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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Neurochem. Author manuscript; available in PMC Oct 1, 2008.
Published in final edited form as:
PMCID: PMC2361098
NIHMSID: NIHMS46061
The expression of L-type voltage-gated calcium channels in retinal photoreceptors is under circadian control
Michael L. Ko,* Yilin Liu,* Stuart E. Dryer, and Gladys Y.-P. Ko*
*Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, USA
Department of Biology, Texas A&M University, College Station, Texas, USA
Department of Biology and Biochemistry, University of Houston, Houston, Texas, USA
Address correspondence and reprint requests to Gladys Y.-P. Ko, PhD, Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, mail stop 4458, College Station, TX 77843-4458, USA. E-mail: gko/at/cvm.tamu.edu
Photoreceptors are non-spiking neurons, and their synapses mediate the continuous release of neurotransmitters under the control of L-type voltage-gated calcium channels (VGCCs). Photoreceptors express endogenous circadian oscillators that play important roles in regulating photoreceptor physiology and function. Here, we report that the L-type VGCCs in chick cone photoreceptors are under circadian control. The L-type VGCC currents are greater when measured during the subjective night than during the subjective day. Using antibodies against the VGCCα1C and VGCCα1D subunits, we found that the immunofluorescence intensities of both VGCCα1C and VGCCα1D in photoreceptors are higher during the subjective night. However, the mRNA levels of VGCCα1D, but not VGCCα1C, are rhythmic. Nocturnal increases in L-type VGCCs are blocked by manumycin A, PD98059, and KN93, which suggest that the circadian output pathway includes Ras, Erk, and calcium-calmodulin dependent kinase II. In summary, four independent lines of evidence show that the L-VGCCs in cone photoreceptors are under circadian control.
Keywords: avian, circadian, photoreceptor, retina, voltage-gated calcium channel
Visual systems must anticipate daily changes in ambient illumination over 10–12 orders of magnitude. Circadian oscillators in the retina provide a mechanism for visual systems to initiate more sustained adaptive changes throughout the course of the day (Cahill and Besharse 1995; Green and Besharse 2004). The circadian oscillators in photoreceptors are endogenous and able to function independently in the absence of other retinal inputs (Cahill and Besharse 1993; Thomas et al. 1993; Ko et al. 2001). Photoreceptor circadian oscillators regulate retinomotor movement (Pierce and Besharse 1985; Burnside 2001), outer segment disc shedding and membrane renewal (LaVail 1980; Besharse and Dunis 1983), morphological changes at synaptic ribbons (Adly et al. 1999), gene expression (Korenbrot and Fernald 1989; Pierce et al. 1993; Haque et al. 2002), and the gating behavior of ion channels (Ko et al. 2001) among other photoreceptor activities. Importantly, photoreceptors are more sensitive to intense light damage at night than during the day, even in animals that have been maintained in constant darkness (DD) for several days after circadian light–dark (LD) cycle entrainment (Vaughan et al. 2002).
Photoreceptors are non-spiking neurons, and they release glutamate continuously in the darkness as a result of depolarization-evoked activation of L-type voltage-gated calcium channels (VGCCs) (Barnes and Kelly 2002). The synthesis and release of the neurohormone melatonin in photoreceptors is also under circadian control (Cahill and Besharse 1993; Bernard et al. 1997; Ivanova and Iuvone 2003b), and melatonin synthesis and release can be blocked by dihydropyridine inhibitors of L-type VGCCs (Iuvone and Besharse 1986; Ivanova and Iuvone 2003a). In this regard, we previously showed that there is a circadian regulation of the apparent affinity of cGMP-gated ion channels (CNGCs) for cGMP, which is higher during the subjective night and lower during the subjective day (Ko et al. 2001, 2003, 2004a). This CNGC affinity rhythm is mediated in part through the Ras, MAP kinase Erk, calcium-calmodulin dependent kinase II (CaMKII) signaling pathway, and the activities of Ras, Erk, and CaMKII are also under circadian control. Increased activity of CNGCs during the subject night causes greater depolarization at those times, which in turn causes activation of VGCCs. In the present study, we show that expression of L-type VGCCα1 subunits transcripts and protein are under circadian control, and the current amplitudes and the VGCCα1 subunit expression are higher during the subjective night (CT 16) than during the subjective day (CT 4).
Cell cultures and circadian entrainment
Fertilized eggs (Gallus gallus) were obtained from the Poultry Science Department, Texas A&M University (College Station, TX). Chick retinas were dissociated at embryonic day 11 (E11) and cultured for 6–7 days as described previously (Ko et al. 2001, 2003, 2004a,Ko et al. b). Cultures prepared in this way in the presence of ciliary neurotrophic factor (R&D Systems, Minneapolis, MN, USA) yield a highly enriched population of cone photoreceptors (Adler et al. 1984; Adler and Hatlee 1989; Belecky-Adams et al. 1996). Cell culture incubators (maintained at 39°C and 5% CO2) were equipped with lights and timers, which allowed for the entrainment of retinal circadian oscillators to 12 h: 12 h LD cycles in vitro. Zeitgeber time zero was designated as when the lights come on, and Zeitgeber time 12 was the time when the lights turn off. The following experiments were made on the second day of DD, after 6 days of prior entrainment to LD cycles as described previously (Ko et al. 2001, 2004b). For in ovo entrainment, retinas were excised from E17 embryos obtained from intact eggs that were exposed to LD 12 h: 12 h for 6 days (from E11 to E16). Retina cells were then dissociated, cultured in the dark, and used for electrophysiology, biochemistry, or molecular biology on the second day of culture in DD. In some experiments, after in ovo LD entrainment for 6 days, eggs were kept in DD for another day. On the second day of DD, retinas were taken out for biochemical analysis at various circadian times (CT) of the day. The reason for choosing chick embryos from E11 + 7 in vitro entrainment, or E17 for in ovo entrainment, is that more than 90% of the retina photoreceptors express functionally mature VGCC currents at the equivalent age of E16 (E8 + 8 days in cultures) in vitro (Gleason et al. 1992).
Quantitative real-time RT-PCR
The methods used for quantitative real-time reverse transcription (RT)-PCR were described previously (Ko et al. 2003, 2004b). Total RNA from cultured cells or intact retinas was collected using a RNA isolation kit (Qiagen, Valencia, CA, USA), with 500 ng of total RNA from each sample used to quantify expression of cPer2 (a clock gene), chick VGCCαsubunit and chick β-actin (loading control) mRNA by quantitative-PCR using the Taqman one-step RT-PCR kit and an ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA). The forward and reverse primers for cPer2 and chick β-actin are listed in Ko et al. (2003, 2004b). The forward and reverse primers for the VGCCα1D subunit were 5′-AAACCTGGAG-GCTTTGATGTCA-3′and 5′-CCGGAGAGGTCGCAATACAC-3′, respectively (GenBank accession number was AF027607). All primers and probes were obtained from Applied Biosystems. Accumulation of PCR products was detected directly by monitoring the increase in fluorescence from 6-carboxy-flourescein. Data were expressed as the ratio of cPer2 to β-actin or VGCCα1 subunit to β-actin. All measurements were repeated four times.
Immunoblot analysis
Samples were collected and prepared as described previously (Ko et al. 2001, 2004b). Briefly, cultured cells were washed in ice-cold phosphate-buffered saline and lysed in ristocetin-induced platelet agglutination buffer. In some experiments, intact retinas were homogenized in ristocetin-induced platelet agglutination buffer. The samples were separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes. The primary antibodies used in the studies were anti-VGCCα1C antibody (Chemicon, Temecula, CA, USA or Alomone (Jerusalem, Israel)), anti-VGCCα1D antibody (Chemicon or Alomone), or a polyclonal antibody insensitive to the phosphorylation state of Erk (Santa Cruz Biochemicals, Santa Cruz, CA, USA). Blots were visualized using anti-mouse or anti-rabbit secondary antibodies conjugated to horseradish peroxidase and an enhanced chemiluminescence detection system (Pierce, Rockford, IL, USA). The ratio of VGCCα1 subunit to total Erk, in each sample was determined by densitometry using Scion Image (NIH, Bethesda, MD, USA). All measurements were repeated 4–6 times.
Immunocytochemistry
Cultured cells on glass coverslips were fixed with Zamboni’s fixative in phosphate buffer (PB; 0.1 mol/L, pH 7.4) for 30 min at 22–24°C followed by three washes in PB containing 0.1% Triton-X, a blocking step with 0.1% triton-X/PB containing 3% normal goat serum for an hour, and then the cells were incubated overnight with primary antibodies against either VGCCα1C or VGCCα1D at a dilution of 1: 100. We observed the same patterns using VGCCα1C and VGCCα1D antibodies obtained from either Chemicon or Alomone. The cells were washed several times with PB and incubated with fluorescent conjugated secondary antibodies (Alexa 488 nm goat anti-rabbit and Alexa 594 nm goat anti-mouse; Molecular Probes, Carlsbad, CA, USA) in PB containing 1.5% normal goat serum for 1 h in the dark. After several washes in PB, the coverslips were mounted on a glass slide and stored at 4°C for later observation on a Zeiss microscope with epi-fluorescence to determine the localization of VGCCs and the Erk signaling pathway. A pair of coverslips from CT 4 and CT 16 along with the control (omitted primary antibodies substituted with the appropriate antiserums) was processed at the same time in each experiment. One single fluorescence image from CT 4, CT 16, or control was taken under identical settings that included the same exposure time and magnification. The averaged fluorescence intensities per pixel (F) of the outlined structures were analyzed without any modification using the luminosity channel of the histogram function in Adobe Photoshop 6.0 software, and the green or red fluorescence intensity was measured on a scale of 0–255. The outlined structures (areas) were identical in all cells measured with Photoshop. Three cells (F1, F2, and F3) from each experiment were measured. The background fluorescence intensity was acquired from an adjacent area without any cells (B). The relative fluorescence intensity from one single image of a particular time point (T) was converted as: T = [(F1 − B) + (F2 − B) + (F3 − B)]/3. There were three repetitions from CT 4 or CT 16, and statistical comparisons were made using Student’s t-test, and p < 0.05 was regarded as significant. All of the fluorescence intensity analyses were carried out blindly.
Perforated patch recordings and statistical analysis
Whole-cell patch clamp configuration of L-type Ca2+ channels was carried out using β-escin based perforated-patch methods (Fan and Palade 1998). For retinal photoreceptors, the external solution was (in mmol/L): NaCl 145, BaCl2 5.4, MgCl2 0.8, KCl 5.3, TEACl 10, HEPES 13, and glucose 10, pH 7.4. The pipette solution was (in mmol/L): CsCl 140, MgCl2 5, EGTA 10, and HEPES 10, pH 7.4 adjusted with CsOH (Ko et al. 2004a). Beta-escin was prepared as a 50 mmol/L stock solution in water and added to the pipette solution to yield a final concentration of 30–50 μmol/L. Cells were held at −65 mV, and ramp voltage commands (−80 to +60 mV in 500 ms) were used to evoke Ba2+ currents in the presence or absence of 3 μmol/L nitrendipine or 3 mmol/L CoCl2. The Ba2+ currents were recorded immediately after the patches were completely perforated (within 5–10 min after gigaOhm seals formed), and then Ba2+ currents were evoked again after perfusion with nitrendipine or CoCl2 for 6 min at the end of each recording. Each group contained 12–19 cells. All of the data were presented as mean ± standard error. Student’s t-test (for two groups) and one-way ANOVA followed by Tukey’s post hoc test for unbalanced n were used for statistical analyses. Throughout, p < 0.05 was regarded as significant.
L-type Ca2+-currents are under circadian control in chick photoreceptors
Gleason et al. (1992) characterized L-type VGCC currents in embryonic chick photoreceptors and showed that more than 90% of chick cones express functionally mature L-type VGCC currents at the equivalent age of E16 (E8 + 8 days in vitro). The cultures used in our studies have an equivalent age of E17 or E18 and expressed an L-type current. These currents were blocked completely by 3 μmol/L nitrendipine or 3 mmol/L CoCl2 (Fig. 1a and b). The current–voltage relationship was similar whether it was derived from ramp commands or 40-ms step commands from a holding potential of −65 mV (−50 to 40 mV at 10 mV increments; Fig. 1c and d). The channels were activated at the voltage range from −50 to −35 mV, while the maximum currents occurred between −10 and 0 mV in all recordings. The maximum amplitudes of L-type VGCCs in 5.4 mmol/L external Ba2+ ranged from 15 to 23 pA. Retinal cells from E11 or E12 were cultured and entrained under LD cycles for 4–5 days and transferred to DD. On the second day of DD, recordings were made using perforated patch configuration in photoreceptors at different times of day. On the second day of DD during the subjective day (CT 4–7, Fig. 1e1–e3) and the subjective night (CT 16–19, Fig. 1f1–f3), the β-escin perforated patch recordings with a ramp command (−80 to +60 mV in 500 ms) were performed. At the end of each experiment, all cells recorded were perfused with 3 mmol/L CoCl2 to block the VGCC currents (Fig. 1e1 and f1), and the net L-type VGCC currents were obtained by the subtraction between Ba2+ and Co2+ currents for each individual photoreceptor recorded (Fig. 1e2 and f2). There was a circadian rhythm in the maximum amplitude of the L-type currents, with a larger current occurring at night than during the subjective day (Fig. 1e, f and g). Figure 1g showed the overall average of currents carried in BaCl2 minus those evoked in the presence of CoCl2. This digital subtraction procedure ensured that we quantitatively isolated L-type Ca2+ currents from other currents that might be present. There was no significant difference in the voltage that activated the maximum currents in cells recorded during the subjective day or the subjective night. In photoreceptor-enriched cultures, the protein expression of the L-type VGCCα1 subunit was also rhythmic as shown by immunoblot (Fig. 1h).
Fig. 1
Fig. 1
The L-type voltage-gated calcium channel (VGCC) currents were present in E17 chick cone photoreceptors in cultures. (a and b) There were L-type VGCC currents present in E17 chick cone photo-receptors, and the currents were blocked by 3 μmol/L (more ...)
The cellular distributions of L-type VGCCα1 subunits in photoreceptors are under circadian regulation
Next, we examined whether there was a circadian regulation of the protein distribution of L-type VGCCs after entrainment in vitro. Chick retinas were cultured at E11 under LD cycles for 5 days and kept in DD for another day. On the second day of DD, cells were fixed with Zamboni’s fixative for immunocytochemistry at CT 4 and CT 16. Chick cone photoreceptors were elongated and bipolar, with one or more prominent oil droplets located at the base of the outer segment, which is the hallmark of chick cone photoreceptors (top panels of Fig. 2a and b; arrows indicated cone photoreceptors). We found that in cultured chick retinas, the VGCCα1C appeared on the membrane boundary and processes of almost all cell types including photoreceptors, other neurons, and glia cells (Fig. 2a), while the VGCCα1D was present mainly in the cell body (inner segment) of photoreceptors as well as other retinal neurons but not glia cells or fibroblasts (Fig. 2b). We used Adobe Photoshop software to quantify the fluorescence intensities of VGCCα1C and VGCCα1D present only in photoreceptors, and found that the fluorescence intensities of both VGCCα1C and VGCCα1D were significantly greater in photoreceptors fixed during the subjective night (CT 16) than during the subjective day (CT 4; Fig. 2c and d).
Fig. 2
Fig. 2
The distribution of voltage-gated calcium channel (VGCC) α1C and α1D subunits was different in photoreceptors. (a) The distribution of VGCCα1C was found on the membrane boundary and processes of almost all cell types, including (more ...)
The mRNA expression and protein levels of L-type VGCCs in chick retinas are under circadian control
We set out to determine whether there was a circadian rhythm in the mRNA expression of VGCCα1C and VGCCα1D in chick retinas. While VGCCα1C exists in almost all the cell types including glia cells in the chick retina, VGCCα1D is mainly present in retinal neurons. Chick embryos (E11) were entrained under LD cycles in ovo for 5 days and kept in DD for another day. On the second day of DD, retinas were dissected out at six different CT points, from CT 0, 4, 8, 12, 16, and 20, and prepared for quantitative real-time RT-PCR. We found that the mRNA of VGCCα1C did not display any circadian rhythmicity (Fig. 3a). The mRNA of VGCCα1D (Fig. 3b) was rhythmic, and the mRNA level was higher and with the peak value at CT 12, which was significantly different from CT 0, 4, 16, and 20 (*p < 0.05), but not CT 8. As the mRNA levels of the VGCCα1D were rhythmic, we next examined whether the protein expression of the L-type VGCCα1D was under circadian control from the embryos entrained in ovo. We observed that the protein expression of the VGCCα1D subunit was higher during the subjective night and lower during the subjective day, and the peak value at CT 16 was significantly different from CT 0 and CT 4 (*p < 0.05) but not from CT 8, 12, and 20 (Fig. 3c). An apparent rhythm in VGCCα1C protein expression in the retina did not achieve statistical significance (n = 4, data not shown).
Fig. 3
Fig. 3
There was a circadian rhythm in the mRNA and protein expression of L-type voltage-gated calcium channel (VGCC) α1D, but not in VGCCα1C. (a) The mRNA levels of the L-type VGCCα1C were not rhythmic; n = 4 for each circadian time (more ...)
The VGCC rhythm is under the control of Ras, MAP kinase Erk, and CaMKII
In chick photoreceptors, the apparent affinity of cGMP to cGMP-gated cation channels is under circadian control. The small GTPase Ras, MAP kinase Erk, and CaMKII are part of the circadian output pathway regulating the rhythmicity of cGMP-gated cation channels (Ko et al. 2001, 2004b). We postulated that this Ras–Erk–CaMKII pathway also controls VGCC rhythmicity. Chick embryos (E11) were entrained in LD, and retinas were excised and cultured in DD. On the second day of DD, photoreceptors were treated with the MEK inhibitor PD98059 (50 μmol/L) for 2 h at CT 2 and CT 14 prior to electrophysiological recordings. MEK phosphorylates and activates Erk. We found that PD98059 decreased the current amplitude when photoreceptors were recorded from during the subjective night (CT 16–19) but not the subjective day (CT 4–7; Fig. 4a). Treatment with the Ras inhibitor manumycin A (1 μmol/L) or CaMKII inhibitor KN-93 (10 μmol/L) for 2 h prior to patch recordings at CT 2 and CT 14 showed the same pattern, as both inhibitors dampened the circadian rhythms of L-type VGCC currents by decreasing current amplitudes during the subjective night (data not shown). Treatment with PD98059, manumycin A, or KN-93 (10 μmol/L) for 2 h decreased the protein expression of VGCCα1 subunits during the subjective night (CT 17) but not the subjective day (CT 5), while treatment with KN-92 (10 αmol/L), the inactive analog of KN-93, had no effect on VGCCα1 subunit expression (Fig. 4b–d). Similar results were observed using anti-VGCCα1D antibody (Fig. 4e and f; n = 4).
Fig. 4
Fig. 4
The circadian regulation of L-type voltage-gated calcium channels (VGCCs) is through Ras, MAPK (Erk), and calcium-calmodulin dependent kinase II (CaMKII). (a) Treatment with the MEK inhibitor PD98059 (50 μmol/L) for 2 h prior to the perforated (more ...)
L-type VGCCs mediate calcium influx into photoreceptor inner segments and synaptic terminals and thereby regulate calcium homeostasis, cell metabolism, cytoskeletal dynamics, gene expression, and cell death (Krizaj and Copenhagen 2002). Among other processes, these channels play a central role in the regulation of melatonin synthesis and secretion, a process that is under circadian control in vertebrate photoreceptors and that drives circadian changes throughout the retina (Iuvone and Besharse 1986; Cahill and Besharse 1993; Bernard et al. 1997; Ivanova and Iuvone 2003a; Ivanova and Iuvone 2003b). Here, we have provided four independent lines of evidence showing the expression of L-type VGCCα1 mRNA transcript and protein in isolated photoreceptors is under circadian control. We observed that the maximum amplitudes of L-type VGCC currents were greater at night than during the day, even in cells maintained in DD after circadian entrainment. However, the gating properties of the channels did not change markedly as a function of the time of the day. The main factor contributing to the circadian regulation of L-type VGCC current amplitudes appears to be the expression of functional VGCCα1 subunits, especially VGCCα1D, as both mRNA levels and protein expression of VGCCα1D are rhythmic. Circadian regulation of L-type VGCCs could play a role in increased light sensitivity at night (Vaughan et al. 2002), as well as in regulating the nocturnal increase of melatonin synthesis and release (Iuvone and Besharse 1986; Ivanova and Iuvone 2003a) in photoreceptors.
Hull et al. (2006) observed a diurnal rhythm of L-type VGCC currents in goldfish retina. The average peak amplitudes of L-type VGCCs recorded from goldfish bipolar terminals were significantly larger at midnight than at midday in cells maintained in LD cycles. As with our studies, Hull et al. did not observe any change in the I–V relationship or activation kinetics of L-type VGCCs, and the calcium-dependent chloride currents remained constant throughout the course of the day (Hull et al. 2006). The results from Hull et al. do not establish a circadian control mechanism, because the cells were isolated from animals kept in LD cycles. However, it is certainly possible that the circadian regulation of L-type VGCCs we observe in photoreceptors may be a more general phenomenon, especially given that several components of the electroretinograms recorded from different species, including humans, display circadian rhythms (Manglapus et al. 1998; Tuunainen et al. 2001; Miranda-Anaya et al. 2002; Ren and Li 2004; Peters and Cassone 2005).
In adult chick retina sections, the distribution of VGCCα1C appears surrounding the cell bodies in the outer nuclear, inner nuclear, and ganglion cell layers with an appearance like Müller glia cells, while the VGCCα1D is distributed mainly on the cell bodies of photoreceptors, bipolar cells, and ganglion cells (Firth et al. 2001). In both mice (Morgans et al. 2005) and rats (Xu et al. 2002), VGCCα1D is distributed in the cell bodies and terminals of photoreceptors, while VGCCα1C is not observed in rat photoreceptors. We found that in dissociated chick retina cultures, VGCCα1C appeared on the membrane boundary and processes of almost all cell types including photoreceptors, glia cells, and fibroblasts, while VGCCα1D was present mainly in the inner segment of photoreceptors as well as other retinal neurons but not in glia cells or fibroblasts. The fluorescence intensities of both VGCCα1C and VGCCα1D were significantly greater when measured in photoreceptors fixed at night than during the day. However, from the whole retina preparation, we only observed the circadian rhythms in mRNA levels and protein expression of VGCCα1D, but not VGCCα1C. This discrepancy could be due to the abundance of VGCCα1C in the glia cells and fibroblasts in the intact retina tissue, while in our photoreceptor enriched cultures, more than 70% of cells were photoreceptors (Ko et al. 2001, 2003, 2004a,Ko et al. b) because of the presence of ciliary neurotrophic factor in the culture medium (Adler et al. 1984; Adler and Hatlee 1989; Belecky-Adams et al. 1996). Therefore, we were able to observe the circadian rhythm in VGCCα1C fluorescence intensities measured in cultured photoreceptors only, but we did not observe the VGCCα1C rhythm in intact retinas. We did not observe the circadian rhythmicity of VGCCα1C fluorescence intensity in cultures when we measured from the glia cells (data not shown). Hence, it also provides indirect evidence that the driving force of the circadian rhythmicity in the retina is in retinal neurons but not in glia cells.
The L-type VGCCs in photoreceptors mediate early stages of visual information and photosensitivity by facilitating the release of glutamate and by receiving inputs that modulate photoreceptor neurotransmission (Barnes and Kelly 2002). The calcium influx through L-type VGCCs is sufficient to trigger sustained glutamate release from photoreceptors (Schmitz and Witkovsky 1997), even though it is evident that cGMP-gated cation channels may play a supporting role in glutamate release (Rieke and Schwartz 1994). Circadian rhythms are evident in electroretinograms recorded from different species, including humans (Manglapus et al. 1998; Tuunainen et al. 2001; Miranda-Anaya et al. 2002; Ren and Li 2004; Peters and Cassone 2005), which indicates that the release of glutamate from photoreceptors and photosensitivity could well be under circadian control. In addition, photoreceptors are more susceptible to intense light damage at night than during the day (Vaughan et al. 2002). Thus, circadian regulation of L-type VGCCs in part contributes to the daily variation of photoreceptor physiology and its photosensitivity.
L-type VGCCs may have dual functionalities as circadian outputs to regulate downstream targets, as well as inputs to the circadian oscillators. In sensory synapses, synaptic ribbons mediate continuous neurotransmitter release in response to graded depolarization, and the shape and number of photoreceptor synaptic ribbons are under circadian control (Vollrath and Spiwoks-Becker 1996; Hull et al. 2006). In mice lacking the α2 subunit of the L-type VGCCs, the photoreceptor ribbons become unanchored or disorganized (Ball et al. 2002). In fish retina bipolar cells, the diurnal regulation of L-type VGCC currents correlates with a higher percentage of organized vesicular halos around the synaptic ribbons at midnight compared to midday (Hull et al. 2006). Hence, L-type VGCCs may contribute to the circadian changes in synaptic ribbon structure.
There is a robust diurnal modulation of L-type VGCC currents in the rat suprachiasmatic nucleus (Pennartz et al. 2002). However, it is controversial as to the precise physiological role of this rhythm in suprachiasmatic nucleus neurons (Pennartz et al. 2002; Cloues and Sather 2003; Jackson et al. 2004). One possibility is that L-type VGCCs could participate in the circadian input pathway to synchronize the rhythmicity of the expression of clock genes such as Per2 and Bmal1 (Nahm et al. 2005). Hence, L-type VGCCs could contribute in coordinating rhythmic clock gene expression through the input pathway, even though the channels themselves are regulated by the circadian clocks and serve as outputs. Such an arrangement would formally similar to the model presented by Roenneberg and Merrow (1999), in which the input pathways that lead to entrainment of the ‘core oscillator’ can themselves be regulated by the oscillators as part of the output pathways. One important feature of this model is that it contains additional feedback loops at the cellular level, which can markedly enhance the stability of the overall oscillator system. Therefore, L-type VGCCs may have a significant impact on maintaining the stability of the circadian oscillators at the cellular level, and such a model has been proposed for invertebrate circadian pacemaker cells (McMahon and Block 1987).
We previously showed that Ras, Erk, and CaMKII are part of the circadian output to regulate the apparent affinity of cGMP to CNGCs (Ko et al. 2001). Here, we show that the same circadian output pathway also regulated the L-type VGCC rhythms. The varying maximum amplitudes of the L-type VGCC currents is in stark contrast to the CNGC maximum currents, which remain constant throughout the day, and which instead exhibit changes in gating properties (Ko et al. 2001). It is possible that this Ras–Erk–CaMKII signaling pathway serves as an essential circadian output component in photoreceptors, while there are different downstream regulators to modulate the different ion-channel activities.
In summary, we have observed a circadian regulation of L-type VGCCs in chick photoreceptors that can be seen in measurements of transcripts, proteins, and macroscopic currents. The circadian regulation of L-type VGCCs is partially through the Ras–Erk–CaMKII output pathway, and this pathway may serve as part of a universal output pathway in the circadian control of photoreceptor physiology and function. The circadian control of L-type VGCCs could have a profound impact in regulation of photoreceptor physiology.
Acknowledgments
We thank Dr Robert Burghardt, Director of Image Analysis Lab, College of Veterinary Medicine and Biomedical Sciences at Texas A&M University, for his help on fluorescence imaging and the usage of the Zeiss Axioplan 2 Microscope. We also thank Drs Paul Hardin and David Earnest and Ms Lily Bartoszek for their critical reading of the manuscript and fruitful discussion. This work was supported by the start-up fund from Texas A&M University and NIHRO1EY017452 to GK.
Abbreviations used
CaMKIIcalcium-calmodulin dependent kinase II
CNGCscGMP-gated ion channels
CTcircadian time
DDconstant darkness
LDlight–dark
PBphosphate buffer
VGCCsvoltage-gated calcium channels

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