. 12 CaBP4 interacts with alpha1F (Cav1.4) L-type voltage-dependent calcium channels in transfected HEK293T cells and shifts their activation to more hyperpolarized voltages. 12 Mutations in the Cabp4 gene have been shown in patients with autosomal recessive incomplete congenital stationary night blindness, 13 a disease associated with mutations in the CACNA1F gene encoding Cav1.4. 14 15 16 CaBPs are also found in the cochlea (e.g., in the inner hair cells) and can modulate Cav1.3 channels that are required for hearing. 7 17  
CaBP5 is expressed in rod and cone bipolar cells in the retina of a variety of mammalian species. In mice, CaBP5 is expressed in rod bipolar cells, in type 5 ON-cone bipolar cells, and in type 3 OFF-cone bipolar cells. 1 18 19 In human retina and monkey retina, rod bipolar cells and ON and OFF cone bipolar cells are immunoreactive for CaBP5. 20 CaBP5 also has been observed in cochlear inner hair cells. 17 The specific function of CaBP5 in vivo remains unclear, but it may modulate various CaM targets in vitro. 1 4 10 CaBP5 modestly suppresses the inactivation of Cav1.3 L-type voltage-activated Ca2+ channels in transfected cells. 17 L-type voltage-activated Ca2+ channels mediate Ca2+ currents in rod bipolar cells. 21 22 23 24 25 26 Low-voltage–activated T-type Ca2+ channels may be expressed in rod bipolar cells, although they may not be directly involved in triggering transmitter release. 21 23 24  
In this study, with the use of mice lacking the expression of CaBP5, we investigated whether CaBP5 is required for vision. We showed that CaBP5 deficiency results in reduced sensitivity of rod-mediated ganglion cell light responses. We also provide evidence that Cav1.2 may be a physiological target for CaBP5. 
Materials and Methods
Animals
Mice were housed in the Department of Comparative Medicine at the University of Washington and treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All procedures for the maintenance and use of animals were approved by the Institutional Animal Care and Use Committee of the University of Washington. 
Antibodies
Commercially available antibodies used were alkaline phosphatase-conjugated anti–mouse and anti–rabbit (Promega, Madison, WI), anti–PKC alpha (Santa Cruz Biotechnology, Santa Cruz, CA), anti–calbindin D-28K (Sigma, St. Louis, MO), anti–calretinin (Chemicon International, Temecula, CA), anti–Cav1.2 (Alomone Laboratories, Jerusalem, Israel), Cy3 goat anti–rabbit and Cy3 goat anti–mouse (Jackson Immunoresearch Laboratories, West Grove, PA), and Alexa Fluor 488 goat anti–mouse and Alexa Fluor 488 goat anti–rabbit (Molecular Probes, Eugene, OR). The development and characterization of rabbit anti–CaBP5 (UW89) was described in Haeseleer et al. 1 To generate the anti–CaBP5 monoclonal antibody, mice were injected with 50 μg purified His-tagged CaBP5 proteins in a RIBI adjuvant system. After two boosts at 2-week intervals, the mouse sera were analyzed using Western blot and immunohistochemistry. One mouse was used for fusion with myeloma. Ninety-six clones were screened for CaBP5 immunoreactivity using Western blot and immunohistochemistry. One clone, A1, which produced antibodies that gave positive signals on retina tissues using Western blot and immunohistochemistry, was selected. 
Generation and Genotyping of Cabp5−/− Mice
A BAC genomic clone originating from DNA of 129/Sv mice and containing the Cabp5 gene was purchased from Genome Systems. Approximately 2 kb, covering the promoter region of the Cabp5 gene upstream of ATG, was amplified by PCR with primer FH576 (5′-GCGGCCGCTGAGACAGTAAACCAGACCC-3′), which was extended with a NotI restriction site, and with primer FH575 (5′-GCTAGCTCAAGCTCTGATGTCAAGATGG-3′), which was extended with a NheI site. The long arm of approximately 6 kb, covering part of intron 2 to intron 5 of the Cabp5 gene, was amplified by PCR with primer FH577 (5′-GGTACCCACCACATTTACTAAG-3′) and primer FH578 (5′-GGTACCTGTAGTCACCTATTACTGCTCTC-3′). Both fragments were cloned in the pCRII-TOPO vector and were partially sequenced. 
The targeting vector was constructed by cloning the 2-kb DNA fragment as a NotI- SpeI fragment between the NotI and NheI sites of the basic targeting vector, 27 introducing this short arm at the 5′-end of the neo gene cassette. The long arm was then cloned between the neo cassette and the HSV-TK (thymidine kinase) as fragment KpnI-KpnI into the site of the targeting vector. Following this strategy, exon 1 and exon 2 of the Cabp5 gene were replaced by the neo gene cassette. To increase the percentage of clones that undergo homologous recombination during the selection of transfected ES cells, we introduced a PGK-DTA (diphtheria toxin) cassette upstream of the 2-kb fragment. A cassette containing the PGK-DTA-bovine growth hormone gene polyadenylation site cassette was amplified with primers FH599 (5′-GCGGCCGCTACCGGGTAGGGGAGGCGCTTTT-3′) and FH600 (5′-GCGGCCGCTACCGGGTAGGGGAGGCGCTTTT-3′) using the PGKneolox2DTA vector (a kind gift from Philippe Soriano 28 ). These primers were extended with a NotI restriction site for cloning of this cassette upstream of the short arm into the NotI site of the targeting vector. 
The PvuI linearized targeting vector was electroporated into 129/Sv-derived embryonic stem (ES) cells. Recombinant clones were selected on medium containing G418 and ganciclovir. Transfected ES cells for homologous recombination events were screened through PCR analysis. To screen for homologous recombination upstream of the neo cassette, we used the primer combination K216 (5′-GAATTATGGTCCCATTAGAGGC-3′), hybridizing approximately 300 bp upstream of the 5′-end of the short arm in the Cabp5 gene, and K204 (5′-GGAGAACCTGCGTGCAATCC-3′), located in the neo cassette and amplifying a fragment of approximately 2.6 kb. A control PCR for the wild-type gene was made with primers K216 and K206 (5′-GATGCAGGCAGGACCCATTGG-3′), located in exon 1 of the Cabp5 gene and amplifying a fragment of approximately 2.2 kb. Selection of homologous recombination events at the 3′ end was analyzed by PCR using primers K214 (5′-TCAACGAGACATCATCTTCAC-3′), which hybridizes in exon 6 of the Cabp5 gene, and K183 (5′-CTTGCCGAATATCATGGTGG-3′), located in the neo cassette. A control PCR for the wild-type gene was carried out with primers K214 and K212 (5′-GAGACCACTGGGACAGGATG-3′), which hybridizes in exon 2 of Cabp5
One targeted ES clone was injected into C57BL/6J blastocysts. One 100% male chimera was crossed with C57BL/6J mice, and agouti offspring were genotyped by PCR to verify germ line transmission. To identify the wild-type allele, the primer pair K212 (5′-GAGACCACTGGGACAGGATGAG-3′, located in exon 2) and K35 (5′-CCCAACTCAGTCAACTCCATCTC-3′, hybridizing in exon 3) was used and gave a PCR product of 620 bp. The targeted Cabp5 allele was identified with primers K183 (5′-CTTGGCGAATATCATGGTGG-3′, located in the neo cassette), and K35, which gave a PCR product of 0.8 kb. Four of 12 agouti pups were carrying the targeted gene. After five-generation backcross to C57Bl/6J, homozygous offspring carrying the targeted gene were selected. Targeted disruption of the Cabp5 gene was confirmed by Southern blot analysis. Twenty micrograms of genomic DNA was digested with HindII and hybridized with a 0.25-kb fragment 5′-end probe located 500 bp upstream of the 5′end gene fragment introduced into the targeting vector. The 5′-end probe hybridized to an approximately 3.8 kb HindII fragment derived from the targeted allele and a 2.8-kb HindII fragment from the wild-type allele. 
Immunohistochemistry
Mouse eyecups were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 1 hour. After fixation, tissues were incubated with a sucrose series to 20% sucrose in phosphate buffer and then embedded in 33% OCT compound (Miles, Elkhart, IN) diluted with 20% sucrose in phosphate buffer. Eye tissue was cut in 12-μm sections. To block nonspecific labeling, retinal sections were incubated with 3% normal goat serum in PBST buffer (136 mM NaCl, 11.4 mM sodium phosphate, 0.1% Triton X-100, pH 7.4) for 20 minutes at room temperature. Sections were incubated overnight at 4°C in a mix of diluted primary antibodies (1:500 for rabbit anti–CaBP5 with 1:1000 for mouse anti–calretinin, 1:200 for rabbit anti–PKCα with 1:500 for mouse anti–calbindin D-28K, 1:200 for mouse anti–PKCα with 1:200 for rabbit anti–Cav1.2, and 1:200 for mouse anti–CaBP5 with 1:200 for rabbit anti–Cav1.2). A mixture of Cy3-conjugated goat anti–rabbit IgG and Alexa 488-conjugated goat anti–mouse IgG or Cy3-conjugated goat anti–mouse IgG and Alexa 488-conjugated goat anti–rabbit was reacted with sections for 1 hour at room temperature. Then the sections were rinsed in PBST and mounted with antifade reagent (Prolong; Molecular Probes) to slow photobleaching. Sections were analyzed under a confocal microscope (LSM510; Carl Zeiss, Thornwood, NY), and immunofluorescent images were obtained with an objective lens (Plan-Neofluar 40×/1.3NA; Carl Zeiss). 
Transmission Electron Microscopy
Mouse eyecups were fixed primarily by immersion in 2.5% glutaraldehyde, 1.6% paraformaldehyde, in pH 7.4, 0.08 M PIPES buffer containing 2% sucrose, initially at room temperature for approximately 1 hour, then at 4°C for the remainder of 24 hours. Eyecups were washed with pH 7.35, 0.13 M phosphate buffer, and secondarily fixed with 1% OsO4 in pH 7.4, 0.1 M phosphate buffer, for 1 hour at room temperature. After another wash with 0.13 M phosphate buffer, the eyecups were dehydrated through a methanol series and transitioned to epoxy embedding medium with propylene oxide. Eyecups were infiltrated (Eponate 812; Ted Pella, Redding, CA) and embedded for sectioning in (Eponate 812; Ted Pella) by hardening at 70°C for 24 hours before ultramicrotomy. Ultrathin sections (60–70 nm) were cut with a diamond knife and mounted on 50 mesh grids coated with film (Pioloform; Ted Pella). Sections were then stained with aqueous-saturated uranium acetate and Reynold formula lead citrate before survey and micrography with an electron microscope (CM10; Philips, Eindhoven, Netherlands). A montage of individual images was created in Adobe Photoshop. 
Electroretinography
Before recording, mice were dark adapted overnight. Under dim red light, mice were anesthetized by intraperitoneal injection using 20 μL/g body weight of 6 mg/mL ketamine and 0.44 mg/mL xylazine diluted in 10 mM sodium phosphate (pH 7.2) containing 150 mM NaCl. Pupils were dilated with 2.5% phenylephrine-HCl and were anesthetized with 0.5% proparacaine-HCl. A contact lens electrode was placed on the eye with a drop of 2.5% hydroxy propyl methylcellulose solution, and a reference electrode and ground electrode were placed in the ear. Electroreginograms (ERGs) were recorded with the universal testing and electrophysiological system (UTAS E-3000; LKC Technologies, Gaithersburg, MD). Mice were placed on temperature-regulated heating pads throughout the recordings. Light intensity was calibrated by the manufacturer and computer controlled. Mice were placed in a Ganzfeld chamber, and scotopic and photopic responses to flash stimuli were obtained from both eyes simultaneously. 
Flash stimuli had a range of intensities (−2.4–2.8 log cd · s/m2). Three to five recordings were made at intervals greater than 10 seconds; for higher intensity, the intervals were 1 minute. For the photopic responses, a steady rod-desensitizing background (2.8 log cd · s/m2) was presented to the mice for 10 minutes before the light-adapted ERG recordings. Five animals were typically used for the recording of each point in all conditions. To analyze the rod photoreceptor component, the amplitude of the a-wave was measured 6 ms after flash stimuli from the prestimulus baseline. 29 The amplitude of the b-wave was measured from the a-wave trough to the peak of the b-wave. In photopic conditions, the amplitude of the slowly rising cone a-wave was analyzed from the prestimulus baseline to the a-wave trough. 29 30 Statistical analysis of the differences between Cabp5 +/+ and Cabp5 −/− ERG data was carried out using the Student t-test for each individual flash intensity (SigmaPlot; SPSS Inc., Chicago, IL). 
Patch-Clamp Recordings
Responses of retinal ganglion cells were measured using whole-cell patch clamp recordings according to previously published procedures. 31 In brief, retinas were isolated from a mouse that had been dark adapted overnight. A small piece of retina was mounted photoreceptor side down in a chamber on the microscope stage. These procedures were performed under infrared (>900 nm) light. ON α-like retinal ganglion cells were identified based on their large somas and characteristic spike responses to a light step. 31 Pipettes for whole-cell voltage-clamp recordings were filled with an internal solution containing 105 mM CsCH3SO3, 10 mM TEA-Cl, 20 mM HEPES, 10 mM EGTA, 5 mM Mg-ATP, 0.5 mM Tris-GTP, and 2 mM QX-314 (pH ∼7.3 with CsOH, ∼280 mOsm). Series resistance (∼10–15 MΩ) was compensated 70%. The chamber was superfused at approximately 8 mL/min with solution (Ames; Sigma Aldrich, St. Louis, MO) warmed to 31° to 34°C. Light stimuli were delivered from a calibrated light-emitting diode with a peak output at 470 nm. Stimuli uniformly illuminated a 630-μm diameter spot centered on the recorded cell. 
Ganglion cell sensitivity was measured from the strength of the flash required to produce a half-maximal response. Half-maximal flash strengths were estimated in two ways. First, the stimulus-response relation for each cell (see 1 2 3 4 5 Fig. 6C ) was fit with a saturating exponential function with the half-maximal flash strength as the (only) free parameter. Second, half-maximal flash strengths were estimated by linearly interpolating between the two nearest responses. These approaches yielded similar results. Mean and SE of the half-maximal flash strengths for ganglion cells from Cabp5 +/+ and Cabp5 −/− mice were compared. 
Cloning and Expression in Bacteria of CaBP5, CaM, CT1, and CT2 Domains of Cav1 Calcium Channels
The C-terminal domain (CT1) containing the CaM-binding domain (CBD) and distal C-terminal domain downstream of the CT1 (CT2) of mouse α1 1.4 and the mouse α1 1.3 CT1 were subcloned and fused to a 6His tag, as described previously. 32 The rat α1 1.2 CT1 domain (amino acid 1523–1668) was amplified with primers FH675 (5′-CACCCTGGATGAATTCAAGAGAATCTGGG-3′) and FH688 (5′-TCACAGCCCCTGCTCTTTTCGCT-3′) from cDNA encoding the rbcII variant of the rat brain α1 1.2. Mouse CaBP5 was amplified by PCR with primers FH700 (5′-CACCATGCAGTTTCCAATGGGTCCTG-3′) and C16 (5′-TCAGCGAGACATCATCTTCACAAAC-3′), and the mouse CaM was amplified by PCR with C208 (5′-caccatggctgatcagctgac-3′) and C209 (5′-TCATTTTGCAGTCATCATCTG-3′) from mouse cDNA 4 and subcloned into the pentr/DTOPO vector (entry clone; Gateway Technology, Invitrogen, Carlsbad, CA). These cDNAs were then transferred by recombination in the pDest17 or pDest15 vectors for expression in bacteria and fused to a His-tag or GST-tag, respectively. Fusion proteins were expressed in BL21(DE3)pLysS Escherichia coli after induction with 0.2 mM IPTG and purified on Ni2+-NTA or a glutathione column according to the manufacturer’s protocol. 
Binding of a Cytoplasmic Fragment of Cav1.2 α1 to CaBP5 Using Affinity Chromatography
Purified His tagged-Cav1.2 α1 CBD (CT1) was loaded onto the CaBP5-Sepharose column equilibrated with PBS buffer (10 mM sodium phosphate, 150 mM NaCl, pH 7.5) containing 2 mM benzamidine and 1 mM CaCl2. The column was then washed with 100 bed volumes of the same buffer. Elution was performed with 5 mM EGTA followed by 0.1 M glycine buffer, pH 2.5. Fractions were collected and aliquots were analyzed by SDS-PAGE, followed by Western blotting with an anti–His tag antibody. 
Gel Overlay Assay
Recombinant 6His-tagged purified proteins (2 μg) were separated on SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. After overnight saturation at 4°C in PBS, 0.1% Tween-20, and 3% nonfat milk, the membranes were incubated in PBS, 0.1% Tween-20, and 2% nonfat milk (blotting buffer) containing 2 μg/mL GST-tagged proteins for 1 hour at room temperature. The blots were washed three times at 5 minutes each wash with 10 mL blot buffer and then were incubated for 1 hour with anti–GST antibody in blotting buffer at room temperature. After a second round of three washes of 5 minutes each, the blots were incubated with an anti–mouse antibody conjugated to alkaline phosphatase for 1 hour at room temperature. Bound recombinant proteins were visualized by incubation with NBT/BCIP (Promega). Interactions were tested in the presence or absence of calcium. For analysis in the absence of Ca2+, 5 mM EGTA was added to the blotting buffer. 
Electrophysiological Recordings of Transfected Cells
HEK293T cells were grown to 70% to 80% confluence and transfected using reagent (Gene Porter; Gene Therapy Systems, San Diego, CA) according to the manufacturer’s protocols. Cells were transfected with approximately 5 μg total DNA (α11.2, 2 μg; β2A, 0.8 μg; α2δ, 0.8 μg; ± CaBP5, 0.1 μg) and GFP expression plasmid (0.01 μg) for fluorescence detection of transfected cells. All electrophysiological data were acquired with EPC-9 patch-clamp amplifier driven by HEKA software (Pulse; HEKA Elektronik, Lambrecht/Pfalz, Germany) and were analyzed with Wavemetrics software (Igor Pro; Wavemetrics, Lake Oswego, OR). Extracellular recording solutions contained 150 mM Tris, 1 mM MgCl2, and 10 mM CaCl2. Intracellular solutions consisted of 140 mM N-methyl-d-glucamine, 10 mM HEPES, 2 mM MgCl2, 2 mM Mg-ATP, and 5 mM EGTA. The pH of intracellular and extracellular recording solutions was adjusted to 7.3 with methane sulfonic acid. Electrode resistances were typically 1 to 2 MΩ in the bath solution, and series resistance was approximately 2 to 4 MΩ, compensated up to 80%. All averaged data are presented as the mean ± SEM. Statistical significance of differences between two groups was determined by Student’s t-test, as indicated (SigmaPlot; SPSS Inc.). 
Results
Generation of CaBP5 Knockout Mice
To study the role of CaBP5 in vivo, we generated CaBP5 knockout mice (Cabp5 −/−) by replacing exon 1 and exon 2 of the Cabp5 gene with a PGK-neo cassette (Fig. 1A) . Agouti offspring obtained from one male chimera were genotyped by PCR to verify germ line transmission (Fig. 1B) . Targeting of the Cabp5 allele was confirmed by the presence of a 3.9-kb HindII fragment using Southern blotting with a 5′ probe (Fig. 1C)
Morphologic Characterization of Cabp5−/− Mouse Retina
CaBP5 expression was analyzed by immunoblotting using antibodies against CaBP5 and PKCα, which served as a marker protein expressed in rod bipolar cells. As expected, CaBP5 was not detected in retinal extract from Cabp5 −/− mice (Fig. 2A) . As reported in previous studies, 1 18 CaBP5 was localized in rod bipolar cells and in type 3 OFF and type 5 ON cone bipolar cells; axon terminals of these cells form three distinct layers in the inner plexiform layer (IPL; Fig. 2B ). No CaBP5 was detected by immunohistochemistry in the Cabp5 −/− mice. However, calretinin immunostaining revealed a normal distribution of ganglion cells and amacrine cell processes, forming three bands in the IPL in Cabp5 +/+ and Cabp5 −/− retinas (Fig. 2B)
To determine whether CaBP5 deficiency triggers structural changes, the morphology and expression of marker proteins were analyzed in 6-week-old mice using electron and confocal microscopy. The thickness and organization of the retinal layers appeared normal in the CaBP5 knockout mice compared with the wild-type mice as analyzed with antibodies against PKCα (rod bipolar cell marker) and calbindin (horizontal cell marker) and DAPI labeling of nuclei (Fig. 3A) . There was no apparent difference in the morphology of the dendrites and axons of rod bipolar cells in wild-type and CaBP5 knockout mice, as shown in higher magnification images (Fig. 3B) . Dendrites were well developed and normally stratified in the OPL of wild-type and CaBP5 knockout mice. The shape and number of bipolar cell terminals were also comparable in both genotypes. Synapse morphology in both the outer plexiform layer (OPL) and the IPL was analyzed using an antibody against the ribbon marker ribeye/CtBP2. Normal horseshoe-shaped and punctuate staining was observed for synaptic ribbons of wild-type and CaBP5 knockout mice located in the OPL and IPL, respectively (Fig. 3C) . The density and morphology of labeled ribbons were similar in wild-type and CaBP5 knockout mice. No changes in retinal morphology were observed in 1-year-old CaBP5 knockout mice (data not shown). 
The structural organization of mouse retina was also analyzed at the ultrastructural level using transmission electron microscopy. No evidence of morphologic changes in any retinal layers was observed in CaBP5 knockout mice (Fig. 4) . The lamination pattern and the thickness of all layers appeared similar in wild-type and CaBP5 knockout mice. Cell density and morphology were also comparable in both genotypes. 
Visual Responses in CaBP5 Knockout Mice Using Electroretinographic Recordings
Because CaBP5 is localized throughout the bipolar cells, including fine processes of the dendrites and in the ramifying axons, we used electroretinography to test whether the deletion of CaBP5 might affect synaptic transmission between photoreceptors and bipolar cells. ERGs of wild-type and CaBP5 knockout mice were recorded under scotopic and photopic conditions to evaluate the effect on rod- and cone-mediated signals. In both conditions, no statistically significant reduction in the a-wave and b-wave amplitudes of CaBP5 knockout mice was observed compared with wild-type for any individual flash intensity (P > 0.1 for all flash intensities; see Materials and Methods; Fig. 5 ). Similarly, no significant difference was observed in the amplitudes of ERGs of either genotype of 1-year-old mice (data not shown). These data argue against a substantial role for CaBP5 in regulating synaptic transmission between photoreceptors and bipolar cells. 
Responses of Cabp5+/+ and Cabp5−/− ON Ganglion Cells
At low light levels, signals traverse the mammalian retina through the specialized rod bipolar pathway. We analyzed the effect of CaBP5 deficiency on signals mediated by this pathway by measuring the sensitivity of an identified class of ON ganglion cells to dim light flashes. Excitatory synaptic inputs were measured by holding a ganglion cell at −70 mV, near the reversal potential for inhibitory inputs. Figures 6A and 6Bsuperimpose responses to a series of light flashes for single ganglion cells from a wild-type and a CaBP5−/− mouse. Figure 6Cplots the relationship between response amplitude and flash strength for a population of cells. Ganglion cells from CaBP5−/− mice were, on average, approximately 50% less sensitive than those from wild-type mice (P < 0.05 for difference in half-maximal flash strengths; see Materials and Methods). Thus, CaBP5 enhances the sensitivity of rod-mediated responses reaching the retinal ganglion cells. 
Analysis of CaBP5 Interaction and Modulation of Cav1.2
CaBP1 and CaBP4, the two CaBPs from the CaBP subfamily whose function has been most extensively investigated to date, differently modulate the activity of voltage-dependent calcium channels. Given the high similarity among CaBPs, we tested the hypothesis that CaBP5 can also interact with and modulate voltage-dependent calcium channels. Because Cav1.2 was previously reported to be expressed in ON bipolar cells, 33 34 35 we analyzed the interaction of CaBP5 with Cav1.2. 
CaBP5 Colocalizes with Cav1.2 in Rod Bipolar Cells.
To be a physiological partner for CaBP5, Cav1must colocalize with CaBP5. Because we had previously shown that CaBP5 is expressed in rod bipolar cells, 1 we first confirmed that Cav1.2 voltage-dependent calcium channel is also expressed in the rod bipolar cells. Cav1.2 is expressed in rod bipolar cells, as shown by the extensive colocalization of Cav1.2 and PKCα, a marker for rod bipolar cells (Fig. 7A) . We then confirmed that CaBP5 is coexpressed with Cav1.2 by double labeling with anti–CaBP5 and anti–Cav1.2 antibodies (Fig. 7A)
CaBP5 Binds to Cav1.2 in a Ca2+-Dependent Manner.
L-type voltage-dependent calcium channels are modulated by CaM, which binds to a domain in the C-terminal domain of the α1 subunit. 36 37 38 We have shown previously that CaBP interacts with and modulates Cav2 6 and Cav1 channels. 12 17 39 We tested whether CaBP5 can directly interact with a cytoplasmic fragment of Cav1.2 α1 containing the CaM binding sequence (CBD) using affinity chromatography. The Cav1.2α1 fragment CT1 domain (CBD; amino acids 1523–1668) was loaded on CaBP5-coupled Sepharose in the presence of Ca2+. Most of the proteins were detected in the fractions first eluted with EGTA, suggesting that CaBP5 binds mostly in a Ca2+-dependent manner to the CBD of Cav1.2α1 (Fig. 7B)
We further investigated the interaction between CaBP5 and Cav1 CT1 using gel overlay assay. Like CaM, GST-CaBP5 bound to 6His-tagged fusion proteins containing the CBD (CT1) of Cav1α1 (Fig. 7C) . Binding was specific in that no binding was detected to a cytoplasmic fragment without the CBD (CT2). The binding of CaBP5 to α1 CT1 was observed in the presence of Ca2+ but was barely detectable in the absence of Ca2+, confirming that CaBP5 requires Ca2+ for binding to Cav1.2α1. 
CaBP5 Modulates Cav1.2.
To test whether CaBP5 can modulate the activity of Cav1.2 channels, we analyzed Ca2+ currents using whole-cell patch-clamp recordings in transfected HEK293 cells. In these experiments, CaBP5 caused a positive shift in the half-activation voltage (V 1/2; 19 ± 2 mV in wild-type mice compared with 28 ± 2 in knockout mice; P = 0.03). CaBP5 also influenced the slope of the I-V relationship (k = −10.0 ± 0.4 for CaBP5 KO vs. −8.9 ± 0.2 for Cav1.2 alone; P = 0.05 ;Fig. 7D ). This effect of CaBP5 on the I-V curve caused a significant increase of the whole-cell Ca2+ current at more depolarized membrane voltages (approximately 20%, 40–70 mV; P < 0.05). In our experimental conditions, the effect of CaBP5 occurs at more depolarized potentials than bipolar cell physiological membrane potentials, but the voltage dependence of the activation of Cav1.2 channels expressed in HEK293 cells is also shifted to more depolarized potentials than of the native Cav1.2 channel. 23 CaBP5 could cause a similar depolarizing shift in the voltage dependence of activation of Cav1.2 in physiological conditions. 
We also studied the effect of CaBP5 on the inactivation of Cav1.2 Ca2+ currents. Cav1.2 Ca2+ currents were evoked by 2-second pulses from −80 to +10 mV (for Cav1.2 alone) or +20 mV (for +CaBP5). Inactivation was measured at these different test voltages because of the approximately 10-mV shift in voltage-dependent activation in cells cotransfected with CaBP5. HEK293T cells were transfected with Cav1.2 (α11.2, β2A, α2δ) alone (black) or were cotransfected with CaBP5 (red). Inactivation was measured as I res/I pk, which was the current amplitude at 1 second normalized to the peak current amplitude. CaBP5 did not affect the kinetics of current decay but significantly suppressed the amount of inactivation (I res/I pk) of the Ca2+ current (P = 0.02; n = 6 each for Cav1.2 alone and +CaBP5; Fig. 7E ). Together, these results indicate that CaBP5 potentiates the activity of Cav1.2 channels through effects on channel activation and inactivation. 
Discussion
We generated and characterized CaBP5 knockout mice to investigate the function of CaBP5 in the retina. Our results suggest that CaBP5 is required for the normal transmission of the rod-mediated responses across the retina. 
The structural organization of the retina appeared normal in CaBP5 knockout mice, suggesting that CaBP5 is not essential for normal bipolar cell development, though it is expressed throughout the postnatal retina development (data not shown). CaBP5 deficiency did not result in any obvious change in the stratification, density, or morphology of rod bipolar cell dendrites, axons, and cell bodies, indicating that structural changes are not likely to account for the changes in light responses observed in CaBP5 knockout mice. 
Rod-mediated responses of retinal ganglion cells in the absence of CaBP5 had a reduced sensitivity compared with wild-type ganglion cells. This effect was modest—an approximately 50% reduction increase in half-saturating flash strength—and the amplitude and kinetics of the ganglion cell responses otherwise appeared normal. The effect of CaBP5 on ganglion cell sensitivity could reflect defects in signal transmission at any previous step in the rod bipolar pathway. Because CaBP5 is expressed primarily in bipolar cells, we propose that the deficiency reflects a change in the presynaptic function of rod bipolar cells. 
CaBP5 could act on the voltage-gated calcium channels that control transmitter release from the rod bipolar cells ( Ref. 42 for recent review). 22 24 26 40 41 More generally, we suggest that CaBPs are cell-type specific proteins that play a key role in Ca2+ signaling through regulation of a common target, the voltage-dependent calcium channel. 5 6 7 12 17 43 This hypothesis is supported by our findings that Cav1.2 colocalizes with CaBP5 in rod bipolar cells and by previous data that Cav1.2 is expressed in ON bipolar cells. 33 34 35 CaBP5 modulates the activation and inactivation of Cav1.2 in transfected cells. Thus, CaBP5 shifted the Ca2+ channel activation curve to more positive potentials and suppressed calcium-dependent inactivation of the Cav1.2 Ca2+ current. Similar changes in the rod bipolar calcium channels could have a substantial effect on the transmission of rod-mediated signals from rod bipolar cells to AII amacrine cells. This synapse has a high gain in darkness and thus may be particularly susceptible to changes in the voltage dependence of calcium entry. 44  
In summary, our results provide the first evidence for a role of CaBP5 in visual function. Although further studies will be needed to establish the molecular mechanisms by which CaBP5 affects the transmission of the light signal, our data suggest that CaBP5 participates in modulating retinal sensitivity, possibly through the modulation of voltage-gated calcium channels. Identifying the role of CaBP5 not only will contribute to our understanding of the basic processes modulated by CaBP5, it may also give insight into the contribution of a specific subset of bipolar cells to the processing of the light stimulus throughout the retinal circuitry. 
 
Figure 1.
 
(A) Generation of CaBP5 knockout mice using gene targeting. Exon and restriction map of the Cabp5 gene locus, targeting vector, and targeted Cabp5 gene. In the targeting vector, the neo cassette replaces exon 1 and exon 2 of the Cabp5 gene. The targeting vector was constructed by using an approximately 2-kb DNA fragment as the short arm that extended upstream of the initial ATG. The long arm is an approximately 6-kb genomic fragment encompassing intron 2 to intron 5 of the Cabp5 gene. The PGK-DTA and HSV-TK cassettes were included for negative selection. Gray box: 5′ probe hybridizing to a 300-bp region located upstream of the short arm of the Cabp5 gene. H, HindII; M, MscI. (B) PCR-based genotyping of wild-type (+/+), heterozygous (+/−), and knockout (−/−) mice. A 620- and 800-bp PCR product is amplified from the wild-type Cabp5 and the targeted loci, respectively. (C) Southern blot analysis of targeted gene. HindII-digested genomic DNA from wild type (+/+), heterozygous (+/−), and knockout (−/−) mice were probed with a 300-bp 5′ probe. A fragment of 2.9 kb, indicative of the wild-type Cabp5 gene, is identified in wild-type and heterozygous mice. Knockout and heterozygous mice show a fragment of 3.9 kb corresponding to the targeted Cabp5 gene.
Figure 1.
 
(A) Generation of CaBP5 knockout mice using gene targeting. Exon and restriction map of the Cabp5 gene locus, targeting vector, and targeted Cabp5 gene. In the targeting vector, the neo cassette replaces exon 1 and exon 2 of the Cabp5 gene. The targeting vector was constructed by using an approximately 2-kb DNA fragment as the short arm that extended upstream of the initial ATG. The long arm is an approximately 6-kb genomic fragment encompassing intron 2 to intron 5 of the Cabp5 gene. The PGK-DTA and HSV-TK cassettes were included for negative selection. Gray box: 5′ probe hybridizing to a 300-bp region located upstream of the short arm of the Cabp5 gene. H, HindII; M, MscI. (B) PCR-based genotyping of wild-type (+/+), heterozygous (+/−), and knockout (−/−) mice. A 620- and 800-bp PCR product is amplified from the wild-type Cabp5 and the targeted loci, respectively. (C) Southern blot analysis of targeted gene. HindII-digested genomic DNA from wild type (+/+), heterozygous (+/−), and knockout (−/−) mice were probed with a 300-bp 5′ probe. A fragment of 2.9 kb, indicative of the wild-type Cabp5 gene, is identified in wild-type and heterozygous mice. Knockout and heterozygous mice show a fragment of 3.9 kb corresponding to the targeted Cabp5 gene.
Figure 2.
 
(A) Western blot analysis of retinal extracts prepared from Cabp5 +/+, Cabp5 +/−, and Cabp5 −/− mice probed with antibodies to CaBP5 and PKCα. CaBP5 proteins are not detected in Cabp5 −/− retinas, confirming targeting of the Cabp5 gene. (B) Immunofluorescence for CaBP5 and calretinin in 6-week-old Cabp5 +/+ (WT) and Cabp5 −/− (KO) mouse retinas. The lack of CaBP5 immunoreactivity in the Cabp5 −/− retina confirmed the loss of CaBP5 protein in the knockout mice. Scale bar, 20 μm.
Figure 2.
 
(A) Western blot analysis of retinal extracts prepared from Cabp5 +/+, Cabp5 +/−, and Cabp5 −/− mice probed with antibodies to CaBP5 and PKCα. CaBP5 proteins are not detected in Cabp5 −/− retinas, confirming targeting of the Cabp5 gene. (B) Immunofluorescence for CaBP5 and calretinin in 6-week-old Cabp5 +/+ (WT) and Cabp5 −/− (KO) mouse retinas. The lack of CaBP5 immunoreactivity in the Cabp5 −/− retina confirmed the loss of CaBP5 protein in the knockout mice. Scale bar, 20 μm.
Figure 3.
 
Morphologic characterization of 6-week-old Cabp5 +/+ (WT) and Cabp5 −/− (KO) mouse retina. (A) Immunolocalization of PKCα (red) and calbindin (green) in mouse retina cross-sections. Cell nuclei were stained with DAPI (blue). (B) Immunofluorescence of PKCα (red) and calbindin (green) in the OPL (A, A′) and in the IPL (B, B′). (C) Immunofluorescence of ribeye (green) in the OPL (A, A′) and in the IPL (B, B′). Scale bars: (A) 20 μm, (B) 5 μm, (C) 2 μm.
Figure 3.
 
Morphologic characterization of 6-week-old Cabp5 +/+ (WT) and Cabp5 −/− (KO) mouse retina. (A) Immunolocalization of PKCα (red) and calbindin (green) in mouse retina cross-sections. Cell nuclei were stained with DAPI (blue). (B) Immunofluorescence of PKCα (red) and calbindin (green) in the OPL (A, A′) and in the IPL (B, B′). (C) Immunofluorescence of ribeye (green) in the OPL (A, A′) and in the IPL (B, B′). Scale bars: (A) 20 μm, (B) 5 μm, (C) 2 μm.
Figure 4.
 
Analysis of 6-week-old retina morphology by transmission electron microscopy. (A) Montage of mouse retina cross-sections analyzed by transmission electron microscopy. (B) Mouse retina cross-sections through the OPL and IPL. Higher magnification of a cross-section through the OPL (upper) and the IPL/ganglion cell layer (bottom). Scale bars: (A) 10 μm, (B) 5 μm.
Figure 4.
 
Analysis of 6-week-old retina morphology by transmission electron microscopy. (A) Montage of mouse retina cross-sections analyzed by transmission electron microscopy. (B) Mouse retina cross-sections through the OPL and IPL. Higher magnification of a cross-section through the OPL (upper) and the IPL/ganglion cell layer (bottom). Scale bars: (A) 10 μm, (B) 5 μm.
Figure 5.
 
Single-flash ERG responses to light stimuli of increasing intensity for 6-week-old Cabp5 +/+ and Cabp5 −/− mice. Serial responses to increasing flash stimuli were obtained for Cabp5 +/+ and Cabp5 −/− for selected intensities under scotopic conditions (A) and photopic conditions (B) and were plotted as a function of a-wave or b-wave amplitude compared with light intensity. Representative ERG waveforms recorded from Cabp5 +/+ and Cabp5 −/− mice in response to flashes of increasing intensity are shown on the left. In scotopic and photopic conditions, no significant differences were observed between the a-wave- and b-wave-amplitudes of Cabp5 +/+ and Cabp5 −/− mice (P > 0.1). SEM bars are shown.
Figure 5.
 
Single-flash ERG responses to light stimuli of increasing intensity for 6-week-old Cabp5 +/+ and Cabp5 −/− mice. Serial responses to increasing flash stimuli were obtained for Cabp5 +/+ and Cabp5 −/− for selected intensities under scotopic conditions (A) and photopic conditions (B) and were plotted as a function of a-wave or b-wave amplitude compared with light intensity. Representative ERG waveforms recorded from Cabp5 +/+ and Cabp5 −/− mice in response to flashes of increasing intensity are shown on the left. In scotopic and photopic conditions, no significant differences were observed between the a-wave- and b-wave-amplitudes of Cabp5 +/+ and Cabp5 −/− mice (P > 0.1). SEM bars are shown.
Figure 6.
 
Flash responses of Cabp5 +/+ and Cabp5 −/− ON ganglion cells. Flash families measured from a Cabp5 +/+ ON ganglion cell (A) and a Cabp5 −/− ON ganglion cell (B). Average responses are superimposed for flashes producing 0.001 to 0.5 photon/μm2. (C) Stimulus-response relationship for Cabp5 +/+ and Cabp5 −/− ON ganglion cells. Error bars are SEM. Half-maximal flash strengths, estimated from saturating exponential fits to the stimulus-response relations, were 0.03045 ± 0.0035 (mean ± SEM; n = 9) for Cabp5 +/+ cells and 0.04971 ± 0.0034 for Cabp5 −/− cells (n = 7).
Figure 6.
 
Flash responses of Cabp5 +/+ and Cabp5 −/− ON ganglion cells. Flash families measured from a Cabp5 +/+ ON ganglion cell (A) and a Cabp5 −/− ON ganglion cell (B). Average responses are superimposed for flashes producing 0.001 to 0.5 photon/μm2. (C) Stimulus-response relationship for Cabp5 +/+ and Cabp5 −/− ON ganglion cells. Error bars are SEM. Half-maximal flash strengths, estimated from saturating exponential fits to the stimulus-response relations, were 0.03045 ± 0.0035 (mean ± SEM; n = 9) for Cabp5 +/+ cells and 0.04971 ± 0.0034 for Cabp5 −/− cells (n = 7).
Figure 7.
 
(A) Colocalization of Cav1.2 voltage-dependent calcium channels with CaBP5 and PKCα in mouse retina. (A′C′) Confocal images of mouse retina sections double labeled with antibodies to PKCα (green, A′) and Cav1.2α1 (red, B′). Extensive colocalization of Cav1.2α1 and PKCα is observed in rod bipolar cells and appears yellow in the merged images (C′). Cav1.2 is expressed only in cells also labeled with the rod bipolar marker PKCα and is thus specifically expressed in rod bipolar cells. Scale bars, 20 μm. (D′F′). Double labeling of mouse retinal sections with CaBP5 (green, D′) and Cav1.2α1 (red, E′). CaBP5 is colocalized with Cav1.2 in rod bipolar cells, predominantly at the axon terminals, and appears yellow in the merged images (F′). Scale bars, 20 μm. (B) Affinity chromatography of purified recombinant Cav1.2 α1 CBD on CaBP5 column. His-tagged Cav1.2α1 CT1 was loaded onto the CaBP5-Sepharose column equilibrated with PBS buffer containing 1 mM CaCl2. After washes with the same buffer, the proteins were eluted with 3 mM EGTA followed by 0.2 M glycine buffer, pH 2.1. Eluted fractions were probed with anti–His antibodies. Lane 1: protein loaded on the column; lane 2: protein present in the flow-trough; lane 3: last wash fraction before elution; lanes 4–6: elution with 5 mM EGTA; lane 7: last fraction before elution with glycine buffer; lanes 8 and 9, further elution with 0.1 M glycine buffer, pH 2.4. (C) Gel overlay assay of recombinant CaBP5 with α1 Cav1 cytoplasmic domains. His-tagged α1 CBD from Cav1.2, Cav1.3, and Cav1.4 (CT1), or C-terminal domain without CBD (CT2), were separated on SDS-PAGE and transferred to PVDF membranes, which were incubated with CaM-GST and CaBP5-GST in the presence or absence of EGTA. Bound proteins were detected with an anti–GST antibody. Ponceau staining (right) shows the relative amount of purified proteins. (D) Effect of CaBP5 on the activation of Cav1.2 Ca2+ currents. Cav1.2 Ca2+ currents were evoked by 50-ms pulses from −80 mV to various voltages in HEK293T cells transfected with Cav1.2 (α11.2, β2A, α2δ) alone or cotransfected with CaBP5 (n = 7 each). Current amplitudes were normalized to the largest in the series (I/I max) and plotted (mean ± SE) against test voltage. Smooth lines represent fits by Boltzmann equation. CaBP5 significantly increased V 1/2 (28.2 ± 2.8 vs. 19.4 ± 2.3 for Cav1.2 alone; P = 0.03) and k (−10.0 ± 0.4 vs. −8.9 ± 0.2 for Cav1.2 alone; P = 0.05). (E) Effect of CaBP5 on inactivation of Cav1.2 Ca2+ currents. Cav1.2 Ca2+ currents were evoked by 2-second pulses from −80 to +10 mV (for Cav1.2 alone) or +20 mV (for +CaBP5). Inactivation was measured at these different test voltages because of the approximately 10-mV shift in voltage-dependent activation in cells cotransfected with CaBP5. HEK293T cells were transfected with Cav1.2 (α11.2, β2A, α2δ) alone (black) or were cotransfected with CaBP5 (red). Inactivation was measured as I res/I pk, which was the current amplitude at 1-second normalized to the peak current amplitude. CaBP5 did not affect the kinetics of current decay but significantly suppressed the inactivation of the Ca2+ current (P = 0.02; n = 6 each for Cav1.2 alone and +CaBP5).
Figure 7.
 
(A) Colocalization of Cav1.2 voltage-dependent calcium channels with CaBP5 and PKCα in mouse retina. (A′C′) Confocal images of mouse retina sections double labeled with antibodies to PKCα (green, A′) and Cav1.2α1 (red, B′). Extensive colocalization of Cav1.2α1 and PKCα is observed in rod bipolar cells and appears yellow in the merged images (C′). Cav1.2 is expressed only in cells also labeled with the rod bipolar marker PKCα and is thus specifically expressed in rod bipolar cells. Scale bars, 20 μm. (D′F′). Double labeling of mouse retinal sections with CaBP5 (green, D′) and Cav1.2α1 (red, E′). CaBP5 is colocalized with Cav1.2 in rod bipolar cells, predominantly at the axon terminals, and appears yellow in the merged images (F′). Scale bars, 20 μm. (B) Affinity chromatography of purified recombinant Cav1.2 α1 CBD on CaBP5 column. His-tagged Cav1.2α1 CT1 was loaded onto the CaBP5-Sepharose column equilibrated with PBS buffer containing 1 mM CaCl2. After washes with the same buffer, the proteins were eluted with 3 mM EGTA followed by 0.2 M glycine buffer, pH 2.1. Eluted fractions were probed with anti–His antibodies. Lane 1: protein loaded on the column; lane 2: protein present in the flow-trough; lane 3: last wash fraction before elution; lanes 4–6: elution with 5 mM EGTA; lane 7: last fraction before elution with glycine buffer; lanes 8 and 9, further elution with 0.1 M glycine buffer, pH 2.4. (C) Gel overlay assay of recombinant CaBP5 with α1 Cav1 cytoplasmic domains. His-tagged α1 CBD from Cav1.2, Cav1.3, and Cav1.4 (CT1), or C-terminal domain without CBD (CT2), were separated on SDS-PAGE and transferred to PVDF membranes, which were incubated with CaM-GST and CaBP5-GST in the presence or absence of EGTA. Bound proteins were detected with an anti–GST antibody. Ponceau staining (right) shows the relative amount of purified proteins. (D) Effect of CaBP5 on the activation of Cav1.2 Ca2+ currents. Cav1.2 Ca2+ currents were evoked by 50-ms pulses from −80 mV to various voltages in HEK293T cells transfected with Cav1.2 (α11.2, β2A, α2δ) alone or cotransfected with CaBP5 (n = 7 each). Current amplitudes were normalized to the largest in the series (I/I max) and plotted (mean ± SE) against test voltage. Smooth lines represent fits by Boltzmann equation. CaBP5 significantly increased V 1/2 (28.2 ± 2.8 vs. 19.4 ± 2.3 for Cav1.2 alone; P = 0.03) and k (−10.0 ± 0.4 vs. −8.9 ± 0.2 for Cav1.2 alone; P = 0.05). (E) Effect of CaBP5 on inactivation of Cav1.2 Ca2+ currents. Cav1.2 Ca2+ currents were evoked by 2-second pulses from −80 to +10 mV (for Cav1.2 alone) or +20 mV (for +CaBP5). Inactivation was measured at these different test voltages because of the approximately 10-mV shift in voltage-dependent activation in cells cotransfected with CaBP5. HEK293T cells were transfected with Cav1.2 (α11.2, β2A, α2δ) alone (black) or were cotransfected with CaBP5 (red). Inactivation was measured as I res/I pk, which was the current amplitude at 1-second normalized to the peak current amplitude. CaBP5 did not affect the kinetics of current decay but significantly suppressed the inactivation of the Ca2+ current (P = 0.02; n = 6 each for Cav1.2 alone and +CaBP5).
The authors thank Amber Jimenez for her outstanding technical assistance in selecting the targeted ES clone, Edward Parker for his expertise with the EM experiments, Jing Huang for her expert work in generating the anti–CaBP5 monoclonal antibody, Philippe Soriano for the PGKneolox2DTA vector, Carola Driessen for the basic targeting vector, Carol Ware and La’Akea Siverts (UW Transgenic Resource Program) for their expert work in transfecting ES cells and injecting mouse embryos, and Artur Cideciyan for helpful comments on the manuscript. 
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Figure 1.
 
(A) Generation of CaBP5 knockout mice using gene targeting. Exon and restriction map of the Cabp5 gene locus, targeting vector, and targeted Cabp5 gene. In the targeting vector, the neo cassette replaces exon 1 and exon 2 of the Cabp5 gene. The targeting vector was constructed by using an approximately 2-kb DNA fragment as the short arm that extended upstream of the initial ATG. The long arm is an approximately 6-kb genomic fragment encompassing intron 2 to intron 5 of the Cabp5 gene. The PGK-DTA and HSV-TK cassettes were included for negative selection. Gray box: 5′ probe hybridizing to a 300-bp region located upstream of the short arm of the Cabp5 gene. H, HindII; M, MscI. (B) PCR-based genotyping of wild-type (+/+), heterozygous (+/−), and knockout (−/−) mice. A 620- and 800-bp PCR product is amplified from the wild-type Cabp5 and the targeted loci, respectively. (C) Southern blot analysis of targeted gene. HindII-digested genomic DNA from wild type (+/+), heterozygous (+/−), and knockout (−/−) mice were probed with a 300-bp 5′ probe. A fragment of 2.9 kb, indicative of the wild-type Cabp5 gene, is identified in wild-type and heterozygous mice. Knockout and heterozygous mice show a fragment of 3.9 kb corresponding to the targeted Cabp5 gene.
Figure 1.
 
(A) Generation of CaBP5 knockout mice using gene targeting. Exon and restriction map of the Cabp5 gene locus, targeting vector, and targeted Cabp5 gene. In the targeting vector, the neo cassette replaces exon 1 and exon 2 of the Cabp5 gene. The targeting vector was constructed by using an approximately 2-kb DNA fragment as the short arm that extended upstream of the initial ATG. The long arm is an approximately 6-kb genomic fragment encompassing intron 2 to intron 5 of the Cabp5 gene. The PGK-DTA and HSV-TK cassettes were included for negative selection. Gray box: 5′ probe hybridizing to a 300-bp region located upstream of the short arm of the Cabp5 gene. H, HindII; M, MscI. (B) PCR-based genotyping of wild-type (+/+), heterozygous (+/−), and knockout (−/−) mice. A 620- and 800-bp PCR product is amplified from the wild-type Cabp5 and the targeted loci, respectively. (C) Southern blot analysis of targeted gene. HindII-digested genomic DNA from wild type (+/+), heterozygous (+/−), and knockout (−/−) mice were probed with a 300-bp 5′ probe. A fragment of 2.9 kb, indicative of the wild-type Cabp5 gene, is identified in wild-type and heterozygous mice. Knockout and heterozygous mice show a fragment of 3.9 kb corresponding to the targeted Cabp5 gene.
Figure 2.
 
(A) Western blot analysis of retinal extracts prepared from Cabp5 +/+, Cabp5 +/−, and Cabp5 −/− mice probed with antibodies to CaBP5 and PKCα. CaBP5 proteins are not detected in Cabp5 −/− retinas, confirming targeting of the Cabp5 gene. (B) Immunofluorescence for CaBP5 and calretinin in 6-week-old Cabp5 +/+ (WT) and Cabp5 −/− (KO) mouse retinas. The lack of CaBP5 immunoreactivity in the Cabp5 −/− retina confirmed the loss of CaBP5 protein in the knockout mice. Scale bar, 20 μm.
Figure 2.
 
(A) Western blot analysis of retinal extracts prepared from Cabp5 +/+, Cabp5 +/−, and Cabp5 −/− mice probed with antibodies to CaBP5 and PKCα. CaBP5 proteins are not detected in Cabp5 −/− retinas, confirming targeting of the Cabp5 gene. (B) Immunofluorescence for CaBP5 and calretinin in 6-week-old Cabp5 +/+ (WT) and Cabp5 −/− (KO) mouse retinas. The lack of CaBP5 immunoreactivity in the Cabp5 −/− retina confirmed the loss of CaBP5 protein in the knockout mice. Scale bar, 20 μm.
Figure 3.
 
Morphologic characterization of 6-week-old Cabp5 +/+ (WT) and Cabp5 −/− (KO) mouse retina. (A) Immunolocalization of PKCα (red) and calbindin (green) in mouse retina cross-sections. Cell nuclei were stained with DAPI (blue). (B) Immunofluorescence of PKCα (red) and calbindin (green) in the OPL (A, A′) and in the IPL (B, B′). (C) Immunofluorescence of ribeye (green) in the OPL (A, A′) and in the IPL (B, B′). Scale bars: (A) 20 μm, (B) 5 μm, (C) 2 μm.
Figure 3.
 
Morphologic characterization of 6-week-old Cabp5 +/+ (WT) and Cabp5 −/− (KO) mouse retina. (A) Immunolocalization of PKCα (red) and calbindin (green) in mouse retina cross-sections. Cell nuclei were stained with DAPI (blue). (B) Immunofluorescence of PKCα (red) and calbindin (green) in the OPL (A, A′) and in the IPL (B, B′). (C) Immunofluorescence of ribeye (green) in the OPL (A, A′) and in the IPL (B, B′). Scale bars: (A) 20 μm, (B) 5 μm, (C) 2 μm.
Figure 4.
 
Analysis of 6-week-old retina morphology by transmission electron microscopy. (A) Montage of mouse retina cross-sections analyzed by transmission electron microscopy. (B) Mouse retina cross-sections through the OPL and IPL. Higher magnification of a cross-section through the OPL (upper) and the IPL/ganglion cell layer (bottom). Scale bars: (A) 10 μm, (B) 5 μm.
Figure 4.
 
Analysis of 6-week-old retina morphology by transmission electron microscopy. (A) Montage of mouse retina cross-sections analyzed by transmission electron microscopy. (B) Mouse retina cross-sections through the OPL and IPL. Higher magnification of a cross-section through the OPL (upper) and the IPL/ganglion cell layer (bottom). Scale bars: (A) 10 μm, (B) 5 μm.
Figure 5.
 
Single-flash ERG responses to light stimuli of increasing intensity for 6-week-old Cabp5 +/+ and Cabp5 −/− mice. Serial responses to increasing flash stimuli were obtained for Cabp5 +/+ and Cabp5 −/− for selected intensities under scotopic conditions (A) and photopic conditions (B) and were plotted as a function of a-wave or b-wave amplitude compared with light intensity. Representative ERG waveforms recorded from Cabp5 +/+ and Cabp5 −/− mice in response to flashes of increasing intensity are shown on the left. In scotopic and photopic conditions, no significant differences were observed between the a-wave- and b-wave-amplitudes of Cabp5 +/+ and Cabp5 −/− mice (P > 0.1). SEM bars are shown.
Figure 5.
 
Single-flash ERG responses to light stimuli of increasing intensity for 6-week-old Cabp5 +/+ and Cabp5 −/− mice. Serial responses to increasing flash stimuli were obtained for Cabp5 +/+ and Cabp5 −/− for selected intensities under scotopic conditions (A) and photopic conditions (B) and were plotted as a function of a-wave or b-wave amplitude compared with light intensity. Representative ERG waveforms recorded from Cabp5 +/+ and Cabp5 −/− mice in response to flashes of increasing intensity are shown on the left. In scotopic and photopic conditions, no significant differences were observed between the a-wave- and b-wave-amplitudes of Cabp5 +/+ and Cabp5 −/− mice (P > 0.1). SEM bars are shown.
Figure 6.
 
Flash responses of Cabp5 +/+ and Cabp5 −/− ON ganglion cells. Flash families measured from a Cabp5 +/+ ON ganglion cell (A) and a Cabp5 −/− ON ganglion cell (B). Average responses are superimposed for flashes producing 0.001 to 0.5 photon/μm2. (C) Stimulus-response relationship for Cabp5 +/+ and Cabp5 −/− ON ganglion cells. Error bars are SEM. Half-maximal flash strengths, estimated from saturating exponential fits to the stimulus-response relations, were 0.03045 ± 0.0035 (mean ± SEM; n = 9) for Cabp5 +/+ cells and 0.04971 ± 0.0034 for Cabp5 −/− cells (n = 7).
Figure 6.
 
Flash responses of Cabp5 +/+ and Cabp5 −/− ON ganglion cells. Flash families measured from a Cabp5 +/+ ON ganglion cell (A) and a Cabp5 −/− ON ganglion cell (B). Average responses are superimposed for flashes producing 0.001 to 0.5 photon/μm2. (C) Stimulus-response relationship for Cabp5 +/+ and Cabp5 −/− ON ganglion cells. Error bars are SEM. Half-maximal flash strengths, estimated from saturating exponential fits to the stimulus-response relations, were 0.03045 ± 0.0035 (mean ± SEM; n = 9) for Cabp5 +/+ cells and 0.04971 ± 0.0034 for Cabp5 −/− cells (n = 7).
Figure 7.
 
(A) Colocalization of Cav1.2 voltage-dependent calcium channels with CaBP5 and PKCα in mouse retina. (A′C′) Confocal images of mouse retina sections double labeled with antibodies to PKCα (green, A′) and Cav1.2α1 (red, B′). Extensive colocalization of Cav1.2α1 and PKCα is observed in rod bipolar cells and appears yellow in the merged images (C′). Cav1.2 is expressed only in cells also labeled with the rod bipolar marker PKCα and is thus specifically expressed in rod bipolar cells. Scale bars, 20 μm. (D′F′). Double labeling of mouse retinal sections with CaBP5 (green, D′) and Cav1.2α1 (red, E′). CaBP5 is colocalized with Cav1.2 in rod bipolar cells, predominantly at the axon terminals, and appears yellow in the merged images (F′). Scale bars, 20 μm. (B) Affinity chromatography of purified recombinant Cav1.2 α1 CBD on CaBP5 column. His-tagged Cav1.2α1 CT1 was loaded onto the CaBP5-Sepharose column equilibrated with PBS buffer containing 1 mM CaCl2. After washes with the same buffer, the proteins were eluted with 3 mM EGTA followed by 0.2 M glycine buffer, pH 2.1. Eluted fractions were probed with anti–His antibodies. Lane 1: protein loaded on the column; lane 2: protein present in the flow-trough; lane 3: last wash fraction before elution; lanes 4–6: elution with 5 mM EGTA; lane 7: last fraction before elution with glycine buffer; lanes 8 and 9, further elution with 0.1 M glycine buffer, pH 2.4. (C) Gel overlay assay of recombinant CaBP5 with α1 Cav1 cytoplasmic domains. His-tagged α1 CBD from Cav1.2, Cav1.3, and Cav1.4 (CT1), or C-terminal domain without CBD (CT2), were separated on SDS-PAGE and transferred to PVDF membranes, which were incubated with CaM-GST and CaBP5-GST in the presence or absence of EGTA. Bound proteins were detected with an anti–GST antibody. Ponceau staining (right) shows the relative amount of purified proteins. (D) Effect of CaBP5 on the activation of Cav1.2 Ca2+ currents. Cav1.2 Ca2+ currents were evoked by 50-ms pulses from −80 mV to various voltages in HEK293T cells transfected with Cav1.2 (α11.2, β2A, α2δ) alone or cotransfected with CaBP5 (n = 7 each). Current amplitudes were normalized to the largest in the series (I/I max) and plotted (mean ± SE) against test voltage. Smooth lines represent fits by Boltzmann equation. CaBP5 significantly increased V 1/2 (28.2 ± 2.8 vs. 19.4 ± 2.3 for Cav1.2 alone; P = 0.03) and k (−10.0 ± 0.4 vs. −8.9 ± 0.2 for Cav1.2 alone; P = 0.05). (E) Effect of CaBP5 on inactivation of Cav1.2 Ca2+ currents. Cav1.2 Ca2+ currents were evoked by 2-second pulses from −80 to +10 mV (for Cav1.2 alone) or +20 mV (for +CaBP5). Inactivation was measured at these different test voltages because of the approximately 10-mV shift in voltage-dependent activation in cells cotransfected with CaBP5. HEK293T cells were transfected with Cav1.2 (α11.2, β2A, α2δ) alone (black) or were cotransfected with CaBP5 (red). Inactivation was measured as I res/I pk, which was the current amplitude at 1-second normalized to the peak current amplitude. CaBP5 did not affect the kinetics of current decay but significantly suppressed the inactivation of the Ca2+ current (P = 0.02; n = 6 each for Cav1.2 alone and +CaBP5).
Figure 7.
 
(A) Colocalization of Cav1.2 voltage-dependent calcium channels with CaBP5 and PKCα in mouse retina. (A′C′) Confocal images of mouse retina sections double labeled with antibodies to PKCα (green, A′) and Cav1.2α1 (red, B′). Extensive colocalization of Cav1.2α1 and PKCα is observed in rod bipolar cells and appears yellow in the merged images (C′). Cav1.2 is expressed only in cells also labeled with the rod bipolar marker PKCα and is thus specifically expressed in rod bipolar cells. Scale bars, 20 μm. (D′F′). Double labeling of mouse retinal sections with CaBP5 (green, D′) and Cav1.2α1 (red, E′). CaBP5 is colocalized with Cav1.2 in rod bipolar cells, predominantly at the axon terminals, and appears yellow in the merged images (F′). Scale bars, 20 μm. (B) Affinity chromatography of purified recombinant Cav1.2 α1 CBD on CaBP5 column. His-tagged Cav1.2α1 CT1 was loaded onto the CaBP5-Sepharose column equilibrated with PBS buffer containing 1 mM CaCl2. After washes with the same buffer, the proteins were eluted with 3 mM EGTA followed by 0.2 M glycine buffer, pH 2.1. Eluted fractions were probed with anti–His antibodies. Lane 1: protein loaded on the column; lane 2: protein present in the flow-trough; lane 3: last wash fraction before elution; lanes 4–6: elution with 5 mM EGTA; lane 7: last fraction before elution with glycine buffer; lanes 8 and 9, further elution with 0.1 M glycine buffer, pH 2.4. (C) Gel overlay assay of recombinant CaBP5 with α1 Cav1 cytoplasmic domains. His-tagged α1 CBD from Cav1.2, Cav1.3, and Cav1.4 (CT1), or C-terminal domain without CBD (CT2), were separated on SDS-PAGE and transferred to PVDF membranes, which were incubated with CaM-GST and CaBP5-GST in the presence or absence of EGTA. Bound proteins were detected with an anti–GST antibody. Ponceau staining (right) shows the relative amount of purified proteins. (D) Effect of CaBP5 on the activation of Cav1.2 Ca2+ currents. Cav1.2 Ca2+ currents were evoked by 50-ms pulses from −80 mV to various voltages in HEK293T cells transfected with Cav1.2 (α11.2, β2A, α2δ) alone or cotransfected with CaBP5 (n = 7 each). Current amplitudes were normalized to the largest in the series (I/I max) and plotted (mean ± SE) against test voltage. Smooth lines represent fits by Boltzmann equation. CaBP5 significantly increased V 1/2 (28.2 ± 2.8 vs. 19.4 ± 2.3 for Cav1.2 alone; P = 0.03) and k (−10.0 ± 0.4 vs. −8.9 ± 0.2 for Cav1.2 alone; P = 0.05). (E) Effect of CaBP5 on inactivation of Cav1.2 Ca2+ currents. Cav1.2 Ca2+ currents were evoked by 2-second pulses from −80 to +10 mV (for Cav1.2 alone) or +20 mV (for +CaBP5). Inactivation was measured at these different test voltages because of the approximately 10-mV shift in voltage-dependent activation in cells cotransfected with CaBP5. HEK293T cells were transfected with Cav1.2 (α11.2, β2A, α2δ) alone (black) or were cotransfected with CaBP5 (red). Inactivation was measured as I res/I pk, which was the current amplitude at 1-second normalized to the peak current amplitude. CaBP5 did not affect the kinetics of current decay but significantly suppressed the inactivation of the Ca2+ current (P = 0.02; n = 6 each for Cav1.2 alone and +CaBP5).
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