Male C57BL/6J mice (WT) were purchased from Harlan (Houston, TX, USA) and used at 2–3 months old in this study. The Ca_v1.3^−/− mice (C57BL/6J background) were originally developed by Dr. Jörg Striessnig (University of Innsbruck, Innrain, Innsbruck, Austria; Platzer et al., 2000). The Ca_v1.3^+/− (heterozygous) breeding pair for generating Ca_v1.3^−/− (homozygous knockout) was from Dr. Amy Lee (University of Iowa, Iowa City, IA, USA). The Ca_v1.3^−/−, Ca_v1.3 ^+/−, and Ca_v1.3^+/+ (WT) littermates used in this study were produced at Texas A&M University (College Station, TX, USA). All animal experiments were approved by the Institutional Animal Care and Use Committee of Texas A&M University. Mice were housed under temperature and humidity-controlled conditions with 12:12 h light-dark cycles.

The human HEK 293 cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in DMEM (BioWhittaker, Walkersville, MD, USA) containing 10% FBS (HyClone, Pittsburgh, PA, USA), 50 u/ml penicillin/50 μg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA), 1 mM sodium pyruvate (Life Technologies, Carlsbad, CA, USA), and 1x non-essential amino acids (Life Technologies) at 37°C under 5% CO₂. Cells were cultured on coverslips and placed in a 24-well culture plate. Cells were seeded in each well to 70–80% confluence 24 h prior to the transfection. Transfections were performed using Lipofectamine 2000 Transfection Reagent (Life Technologies) according to the manufacturer's protocol. The calcium channel α2δ1 subunit (rat) expression vector was a gift from Dr. Terrance P. Snutch (University of British Columbia, Vancouver, Canada). The pCDNA-Ca_v1.2 α1 subunit was originally generated by Dr. Diane Lipscombe (Brown University, Providence, Rhode Island, USA) and distributed through Addgene (Cambridge, MA, USA). The pCDNA-Ca_v1.3 α1 subunit (mouse) was from Dr. Amy Lee. The pCMV-Sport-β2 subunit (mouse) was purchased from MGC cDNA clones collection (Dharmacon, GE, Lafayette, CO). Up to 500 ng DNA (150 ng for each plasmid) was transfected into the cultured HEK cells, and culture media was exchanged 12 h after transfection. Electrophysiological recordings were carried out 60 h after the transfection.

The whole cell patch-clamp recordings for LTCCs were carried out as previously described (Ko et al., 2007; Shi et al., 2009). The external solution was (in mM): 145 TEACl, 9 BaCl₂, 0.5 MgCl₂, 5.5 glucose, 0.1 NiCl₂, and 5 HEPES, pH 7.4 adjusted with TEAOH. The pipette solution was (in mM): 125 Cs acetate, 20 CsCl, 3 MgCl₂, 10 EGTA, and 5 HEPES, pH adjusted with CsOH. The holding potential for transfected HEK cells was set at −65 mV. Currents were recorded at room temperature using an A-M Systems model 2400 patch-clamp amplifier (Sequim, WA, USA). Signals were low-pass filtered at 2 kHz and digitized at 5 kHz with Digidata 1550A interface and pCLAMP 10.5 software (Molecular Devices, Sunnyvale, CA, USA). The Ba²⁺ current was recorded immediately after the whole-cell configuration was formed by gentle suction, and the ramp-voltage command (−80 to +60 mV in 500 ms) was applied to elicit Ba²⁺ currents. D-cis-diltiazem (diltiazem; Sigma-Aldrich) was first dissolved in water and further diluted in external recording solutions to the appropriate final concentrations as denoted in the results. The cells were first recorded in normal external solution for baseline currents, followed by perfusion with diltiazem (DIL), and the ramp-voltage command was elicited once per minute after perfusion with DIL. The controls were recorded following the same protocol but perfused with an external solution without DIL. The peak current amplitudes were normalized as the percentage (%) to the original baseline amplitude (set at 100%) for each cell recorded.

Mice were dark adapted for at least 3 h prior to the recordings. All experiments were performed under dim red light as previously described (Kolesnikov and Kefalov, 2012). This ex vivo ERG recording technique and the configuration of the recording chamber were originally designed by Dr. Vladimir Kefalov (Washington University, St. Louis, MO, USA). Mouse retinas were dissected out in oxygen saturated dissection medium containing 1 mg/ml BSA and 13.6 mg/ml L-15 (Sigma-Aldrich) at 37°C. Retinas were transferred to an ex vivo ERG recording chamber (OcuScience, Henderson, NV, USA) and perfused with a buffer containing (in mM): 112 NaCl, 3.6 KCl, 2.4 MgCl₂, 20 NaHCO₃, 3 Na succinate, 0.02 EDTA, 10 Glucose, 10 HEPES (pH 7.4), 0.72 mg/ml L-15, 0.1% MEM vitamins, and MEM non-essential amino acids (Sigma-Aldrich) at 37°C. The electrode solution in the recording chamber contained (in mM): 140 NaCl, 2.4 MgCl₂, 1.2 CaCl₂, 3 HEPES (pH 7.4). In order to isolate the ERG a-wave and observe photoreceptor responses, the perfusion solution was supplemented with 2 mM L-glutamate and 10 μM DL-AP-4, and the electrode solution was supplemented with 2 mM L-glutamate and 10 mM BaCl₂ to block the higher order photo-responses (Kolesnikov and Kefalov, 2012)_. Nitrendipine (EMD Millipore, Billerica, MA) was first dissolved in dimethylsulfoxide (DMSO), and further diluted in the external recording solution to 10 μM as the final concentration. A portable ERG device (OcuScience) was used for ex vivo ERG recordings. The ERG measurements were carried out sequentially at light intensities of 0.1, 0.3, 1.0, and 3.0 cd·s/m². Each ERG response was an average of 4 light flashes at a specific light intensity. A 1-min recovery period was allowed between different intensities. The amplitudes and implicit times of a- and b-waves were recorded and analyzed by using the ERGView 4.4 software (OcuScience). Ex vivo results were normalized to the original baseline amplitude prior to the perfusion with the calcium channel inhibitors (diltiazem or nitrendipine) and reported as a change in percentage from the original baseline amplitude.

Male mice at 3 months old were used for in vivo ERG recordings (performed as previously described; Chang et al., 2015). Mice were dark-adapted for at least 8 h then anesthetized with an intraperitoneal injection of Avertin (0.5 mL/25 g-body weight of 2% 2,2,2-tribromoethanol, 1.25% tert-amyl alcohol; Fisher Scientific, Pittsburgh, PA, USA). Pupils were dilated using a single drop of a 1% tropicamide and 2.5% phenylephrine mixture for 5 min. Mice were placed on a heating pad to maintain body temperatures at 37°C. The ground electrode was placed on the tail and the reference electrode placed under the skin in the cheek below the eye. A drop of Goniovisc (Hub Pharmaceuticals, Rancho Cucamonga, CA, USA) was applied on the surface of the cornea to keep it moist, and the threaded recording electrode conjugated to a mini contact lens (Ocuscience) was placed on top of the cornea. A dim red light was used for all preparatory procedures but was turned off during the ERG recording. A portable ERG device was used for measurements of light responses from a series of light stimulations at 0.1, 0.3, 1, 3, 10, and 25 cd·s/m². Low light intensities (0.1, 0.3, 1.0, and 3.0 cd·s/m²) were flashed four times at 10 s intervals and the traces were averaged for a final ERG measurement. High light intensities (10 and 25 cd·s/m²) only had one flash. A 1 min recovery period was programmed between each light intensity. Amplitudes and implicit times of a- and b-waves were recorded and analyzed, and the oscillatory potentials were band-pass filtered between 100 and 300 Hz using the ERGView 4.4 software. The a-wave implicit time was measured from the onset of the light stimulus to the most hyperpolarized point (trough) of the a-wave, while the b-wave implicit time was measured from the onset of the light stimulus to the highest peak of the b-wave.

Ca_v1.3^−/− and Ca_v1.3^+/+ (WT) littermate mice were first anesthetized with isoflurane followed by cervical dislocation. The eyes were excised and fixed in Zamboni fixative (American Matertech Scientific Inc, Lodi, CA, USA) then cryo-protected in a 30% sucrose-PBS solution. Ca_v1.3^−/− and Ca_v1.3^+/+ eyes were embedded side by side in Tissue-Tek O.C.T. Compound (Sakura Finetek Inc, Torrance, CA, USA) and stored at −80°C. The frozen eye sections (10 μm) were cut using a cryostat (Leica Biosystem, Buffalo Grove, IL, USA) and mounted on glass slides. The sections were washed with PBS and incubated with a blocking solution containing 10% goat serum for 2 h at room temperature, followed by incubation with the primary antibodies (anti-Ribeye+anti-Ca_v1.4 or anti-Ca_v1.3 alone) at 4°C overnight. Sections were then washed three times with PBS containing 0.1% Triton 100 (PBST), incubated with a secondary antibody at room temperature for 2 h in a dark chamber, washed with PBST, and covered with the ProLong Gold antifade reagents containing 4′,6-diamidino-2-phenylindole (DAPI; Life Technologies) and a glass coverslip. The primary antibodies used were rabbit anti-Ca_v1.3 (1:100; Chemicon/Millipore Sigma, St. Louis, MO, USA), mouse anti-ribeye (1:100; Millipore) and rabbit anti-Ca_v1.4 (1:1000; a generous gift from Dr. Amy Lee, University of Iowa, Iowa City, IA). The secondary antibodies used were Alexa fluor 488 goat anti-rabbit IgG (1:200; Life Technologies) and Cy5 goat anti-mouse IgG (1:200; Abcam, Cambridge, MA, USA). The images were captured with a Zeiss LSM 780 NLO Multiphoton Microscope (Carl Zeiss AG, Oberkochen, Germany). The images from WT or Ca_v1.3^−/− were taken under the identical setting (light intensity, magnification, and capture time) for Ribeye or Ca_v1.4.

Quantification of Ribeye positive and Ca_v1.4 positive synaptic terminals: After images were captured, we used “Fiji,” an image processing package that is an open-source platform for biological image analysis (Schindelin et al., 2012) to analyze our images. The “analyze particle” function in the Image J (software in “Fiji”) to quantify all fluorescent positive structures (as in pixels). Briefly, the threshold of individual fluorescent channel (red for Ribeye positive and green for Ca_v1.4 positive) was automatically adjusted. The setting in the Image J software: particle size was from 0 to infinity, and circularity was from 0 to 1.0. We also used the “Coloc 2” (available at https://imagej.net/Coloc_2), a plugin utility to determine the colocalization of Ribeye and Ca_v1.4. One stained retinal section was used per mouse. Three areas per retinal section were randomly selected. Channel 1 was assigned as red fluorescence (Ribeye positive), and channel 2 was assigned as green fluorescence (Ca_v1.4 positive). The Li's Intensity Correlation Quotient (ICQ) value (Li et al., 2004) was generated to determine the degree of Ribeye and Ca_v1.4 colocalization: for colocalized staining 0 < ICQ ≤ 0.5; ICQ = ~0 for random staining; for segregated staining 0 > ICQ ≥ −0.5.

Samples were collected and prepared as described previously (Ko M. L. et al., 2009; Huang et al., 2013; Lin et al., 2015). Retinas were homogenized in a Tris lysis solution (50 mM Tris, 1 mM EDTA, 150 mM NaCl, 1% NP-40) including phosphatase (50 mM NaF, 1 mM Na₃VO₄) and protease inhibitors (Sigma-Aldrich). After centrifugation to remove cellular debris, an equal volume of 2x Laemmli buffer was added to each sample lysate, then the samples were heated at 95°C for 5 min. Proteins were separated by SDS-PAGE (10% gels) for 1–2 h. Proteins were then transferred to nitrocellulose membranes and probed by primary antibodies. The primary antibodies used were rabbit anti-Ca_v1.3 antibody (1:1,000; Chemicon/ Millipore Sigma) and rabbit anti-actin antibody (1:1,000; Cell Signaling Technology, Danvers, MA, USA). Actin was used for loading controls. The secondary antibody (goat anti-rabbit) conjugated to horseradish peroxidase (1:1,000; Cell Signaling Technology) and the Femto and Pico electrochemiluminescense (ECL) kits (Pierce ThermoFisher Scientific, Waltham, MA, USA) were used to visualize the blots.

All data are presented as mean ± SEM (standard error of mean). Based on this study, as well as previously published data by us and our unpublished results, the data for our patch-clamp electrophysiological recordings, in vivo ERG, ex vivo ERG, and morphological analyses were all in normal distribution. One-way analysis of variance (ANOVA) with Tukey post hoc tests were used for statistical analyses between the various treatment groups or between the WT, Ca_v1.3^+/− (heterozygous mutant), and Ca_v1.3^−/− homozygous mutant groups. The Student's t-test was used to compare between WT and Ca_v1.3 ^−/−. Throughout, p < 0.05 was regarded as significant.

We first determined which pharmacological inhibitor could effectively differentiate Ca_v1.2 from Ca_v1.3, since there is no commercially available inhibitor that will selectively block Ca_v1.2 but not Ca_v1.3 (Cooper et al., 1987; Xu and Lipscombe, 2001). Among the LTCC inhibitors, dihydropyridines, phenylalkylamines, and benzothiazepines have lower affinities for Ca_v1.3 compared to Ca_v1.2 (Cai et al., 1997; Hockerman et al., 2000; Schnee and Ricci, 2003; Baumann et al., 2004; Tarabova et al., 2007; Bissig et al., 2013; Berkowitz et al., 2014). Particularly, the half maximal inhibitory concentration (IC50) of diltiazem (DIL) for Ca_v1.3 is more than 10 times higher than Ca_v1.2 (Cai et al., 1997; Hockerman et al., 2000; Schnee and Ricci, 2003; Baumann et al., 2004; Tarabova et al., 2007; Bissig et al., 2013; Berkowitz et al., 2014), so we took advantage of using DIL at a lower concentration to inhibit Ca_v1.2 with minimal effects on Ca_v1.3. However, a caveat we faced was that the effectiveness of DIL on Ca_v1.2 vs. Ca_v1.3 was not compared in the same cell type or preparation in previous reports (Cai et al., 1997; Hockerman et al., 2000; Schnee and Ricci, 2003; Baumann et al., 2004; Tarabova et al., 2007; Bissig et al., 2013; Berkowitz et al., 2014). Hence, we first set forth using HEK 293 cells transfected with Ca_v1.2 or Ca_v1.3 and recorded the LTCC currents to identify a concentration of DIL that would inhibit Ca_v1.2 without affecting Ca_v1.3.

The HEK 293 cells were co-transfected with Ca_v1.2 or Ca_v1.3 and β2+α2δ1 auxiliary subunits. Ba²⁺ currents were recorded in the absence or presence of DIL. After 5 min of perfusion, the control cells (extracellular solution added H₂O, the vehicle) had a decrease of 20% from the baseline, which was due to the run-down of the current. After 5 min of perfusion with extracellular solution containing DIL (10, 100, or 400 μM), the current amplitudes all decreased to <50% of baseline (data not shown). Since perfusion for 2 min did not elicit any current run-down in the control cells, we chose to compare the inhibitory effects of DIL after 2 min of perfusion to avoid current run-down issues. Extracellular perfusion with 10 or 100 μM DIL for 2 min significantly decreased the Ca_v1.2-LTCC currents by 27 and 30%, respectively, while perfusion of 400 μM DIL for 2 min further decreased Ca_v1.2 currents by 71% (Figures 1A–C; Control: 96 ± 4%; DIL 10 μM: 73 ± 6%; DIL 100 μM: 70 ± 4%; DIL 400 μM: 29 ± 4%). However, extracellular perfusion with 10 μM DIL did not inhibit Ca_v1.3-LTCC currents, while perfusion with 400 μM DIL did (Figures 1D–F; Control: 95 ± 4%; DIL 10 μM: 98 ± 5%; DIL 400 μM: 42 ± 9%). This result indicates that Ca_v1.2 is more sensitive to DIL inhibition compared to Ca_v1.3 in transfected HEK cells, so treatments with 10 μM DIL could selectively dampen Ca_v1.2 with minimal impact on Ca_v1.3.

The ERG has long been used to determine overall retinal light sensitivities (Newman and Odette, 1984). The ERG a-wave reflects the photoreceptor responses to light stimulation, while the ERG b-wave represents the secondary light-evoked inner retinal responses, which reflects the summation from photoreceptor-bipolar cell synaptic transmission as well as responses from bipolar, amacrine, and Müller cells (Newman and Odette, 1984; Pinto et al., 2007). We employed the ex vivo transretinal ERG technique originally developed by Dr. Vladimir J. Kefalov (Vinberg et al., 2014), which allowed us to further dissect the contributions of LTCCs in retinal light responses from isolated mouse retinas. These ex vivo ERG recordings measure the light-induced voltage changes across the isolated retina from the photoreceptors to the ganglion cell layer (Vinberg et al., 2014), and it significantly improves the signal-to-noise ratio compared to in vivo ERGs and allows for easy assessments of pharmacological treatments in the isolated retina through extracellular perfusion (Kolesnikov and Kefalov, 2012; Vinberg et al., 2014).

Both retinas were isolated from a dark-adapted mouse and placed in a dual-recording chamber. The ex vivo ERG responses elicited at various light intensity flashes were recorded before and after perfusion with LTCC blockers under dim red lighting. We first tested the inhibitory effect of DIL and found that perfusion with 10 μM DIL for 10 min reached its maximal inhibition of the ERG responses, since continuous perfusion at this concentration for 30 min, or perfusion with higher concentrations of DIL (20, 50, or 100 μM), did not further decrease the ERG amplitudes. After ERG responses were first recorded under normal perfusion buffer, 10 μM DIL was perfused for 10 min to inhibit Ca_v1.2 followed by another round of ERG recordings. Subsequently, after perfusion with 10 μM nitrendipine (NIT) for another 10 min to further inhibit both Ca_v1.2 and Ca_v1.3, a third round of ERG responses were recorded. The representative ex vivo ERG waveforms are presented in Figures 2A–D. We found that both DIL and NIT did not significantly affect the ERG a-wave responses (Figures 2E). Inhibition of LTCCs with DIL or NIT largely decreased the ERG b-wave responses (Figures 2F), indicating that Ca_v1.2 and Ca_v1.3 were involved in post-photoreceptor and inner retinal light responses.

While the ERG a-wave represents the photoreceptor light responses, under higher light intensity stimulations, the a-wave is often contaminated by the rising phase of the b-wave responses. Without pharmacological isolation of the a-wave, there was no specific effect elicited by NIT or DIL determined. In order to assess the role of LTCCs in photoreceptor light responses, we isolated the photoreceptor responses (ERG a-wave) with a perfusion solution containing 2 mM L-glutamate and 10 μM DL-AP-4 and an electrode solution containing 2 mM L-glutamate and 10 mM BaCl₂ to block the higher order retinal light responses (Kolesnikov and Kefalov, 2012). After a-wave isolation, we found that both DIL (10 μM) and NIT (10 μM) were able to reduce the ERG a-wave amplitudes but NIT caused a larger decrease (Figures 3A–D), indicating that both Ca_v1.2 and Ca_v1.3 were involved in photoreceptor light-evoked responses. In addition, at a lower light intensity stimulation (0.3 cd.s/m²), DIL did not significantly affect the ERG a-wave as it did at higher light intensity stimulations (1 and 3 cd.s/m²), but NIT significantly decreased the ERG a-wave at all three light intensity stimulations. This suggests that Ca_v1.3 in photoreceptors might be more sensitive to changes in ambient light intensities, which echoes the notion that Ca_v1.3 is “plastic” in responding to external stimulations. Since NIT and DIL were able to dampen isolated a-wave, this could partially explain the reduction of ex vivo ERG b-wave by NIT and DIL shown in Figure 2.

Since all Ca_v (Ca_v1.2, 1.3, and 1.4) channels are expressed in the retina, our pharmacological studies (Figures 2, 3) could not exclude the possibility that DIL and NIT might inhibit Ca_v1.4. Thus, to further verify the role of Ca_v1.3 in retinal light sensitivities, we recorded retinal light responses using in vivo ERG recordings from Ca_v1.3^−/− homozygous null, Ca_v1.3^+/− heterozygous, and wild type (WT) mice at 2.5 months old. Mice were dark adapted overnight for at least 8 h prior to ERG recordings with various light intensities at 0.1, 0.3, 1, 3, 10, and 25 cd·s/m² (Figure 4A). There were 3 Ca_v1.3^+/+ (WT) mice from the littermates and 5 WT mice purchased from the vendor. There was no statistical difference in the ERG amplitudes and implicit times recorded between these two WT groups, so we merged the data as a single WT group. But both Ca_v1.3^+/− and Ca_v1.3^−/− mice had significantly decreased ERG a-wave amplitudes (Figure 4B) and delayed implicit times (Figure 4C) compared to that of WT mice. Similarly, the ERG b-wave amplitudes were decreased (Figure 4D) and implicit times delayed (Figure 4E) in Ca_v1.3^+/− and Ca_v1.3^−/− mice compared to WT mice (Table 1).

We further analyzed the ERG oscillatory potentials (OP1-OP4), which largely represent the inner retinal responses especially from amacrine cells (Wachtmeister and Dowling, 1978; Wachtmeister, 1998; Pinto et al., 2007). Both Ca_v1.3^+/− and Ca_v1.3^−/− mice had decreased OP1-4 amplitudes (Figures 5A,C,E,G) and delayed OP1-4 implicit times (Figures 5B,D,F,H) compared to WT mice (Table 2). Hence, these in vivo ERG data clearly demonstrate that Ca_v1.3 contributes to both outer and inner retinal light responses.

Our ERG results revealed that deletion of Ca_v1.3 had an impact on the retinal light responses, mainly on the b-wave amplitude, suggesting that the light signal from the photoreceptors to the inner retina, as well as the light responses in the inner retinal neurons were dampened. The Ca_v1.3^−/− retina (−/−) had the least protein expression of Ca_v1.3 compared to the WT or Ca_v1.3^+/− (+/−) retina as shown by Western blots (Figure 6A, right panel, arrow head). Hence, we carried out immunostaining to examine any potential morphological changes in the Ca_v1.3^−/− retina.

There was no noticeable morphological change of the overall retinal organization in the Ca_v1.3^−/− retina compared to the WT (Figure 6A). In the WT retina, Ca_v1.3 was present in the photoreceptor inner segment (IS), outer nuclear layer (ONL), OPL, inner nuclear layer (INL), inner plexiform layer (IPL), and the retinal ganglion cells (Figure 6A). Even though Ca_v1.3 existed in the OPL, it was not exclusively co-localized with Ribeye at the ribbon synapses (Figure 6B), indicating that Ca_v1.3 is present at the synaptic terminals but maybe not specifically at the ribbon structure. The Ca_v1.3 fluorescence in the Ca_v1.3^−/− retinal section could be the background staining.

Since one of the essential roles of LTCCs is governing the tonic release of neurotransmitters from photoreceptors, we focused on the OPL and detected two major proteins (Ribeye and Ca_v1.4) that are present in the photoreceptor ribbon synapses. Ca_v1.4 is largely expressed in the OPL, which contains the synaptic terminals of rod and cone photoreceptors (Figure 6A). Staining with Ribeye, ribbon synapses have a horseshoe-like shape in the OPL, and Ca_v1.4 is mostly co-localized within the synaptic ribbons (Figure 6C), which is consistent with a previous report (Lee et al., 2015). We quantified both Ribeye positive and Ca_v1.4 positive staining at the OPL using “Fiji” with “Coloc 2” to analyze their colocalization. “Fiji” is an image processing package that is an open-source platform for biological image analysis (Schindelin et al., 2012). The Li's Intensity Correlation Quotient (ICQ) value (Li et al., 2004) was generated in this software package to determine the degree of Ribeye and Ca_v1.4 colocalization: for colocalized/dependent staining 0 < ICQ ≤ +0.5; ICQ = ~0 for random staining; for segregated staining 0 > ICQ ≥ −0.5. In WT and Ca_v1.3^−/− mouse retinas, Ribeye and Ca_v1.4 were highly colocalized, with Li's ICQ at ~0.35 for both. With further quantification using Image J to analyze the Ribeye or Ca_v1.4 positive structure, we found that WT retinas had significantly higher Ribeye positive and Ca_v1.4 positive structures at the OPL compared to that in the Ca_v1.3^−/− mouse retinas (Table 3, Supplementary Figure 1). Thus, deletion of Ca_v1.3 had a negative impact on the photoreceptor ribbon synapses.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.