|Home | About | Journals | Submit | Contact Us | Français|
The L-type voltage-gated calcium channels (L-VGCCs) in avian retinal cone photoreceptors are under circadian control, in which the protein expression of the α1 subunits and the current density are greater at night than during the day. Both Ras-mitogen-activated protein kinase (MAPK) and Ras-phosphatidylionositol 3 kinase-protein kinase B (PI3K-AKT) signaling pathways are part of the circadian output that regulate the L-VGCC rhythm, while cAMP-dependent signaling is further upstream of Ras to regulate the circadian outputs in photoreceptors. However, there are missing links between cAMP-dependent signaling and Ras in the circadian output regulation of L-VGCCs. In this study, we report that calcineurin, a Ca2+/calmodulin-dependent serine (ser)/threonine (thr) phosphatase, participates in the circadian output pathway to regulate L-VGCCs through modulating both Ras-MAPK and Ras-PI3K-AKT signaling. The activity of calcineurin, but not its protein expression, was under circadian regulation. Application of a calcineurin inhibitor, FK-506 or cyclosporine A, reduced the L-VGCC current density at night with a corresponding decrease in L-VGCCα1D protein expression, but the circadian rhythm of L-VGCCα1D mRNA levels were not affected. Inhibition of calcineurin further reduced the phosphorylation of ERK and AKT (at thr 308) and inhibited the activation of Ras, but inhibitors of MAPK or PI3K signaling did not affect the circadian rhythm of calcineurin activity. However, inhibition of adenylate cyclase significantly dampened the circadian rhythm of calcineurin activity. These results suggest that calcineurin is upstream of MAPK and PI3K-AKT but downstream of cAMP in the circadian regulation of L-VGCCs.
Circadian oscillators regulate functional and physiological activities in vertebrates (Cohen and Albers, 1991; Eastman et al., 1984; Boden et al., 1996), and those in retinal photoreceptors control the daily oscillation in their physiological and morphological changes (Pierce and Besharse, 1985; Burnside, 2001; LaVail, 1980; Adly et al., 1999; Korenbrot and Fernald, 1989; Pierce et al., 1993; Haque et al., 2002; Ko et al., 2001; Ko et al., 2007). In photoreceptors, the continuous release of neurotransmitters is an L-type voltage-gated calcium channel (L-VGCC) dependent process (Barnes and Kelly, 2002), and cone L-VGCCs are under circadian control (Ko et al., 2007). The circadian regulation of L-VGCCs is mediated through two parallel signaling pathways, Ras-mitogen-activated protein kinase (MAPK) and Ras-phosphatidylionositol 3 kinase-protein kinase B (PI3K-AKT), and both are downstream of cAMP signaling (Ko et al., 2007; Ko et al., 2009).
Calcineurin, also known as protein phosphatase 2B (PP2B), is a Ca2+/calmodulin-dependent ser/thr phosphatase, which often dephosphorylates the targets of Ca2+/calmodulin-dependent kinase II (CaMKII; Wang and Kelly, 1996; Ghetti and Heinemann, 2000; Wen et al., 2004; Gerges et al., 2005). Calcineurin contains a 58–64 kDa calmodulin-binding catalytic subunit and a 19 kDa Ca2+-binding regulatory subunit (Klee et al., 1979; Klee et al., 1988). In the retina, calcineurin is expressed in various neurons including photoreceptors (Cooper et al., 1985; Nakazawa et al., 2001). Glutamate induces hyperpolarization of postsynaptic ON bipolar cells by binding to metabotropic glutamate receptors (mGluR6), and this depression of postsynaptic response is mediated through calcineurin (Snellman and Nawy, 2002). However, little is known about the role of calcineurin in the retina, or whether it participates in the visual process. Therefore, in this study, we set forth to investigate the role of calcineurin in the regulation of retinal photoreceptors.
Both FK-506 and cyclosporine A are widely used as immunosuppressants in post-organ transplantation and autoimmune diseases, partially through their actions as calcineurin inhibitors (Emmel et al., 1989; Tocci et al., 1989; Shapiro et al., 1991; Fruman et al., 1992). FK-506 and cyclosporine A inhibit calcineurin through interactions with FK-506 binding protein 12 (FKBP12) and cyclophilin A, respectively (Liu et al., 1991). Inhibition of calcineurin causes circadian phase shifts in mammals, which indicates that calcineurin is involved in the circadian input pathway to reset or entrain the circadian clock in the suprachiasmatic nucleus (SCN; Ding et al., 1998; Katz et al., 2008). In addition, other ser/thr phosphatase families, such as PP2A and PP1, are known to directly regulate the circadian clock mechanism in Drosophila and Neurospora (Sathyanarayanan et al., 2004; Yang et al., 2004; Schafmeier et al., 2005; Fang et al., 2007). Therefore, it is possible that calcineruin may have circadian phase-dependent actions in vertebrates. Here, we report that calcineurin served as part of the circadian output pathway, downstream from cAMP but upstream of Ras, to regulate photoreceptor L-VGCCs. These results suggest that in addition to phase-shifting as previously reported, calcineurin also serves in the circadian output pathway to regulate downstream targets.
Fertilized eggs (Gallus gallus) were obtained from the Poultry Science Department, Texas A&M University (College Station, TX, USA). Chicken retinas were dissociated at embryonic day 12 (E12) and cultured for 6 days as described previously (Ko et al., 2007; Ko et al., 2009). Cultures were prepared in the presence of 20 ng/ml ciliary neurotrophic factor (CNTF; R&D Systems, Minneapolis, MN, USA), which yields cultures highly enriched with cone photoreceptors (Adler and Hatlee, 1989; Adler et al., 1984; Belecky-Adams et al., 1996) and 10% heat-inactivated horse serum. 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 12h: 12 h light-dark (LD) cycles in vitro. Zeitgeber time zero (ZT 0) was designated as the time when the lights turned on and ZT 12 was the time when the lights went off. For in ovo entrainment, intact eggs at E10 were exposed to LD 12h: 12 h for 7 days. Retina cells were then dissociated, cultured, kept in constant darkness (DD), and used for biochemical and molecular biological assays on the second day of 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 collected at different circadian time (CT) points throughout a day for biochemical assays (Ko et al., 2007; Ko et al., 2009). The reason for using chick embryos from E12+6 for in vitro entrainment or E18 for in ovo entrainment is that more than 90% of the retina photoreceptors express functionally mature VGCC currents by E18 (Gleason et al., 1992).
Samples were collected and prepared as described previously (Ko et al., 2009; Ko et al., 2007). Briefly, intact retinas were homogenized in Tris lysis buffer including (in mM): 50 Tris,1 EGTA, 150 NaCl, 1% Triton X-100, 1% β-mercaptoethanol, 50 NaF, 1 Na3VO4; pH 7.5. Samples were separated on 10% sodium dodecyl sulfate–polyacrylamide gels by electrophoresis and transferred to nitrocellulose membranes. The primary antibodies used in this study were anti-pan calcineurin A (Cell Signaling Technology, Danvers, MA, USA), an antibody specific for di-phospho-ERK (pERK; Sigma, St. Louis, MO, USA), an antibody insensitive to the phosphorylation state of ERK (total ERK, used for loading control; Santa Cruz Biochemicals, Santa Cruz, CA, USA), anti-VGCCα1D subunit (Alomone, Jerusalem, Israel), and anti-Ras (Millipore, Temecula, CA, USA). Blots were visualized using appropriate secondary antibodies conjugated to horseradish peroxidase (Cell Signaling Technology) and an enhanced chemiluminescence (ECL) detection system (Pierce, Rockford, IL, USA). Relative protein expressions for all proteins involved in this study are reported as a ratio to total ERK, since total ERK remains constant throughout the day. Band intensities were quantified by densitometry using Scion Image (NIH, Bethesda, MD, USA). All measurements were repeated at least 3 times.
Retina samples were lysed in a phosphatase lysis buffer including (in mM): 50 Tris, pH 7.5, 1 EGTA, 150 NaCl, 1%Triton X-100, and 1% β-mercaptoethanol. Calcineurin activities were assayed using a commercially available ser/thr phosphatase assay kit (Promega, Madison, WI, USA). This kit can distinguish between tyrosine (tyr) and ser/thr phosphatases by using a synthetic polypeptide, RRA(pT)VA, that is compatible with ser/thr phosphatases but is structurally incompatible for tyr phosphatases. To differentiate between PP2A, 2B, and 2C, the reaction buffer is made to favor one over the others since this class of enzyme has a diverse range of optimum conditions. For calcineurin (PP2B), the reaction buffer contained 250 mM imidazole (pH 7.2), 1 mM EGTA, 50 mM MgCl2, 5 mM NiCl2, 250 ug/ml calmodulin, and 0.1% β-mercaptoethanol, as described in the manufacturer’s protocol. Free cytoplasmic phosphate was first removed from the samples then dephosphorylation of the kit’s calcineurin substrate proceeded for 30 min at room temperature (RT). This system determines the amount of free phosphate generated in a reaction by measuring the absorbance (600 nm) of a molybdate/ malachite green/ phosphate complex.
Whole cell patch-clamp configuration of L-VGCC current recordings were carried out using mechanically ruptured patches. For retinal photoreceptors, the external solution was (in mM): 110 NaCl, 10 BaCl2, 0.4 MgCl2, 5.3 KCl, 20 TEA-Cl, 10 HEPES, and 5.6 glucose, pH 7.35 with NaOH. The pipette solution was (in mM): 135 Cs acetate, 10 CsCl, 1 NaCl, 2 MgCl2, 0.1 CaCl2, 1.1 EGTA, and 10 HEPES, pH 7.3 adjusted with CsOH. Recordings were made only from cells with elongated cell bodies with one or more prominent oil droplets (hallmark of avian cone photoreceptors). Currents were recorded at RT (23°C) using an Axopatch 200B (Axon Instruments/Molecular Devices, Union City, CA, USA) or A-M 2400 amplifier (A-M Systems Inc., Carlsborg, WA, USA). Signals were low-pass filtered at 2 kHz and digitized at 5 kHz with Digidata 1440A interface and pCLAMP 10.0 software (Molecular Devices). After Gigaohm seals were formed, the electrode capacitance was compensated. Cells were held at −65mV, and ramp voltage commands from −80 to +60 mV in 500 ms were used to evoke Ba2+ currents. Current–voltage (I–V) relations were also elicited from a holding potential of −65 mV in 200 ms steps (5 s between steps) to test potentials over a range of −80 to +60 mV in 10 mV increments. The maximal currents were obtained when the steps depolarized to 0 ~ +10 mV. The membrane capacitance, series resistance, and input resistance of the recorded photoreceptors were measured by applying a 5 mV (100 ms) depolarizing voltage step from a holding potential of −65 mV. Cells with an input resistance smaller than 1 GΩ were discarded. The membrane capacitance reading was used as the value for whole cell capacitance. The current densities (pA/pF) were obtained by dividing current amplitudes by membrane capacitances. FK-506 and cyclosporine A were obtained from A.G. Scientific (San Diego, CA, USA). The concentrations of FK-506 (Mukherjee et al., 2010; Okazawa et al., 2009; Wilson et al., 2001) and cyclosporine A (Bambrick et al., 2006; Chen et al., 2009; McDonald et al., 1996; Rana et al., 2009; Tan et al., 2011) used in this report were based on previous studies using these inhibitors in various neuronal tissue or cell preparations.
The method used for Q-PCR analysis was described previously (Ko et al., 2004; Ko et al., 2007). Total RNA was isolated using a commercially available kit (Qiagen, Valencia, CA, USA). Three hundred ng of total RNA was used to quantify VGCCα1D and β- actin (loading control) mRNA by Q-PCR using the Taqman one-step RT-PCR kit and an ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA). All primers and probes were purchased from Applied Biosystems and sequences were listed previously (Ko et al., 2004; Ko et al., 2007). All measurements were repeated 6 times.
The amount of cAMP in retina samples was determined by a commercially available immunoassay kit (Arbor Assays, Ann Arbor, MI, USA). Whole retina (for time point analysis) or cultured retina cells (for FK-506 treatment) were lysed with a small portion being saved for protein concentration determination (Bradford method; Bio-Rad, Hercules, CA, USA). Samples were incubated at RT for 30 min in the microplate wells provided. Reactions were then stopped, and the optical density of each well was determined at 450 nm. Cyclic AMP amount was calculated by comparing sample absorbance readings to a standard curve. n=4–5.
Ras activity was determined by a commercially available kit (Millipore), and the procedure was outlined previously (Ko et al., 2004). The procedure takes advantage of the fact that only activated Ras binds to the Ras binding domain of Raf-1 (Raf-1 RBD). The Raf-1 RBD is a GST (glutathione S-transferase) fusion-protein bound to glutathione agarose. Cultured retina cells (control and FK-506 treated) were lysed in a Mg2+ lysis buffer. A small portion (20 µl) of the supernatant was saved for total ERK (loading control) analysis by Western blotting. The remaining supernatant was incubated with Raf-1 RBD agarose for 45 min at 4°C. Subsequently, the agarose beads were pelleted, washed, and boiled in 2× Lamelli buffer (20 µl). Samples were then subjected to Western immunoanalysis as described above. n=4.
All data are presented as mean ± SEM (standard error of mean). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for unbalanced n was used for statistical analyses. Throughout, * p<0.05 was regarded as significant. Any defined rhythmic expression had to exhibit at least a 1.5 fold change in rhythmic amplitude (Karaganis et al., 2008).
Calcineurin is known to be involved in circadian phase-shifting in mammals (Katz et al., 2008), but it is not clear whether calcineurin itself is expressed in circadian oscillations. We first examined whether the protein expression or the activity of calcineurin was under circadian control. Chick embryos were entrained to LD cycles for 7 days in ovo then kept in DD. On the second day of DD, retinas were collected at six different CT points for immunoblotting or calcineurin activity assay. Previously, we showed that the total amount of ERK protein is constant throughout the day, while the phosphorylation status of ERK (phosphorylated ERK) is under circadian control (Ko et al., 2001; Ko et al., 2007). Therefore, in this study, we used total ERK as the loading control. We found that calcineurin protein expression was constant throughout the day (Fig. 1A), but its activity was under circadian control with peak activity during the middle of the subjective night (CT16) with a threefold difference between apex and trough values (Fig. 1B). Hence, there was a circadian regulation of calcineurin activity at the post-translational level in the chick retina.
We previously found that L-VGCCs are under circadian control in cone photoreceptors (Ko et al., 2007; Ko et al., 2009), with maximal current density elicited at 0 mV significantly larger when cells are recorded during the subjective night than during the subjective day (Fig. 2; Ko et al., 2007; Ko et al., 2009). The underlying mechanism of the L-VGCC circadian rhythm is in part attributed to the circadian regulation of both mRNA and protein expression of the L-VGCCα1 subunit (Ko et al., 2007), as well as α1 subunit trafficking and insertion / retention into the plasma membrane (Ko et al., 2007; Ko et al., 2009; Shi et al., 2009). Various signaling pathways are involved in the circadian regulation of L-VGCCs, including CaMKII, Ras-MAPK, and Ras-PI3K-AKT (Ko et al., 2007; Ko et al., 2009). Since CaMKII is involved in the circadian regulation of L-VGCCs, we hypothesized that calcineurin, a Ca2+-calmodulin dependent phosphatase that often dephosphorylates the same targets of CaMKII, might also participate in the circadian regulation of L-VGCCs.
We examined whether inhibitors of calcineurin might have a circadian phase-dependent effect on L-VGCC currents. Whole-cell patch recordings were performed from cultured cone photoreceptors at either ZT (or CT) 4–8 or 16–20. We observed that calcineurin inhibitors evoked a circadian phase-dependent modulation of L-VGCCs. Application of the calcineurin inhibitor FK- 506 (10 µM) for 2 hr prior to recordings decreased L-VGCC current density when cells were recorded at night (ZT 16–20), but did not affect L-VGCC recordings during the day (ZT 4–8; Fig. 2B, 2C). Similarly, application of a structurally unrelated calcineurin inhibitor, cyclosporine A (2 µM), for 2 hr prior to recordings also caused a significant decrease in photoreceptor L-VGCC current densities when recorded during the subjective night (CT16–20; Fig. 2D). Hence, treatment with a calcineurin inhibitor decreased L-VGCC currents at night under both LD (ZT 16–20) and DD (constant darkness, CT 16–20) conditions.
Since calcineurin is known to cause circadian phase shifting in mammals (Katz et al., 2008), it is possible that the circadian phase-dependent regulation of L-VGCCs by calcineurin could be due to phase-shifting, in which L-VGCCα1 subunit mRNA would be affected after calcineurin inhibitor treatments. However, we found that while FK-506 dampened the circadian rhythm of L-VGCCα1D protein expression (Fig. 3A), it had no effect on the circadian rhythm of L-VGCCα1D mRNA levels (Fig. 3B). Hence, the circadian phase-dependent action of calcineurin was not as a circadian input to shift the circadian phase of L-VGCCs, since affecting the circadian input pathway or the molecular clock itself would alter the circadian rhythm/phase of L-VGCCα1D mRNA levels. Thus, we set forth to examine the role of calcineurin as part of the circadian output to regulate L-VGCCs.
Both MAPK and PI3K-AKT signaling pathways are known to regulate ion channel trafficking and insertion into the plasma membrane (Lhuillier and Dryer, 2000; Lhuillier and Dryer, 2002; Keifer et al., 2007; Le Blanc et al., 2004). We showed that both MAPK and PI3K-AKT signaling serve as parallel circadian outputs to regulate L-VGCC trafficking and membrane insertion in photoreceptors (Ko et al., 2007; Ko et al., 2009). Since the circadian phase-dependent action of calcineurin on L-VGCCs was through post-translational modulation, we next examined whether calcineurin interacted with MAPK and/or PI3K-AKT signaling pathways. Chick embryos were entrained in LD cycles, and on the last day of LD, retinal cells were cultured and kept in DD. On the second of DD, cells were treated with the calcineurin inhibitor FK-506 (10 uM) or cyclosporine A (2 uM) for 2 hr prior to harvest at CT 4 and CT 16 for Western blotting or PP2B activity assay. As shown previously (Ko et al., 2007; Ko et al., 2009) both phosphorylated ERK (pERK) and pAKT at thr308 (pAKTthr308) are under circadian control and significantly higher during the subjective night than during the subjective day (pERK rhythm, Fig. 4A and 4C; pAKTthr308 rhythm, Fig. 4B, 4D). Treatment with FK-506 or cyclosporine A dampened the circadian rhythm of pERK (Fig. 4A and 4C) and pAKTthr308 (Fig. 4B and 4D). However, inhibition of MAPK signaling using a MEK1 inhibitor PD98059 (50 µM) or the PI3K-AKT pathway with a PI3K inhibitor LY294002 (50 µM) did not alter the circadian rhythm of calcineurin activity (Fig. 5A and B). Hence, calcineurin was upstream of both MAPK and PI3K-AKT signaling as part of the circadian output pathway to regulate L-VGCCs.
In the retina, the activity of Ras is under circadian control with higher activity at night (Ko et al., 2004). Both MAPK and PI3K-AKT signaling pathways are downstream of Ras, since inhibition of Ras abolishes the circadian rhythm of pERK and pAKT (Ko et al., 2009), while inhibition of cAMP signaling dampens the circadian rhythm of Ras activity (Ko et al., 2004). Since calcineurin was also upstream of both MAPK and PI3K-AKT signaling, we next investigated the potential interaction among calcineurin, Ras, and cAMP signaling. Using a commercially available cAMP assay kit, we found that cAMP content in the chick retina was rhythmic with its peak during the subjective night (Fig. 6A). This result was similar to previous reports (Ivanova and Iuvone, 2003; Nikaido and Takahashi, 1998). Treatment with the calcineurin inhibitor FK-506 did not alter the circadian rhythm of cAMP (Fig. 6B), but the adenylate cyclase inhibitor MDL-12330A (50 µM) significantly dampened the circadian rhythm of calcienurin activity (Fig. 6C). Furthermore, inhibition of calcineurin with FK-506 abolished the circadian rhythm of Ras activity (Fig. 6D). Therefore, calcineurin was downstream of cAMP but upstream of Ras to serve as a circadian output to regulate L-VGCCs.
Calcineurin is involved in diverse biological processes, including regulating nuclear factor of activated T cells (NFAT) transcriptional activation (O'Keefe et al., 1992; Jain et al., 1993) and apoptosis (Yazdanbakhsh et al., 1995; Wang et al., 1999). In neurons, calcineruin participates in the modulation of synaptic plasticity (Mulkey et al., 1994; Zhuo et al., 1999; Wang and Kelly, 1997), neurotransmitter release (Halpain et al., 1990; Renstrom et al., 1996; Nishi et al., 1997), and gating of ion channels (Chen et al., 1995; Marrion, 1996; Marcaida et al., 1996; Oliveria et al., 2007). Since the visual system must anticipate large daily changes in ambient illumination, circadian oscillators in the retina provide a mechanism for the visual system to initiate more sustained adaptive changes throughout the course of a day (Cahill and Besharse, 1995; Green and Besharse, 2004). In this study, we demonstrated that calcineurin was involved in the circadian phase-dependent modulation of L-VGCCs in the retina. We focus on the circadian regulation of the L-VGCCα1D subunit since in the avian retina, α1D is distributed mainly on the cell bodies of photoreceptors, bipolar cells, and ganglion cells, while L-VGCCα1C is abundant in Müller glia cells (Firth et al., 2001; Ko et al., 2007). In addition, L-VGCCα1D is present in the cell bodies and terminals of rodent photoreceptors, while VGCCα1C is not observed in rat photoreceptors (Morgans et al., 2005; Xu et al., 2002). Hence, α1D, but not α1C, is the dominant L-VGCCα1 subunit in retinal photoreceptors across several vertebrate species.
We found that calcineurin activity was under circadian control (significantly higher during the subjective night than the subjective day), but its protein expression remained constant throughout the day (Fig. 1). Application of a calcineurin inhibitor, FK-506 or cyclosporine A, for 2 hr at night decreased L-VGCC current density in cone photoreceptors (Fig. 2) corresponding with a decrease in the protein expression of the L-VGCCα1D subunit in cultured retinal cells, but the circadian rhythm of L-VGCCα1D mRNA was not affected (Fig. 3). Therefore, the circadian phase-dependent action of calcineurin was not due to circadian phase-shifting of L-VGCCs. Instead, calcineurin served in the circadian output pathway to regulate L-VGCCs. We previously demonstrated that the circadian rhythm of L-VGCCs is in part through both Ras-ERK and Ras-PI3K-AKT signaling, both of which are involved in the protein trafficking and membrane insertion of L-VGCCα1 subunits (Ko et al., 2007; Ko et al., 2009). Here, we showed that calcineurin regulated L-VGCCs in a circadian-phase dependent manner through modulation of ERK and PI3K-AKT signaling, since calcineurin inhibitors dampened the circadian rhythms of phosphorylated ERK and AKTthr308 (Fig. 4), while inhibition of either signaling pathway did not alter calcineurin activity (Fig. 5).
In the chick retina, cAMP content is under circadian control as previously shown by others (Nikaido and Takahashi, 1998; Ivanova and Iuvone, 2003; Chaurasia et al., 2006) and in this study (Fig. 6A), and the activity of Ras is also under circadian control and is downstream of cAMP signaling (Ko et al., 2004). We found that the adenylate cyclase inhibitor MDL-12230A dampened the circadian rhythm of calcineurin activity, but the calcineurin inhibitor FK-506, while having the ability to inhibit the Ras rhythm, had no effect on the circadian rhythm of retinal cAMP content (Fig. 6). Hence, calcineurin acted downstream of cAMP and upstream of Ras to regulate the circadian rhythm of L-VGCCs through regulating ion channel trafficking and membrane insertion (Fig. 7).
Photoreceptors are non-spiking neurons, and its neurotransmitter release is continuous in the dark through voltage-dependent activation of L-VGCCs (Barnes and Kelly, 2002). The circadian regulation of L-VGCCs has been shown in gold fish retinal bipolar cells (Hull et al., 2006) and avian cone photoreceptors (Ko et al., 2007). In each case, the L-VGCC current density is greater at night than during the day. The mechanism of this circadian rhythm is in part through the circadian regulation of mRNA and protein expression of the L-VGCCα1 subunits (Ko et al., 2007), as well as channel subunit trafficking and insertion / retention into the plasma membrane (Ko et al., 2007; Ko et al., 2009; Shi et al., 2009). There are two parallel signaling pathways that take part in the circadian regulation of L-VGCCs, Ras-MAPK and Ras-PI3K-AKT (Ko et al., 2007; Ko et al., 2009), and both pathways are known to regulate ion channel trafficking (Lhuillier and Dryer, 2000; Lhuillier and Dryer, 2002; Keifer et al., 2007; Le Blanc et al., 2004). Since calcineurin was upstream of Ras, we concluded that the circadian phase-dependent regulation of L-VGCCs by calcineurin was also through the regulation of channel trafficking and insertion of VGCCα1.
Calcineurin is also involved in circadian phase-shifting in mammals, since in vivo administration of calcineurin inhibitors blocks circadian responses to light at night, produces circadian phase advances when applied during the subjective day, and disrupts circadian locomotor behavior rhythms when applied chronically in hamsters (Katz et al., 2008). These effects of calcineurin inhibitors on circadian phase-shifting are in part attributed to their interference with intracellular Ca2+ storage and release in SCN neurons (Ding et al., 1998). In addition, calcineurin is an important regulator of casein kinase-I (CKI) and glycogen synthase kinase 3β (GSK3β; Cegielska et al., 1998; Lowrey et al., 2000; Liu et al., 2002; Kim et al., 2009). CKI and GSK3β are able to regulate the circadian core oscillator by phosphorylating circadian clock proteins (Vielhaber et al., 2000; Eide and Virshup, 2001; Iitaka et al., 2005; Yin et al., 2006). Therefore, calcineurin may well be involved in the circadian core oscillator mechanism through dephosphorylation of CKI and GSK3β in the mammalian SCN. Since we did not observe any changes in the circadian rhythm of L-VGCCα1D mRNA levels after inhibition of calcineurin, we concluded that calcineurin is part of the circadian output pathway to regulate L-VGCCs post-translationally. However, we cannot rule out the possibility that calcineurin might also serve in the circadian input pathway of retinal circadian oscillators, which will require further investigation.
In addition, there is a circadian oscillation of calcineurin activity in the mouse heart, with a gradual increase throughout the night when these animals are active and decrease when these animals are at rest (Sachan et al., 2011). We also observed a circadian rhythm of calcineurin activity in the retina when its protein level remained constant. Hence, it is possible that while calciuneurin participates in the modulation of the circadian core oscillator, calcineurin activity is also subject to circadian control. Other examples, such as MAPK, CaMKII, and L-VGCCs, are all under circadian regulation (Sanada et al., 2000; Ko et al., 2001; Ko et al., 2007; Ko et al., 2009; Hull et al., 2006; Pennartz et al., 2002), and yet they all can shift the circadian phase (Obrietan et al., 1998; Butcher et al., 2002; Nahm et al., 2005). This phenomenon seems to reinforce the model proposed by Roenneberg and Merrow (1999): pathways that lead to entrainment of the core oscillator (the circadian inputs) can themselves be regulated by the oscillator and serve as components of the physiologically relevant circadian output pathways. Hence, these additional feedback loops (the output components feeding back to the inputs) can markedly enhance the stability of the overall oscillator system at the cellular level (Roenneberg and Merrow, 1999).
Calcineurin is also an important regulator of L-VGCCs in various cell types. However, the effect of calcineurin on L-VGCCs varies, as both inhibition (Chad and Eckert, 1986; Armstrong, 1989; Victor et al., 1997; Schuhmann et al., 1997) and enhancement (Norris et al., 2002; Tandan et al., 2009) have been observed. The L-VGCCα1C (Cav1.2) subunit can form macromolecular signaling complexes that comprise the β-adrenergic receptor, G(s) protein, adenylate cyclase, protein kinase A (PKA), as well as PP2A and calcineurin in the heart and brain (Xu et al., 2010). Calcineurin is capable of binding the C-terminus of the cardiac L-VGCCα1C (Xu et al., 2010). In the present study, we found that inhibition of calcineurin decreased L-VGCC currents in cone photoreceptors only at night (Fig 2), and this inhibition was due to decreased L-VGCCα1D protein expression (Fig 3A). Therefore, we rule out direct dephosphorylation as the circadian phase-dependent action of calcineurin on L-VGCCs in cone photoreceptors. Even though we demonstrated that calcineurin was downstream of cAMP signaling and upstream of Ras, it is still not known which molecule is the direct target of calcineurin. Missing links remain between cAMP signaling and calcineurin, as well as between calcineurin and Ras, and the complexity of the signaling network in the circadian regulation of L-VGCCs are not completely understood (Fig. 7). Thus far, we have shown that at the post-translational level, the trafficking and membrane insertion / retention of L-VGCCs are under circadian control (Ko et al., 2007; Ko et al., 2009), in which calcineurin was part of the output pathway as shown in this study. However, we do not know whether the internalization and recycling of the channel subunits are also under circadian control. The circadian rhythm of L-VGCCs could be the focal point between channel protein expression, insertion / retention into the plasma membrane, and sequestering / recycling of the channel subunits. In conclusion, our present study provides new insight on the mechanism underlying the circadian regulation of L-VGCCs in chick cone photoreceptors.
We thank fruitful comments and discussion from Drs. Kirill Grushin and Liheng Shi. This project is supported by NIHRO1 EY017452 to G. K.