Michael L. Ko

Circadian regulation of ion channels and their functions.

Ion channels are the gatekeepers to neuronal excitability. Retinal neurons of vertebrates and invertebrates, neurons of the suprachiasmatic nucleus (SCN) of vertebrates, and pinealocytes of non‐mammalian vertebrates display daily rhythms in their activities. The interlocking transcription–translation feedback loops with specific post‐translational modulations within individual cells form the molecular clock, the basic mechanism that maintains the autonomic ∼24‐h rhythm. The molecular clock regulates downstream output signaling pathways that further modulate activities of various ion channels. Ultimately, it is the circadian regulation of ion channel properties that govern excitability and behavior output of these neurons. In this review, we focus on the recent development of research in circadian neurobiology mainly from 1980 forward. We will emphasize the circadian regulation of various ion channels, including cGMP‐gated cation channels, various voltage‐gated calcium and potassium channels, Na+/K+‐ATPase, and a long‐opening cation channel. The cellular mechanisms underlying the circadian regulation of these ion channels and their functions in various tissues and organisms will also be discussed. Despite the magnitude of chronobiological studies in recent years, the circadian regulation of ion channels still remains largely unexplored. Through more investigation and understanding of the circadian regulation of ion channels, the future development of therapeutic strategies for the treatment of sleep disorders, cardiovascular diseases, and other illnesses linked to circadian misalignment will benefit.

The expression of L-type voltage-gated calcium channels in retinal photoreceptors is under circadian control

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.

Rhythmic Expression of MicroRNA-26a Regulates the L-type Voltage-gated Calcium Channel α1C Subunit in Chicken Cone Photoreceptors

MicroRNAs (miRNAs) modulate gene expression by degrading or inhibiting translation of messenger RNAs (mRNAs). Here, we demonstrated that chicken microRNA-26a (gga-mir-26a) is a key posttranscriptional regulator of photoreceptor L-type voltage-gated calcium channel α1C subunit (L-VGCCα1C) expression, and its own expression has a diurnal rhythm, thereby explaining the rhythmic nature of L-VGCCα1Cs. Circadian oscillators in retinal photoreceptors provide a mechanism that allows photoreceptors to anticipate daily illumination changes. In photoreceptors, L-VGCC activities are under circadian control, which are higher at night and lower during the day. Interestingly, the mRNA level of VGCCα1D oscillates, but those for VGCCα1C do not. However, the protein expression of both VGCCα1C and α1D are higher at night in cone photoreceptors. The underlying mechanism regulating L-VGCCα1C protein expression was not clear until now. In vitro targeting reporter assays verified that gga-mir-26a specifically targeted the L-VGCCα1C 3′-untranslated region, and gga-mir-26a expression in the retina peaked during the day. After transfection with gga-mir-26a, L-VGCCα1C protein expression and L-VGCC current density decreased. Therefore, the rhythmic expression of gga-mir-26a regulated the protein expression of the L-VGCCα1C subunit. Additionally, both CLOCK (circadian locomoter output cycles kaput) and CREB (cAMP-response element-binding protein-1) activated gga-mir-26a expression in vitro. This result implies that gga-mir-26a might be a downstream target of circadian oscillators. Our work has uncovered new functional roles for miRNAs in the regulation of circadian rhythms in cone photoreceptors. Circadian regulated miRNAs could serve as the link between the core oscillator and output signaling that further govern biological functions.

Retinoschisin, a New Binding Partner for L-type Voltage-gated Calcium Channels in the Retina

The L-type voltage-gated calcium channels (L-VGCCs) are activated under high depolarization voltages. They are vital for diverse biological events, including cell excitability, differentiation, and synaptic transmission. In retinal photoreceptors, L-VGCCs are responsible for neurotransmitter release and are under circadian influences. However, the mechanism of L-VGCC regulation in photoreceptors is not fully understood. Here, we show that retinoschisin, a highly conserved extracellular protein, interacts with the L-VGCCα1D subunit and regulates its activities in a circadian manner. Mutations in the gene encoding retinoschisin (RS1) cause retinal disorganization that leads to early onset of macular degeneration. Since ion channel activities can be modulated through interactions with extracellular proteins, disruption of these interactions can alter physiology and be the root cause of disease states. Co-immunoprecipitation and mammalian two-hybrid assays showed that retinoschisin and the N-terminal fragment of the L-VGCCα1 subunit physically interacted with one another. The expression and secretion of retinoschisin are under circadian regulation with a peak at night and nadir during the day. Inhibition of L-type VGCCs decreased membrane-bound retinoschisin at night. Overexpression of a missense RS1 mutant gene, R141G, into chicken cone photoreceptors caused a decrease of L-type VGCC currents at night. Our findings demonstrate a novel bidirectional relationship between an ion channel and an extracellular protein; L-type VGCCs regulate the circadian rhythm of retinoschisin secretion, whereas secreted retinoschisin feeds back to regulate L-type VGCCs. Therefore, physical interactions between L-VGCCα1 subunits and retinoschisin play an important role in the membrane retention of L-VGCCα1 subunits and photoreceptor-bipolar synaptic transmission.

Phosphatidylinositol 3 kinase–Akt signaling serves as a circadian output in the retina

The daily rhythm of L‐type voltage‐gated calcium channels (L‐VGCCs) is part of the cellular mechanism underlying the circadian regulation of retina physiology and function. However, it is not completely understood how the circadian clock regulates L‐VGCC current amplitudes without affecting channel gating properties. The phosphatidylinositol 3 kinase–protein kinase B (PI3K–Akt) signaling pathway has been implicated in many vital cellular functions especially in trophic factor‐induced ion channel trafficking and membrane insertion. Here, we report that PI3K–Akt signaling participates in the circadian phase‐dependent modulation of L‐VGCCs. We found that there was a circadian regulation of Akt phosphorylation on Thr308 that peaked at night. Inhibition of PI3K or Akt significantly decreased L‐VGCC current amplitudes and the expression of membrane‐bound L‐VGCCα1D subunit only at night but not during the subjective day. Photoreceptors transfected with a dominant negative Ras had significantly less expression of phosphorylated Akt and L‐VGCCα1D subunit compared with non‐transfected photoreceptors. Interestingly, both PI3K–Akt and extracellular signal‐related kinase were downstream of Ras, and they appeared to be parallel and equally important pathways to regulate L‐VGCC rhythms. Inhibition of either pathway abolished the L‐VGCC rhythm indicating that there were multiple mechanisms involved in the circadian regulation of L‐VGCC rhythms in retina photoreceptors.

CIRCADIAN PROFILES IN THE EMBRYONIC CHICK HEART: L-TYPE VOLTAGE-GATED CALCIUM CHANNELS AND SIGNALING PATHWAYS

Circadian clocks exist in the heart tissue and modulate multiple physiological events, from cardiac metabolism to contractile function and expression of circadian oscillator and metabolic-related genes. Ample evidence has demonstrated that there are endogenous circadian oscillators in adult mammalian cardiomyocytes. However, mammalian embryos cannot be entrained independently to light-dark (LD) cycles in vivo without any maternal influence, but circadian genes are well expressed and able to oscillate in embryonic stages. The authors took advantage of using chick embryos that are independent of maternal influences to investigate whether embryonic hearts could be entrained under LD cycles in ovo. The authors found circadian regulation of L-type voltage-gated calcium channels (L-VGCCs), the ion channels responsible for the production of cardiac muscle contraction in embryonic chick hearts. The mRNA levels and protein expression of VGCCα1C and VGCCα1D are under circadian control, and the average L-VGCC current density is significantly larger when cardiomyocytes are recorded during the night than day. The phosphorylation states of several kinases involved in insulin signaling and cardiac metabolism, including extracellular signal-regulated kinase (Erk), stress-activated protein kinase (p38), protein kinase B (Akt), and glycogen synthase kinase-3β (GSK-3β), are also under circadian control. Both Erk and p38 have been implicated in regulating cardiac contractility and in the development of various pathological states, such as cardiac hypertrophy and heart failure. Even though both Erk and phosphoinositide 3-kinase (PI3K)-Akt signaling pathways participate in complex cellular processes regarding physiological or pathological states of cardiomyocytes, the circadian oscillators in the heart regulate these pathways independently, and both pathways contribute to the circadian regulation of L-VGCCs. (Author correspondence: gko@cvm.tamu.edu)

High-Fat Diet-Induced Retinal Dysfunction.

PURPOSE The purpose of this study was to investigate the impact of obesity-induced prediabetes/early diabetes on the retina to provide new evidence on the pathogenesis of type 2 diabetes-associated diabetic retinopathy (DR). METHODS A high-fat diet (HFD)-induced obesity mouse model (male C57BL/6J) was used in this study. At the end of the 12-week HFD feeding regimen, mice were evaluated for glucose and insulin tolerance, and retinal light responses were recorded by electroretinogram (ERG). Western immunoblot and immunohistochemical staining were used to determine changes in elements regulating calcium homeostasis between HFD and control retinas, as well as unstained human retinal sections from DR patients and age-appropriate controls. RESULTS Compared to the control, the scotopic and photopic ERGs from HFD mice were decreased. There were significant decreases in molecules related to cell signaling, calcium homeostasis, and glucose metabolism from HFD retinas, including phosphorylated protein kinase B (pAKT), glucose transporter 4, L-type voltage-gated calcium channel (L-VGCC), and plasma membrane calcium ATPase (PMCA). Similar changes for pAKT, PMCA, and L-VGCC were also observed in human retinal sections from DR patients. CONCLUSIONS Obesity-induced hyperglycemic and prediabetic/early diabetic conditions caused detrimental impacts on retinal light sensitivities and health. The decrease of the ERG components in early diabetes reflects the decreased neuronal activity of retinal light responses, which may be caused by a decrease in neuronal calcium signaling. Since PI3K-AKT is important in regulating calcium homeostasis and neural survival, maintaining proper PI3K-AKT signaling in early diabetes or at the prediabetic stage might be a new strategy for DR prevention.

Cardiac-Specific Mutation of Clock Alters the Quantitative Measurements of Physical Activities without Changing Behavioral Circadian Rhythms

Even though peripheral circadian oscillators in the cardiovascular system are known to exist, the daily rhythms of the cardiovascular system are mainly attributed to autonomic or hormonal inputs under the control of the central oscillator, the suprachiasmatic nucleus (SCN). In order to examine the role of peripheral oscillators in the cardiovascular system, we used a transgenic mouse where the Clock gene is specifically disrupted in cardiomyocytes. In this cardiomyocyte-specific CLOCK mutant (CCM) mouse model, the circadian input from the SCN remains intact. Both CCM and wild-type (WT) littermates displayed circadian rhythms in wheel-running behavior. However, the overall wheel-running activities were significantly lower in CCM mice compared to WT over the course of 5 weeks, indicating that CCM mice either have lower baseline physical activities or they have lower physical adaptation abilities because daily wheel running, like routine exercise, induces physical adaptation over a period of time. Upon further biochemical analysis, it was revealed that the diurnal oscillations of phosphorylation states of several kinases and protein expression of the L-type voltage-gated calcium channel (L-VGCC) α1D subunit found in WT hearts were abolished in CCM hearts, indicating that in mammalian hearts, the daily oscillations of the activities of these kinases and L-VGCCs were downstream elements of the cardiac core oscillators. However, the phosphorylation of p38 MAPK exhibited robust diurnal rhythms in both WT and CCM hearts, indicating that cardiac p38 could be under the influence of the central clock through neurohormonal signals or be part of the circadian input pathway in cardiomyocytes. Taken together, these results indicate that the cardiac core oscillators have an impact in regulating circadian rhythmicities and cardiac function.

Calcineurin serves in the circadian output pathway to regulate the daily rhythm of L-type voltage-gated calcium channels in the retina

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. J. Cell. Biochem. 113: 911–922, 2012.

Circadian Regulation of Retinoschisin in the Chick Retina

PURPOSE To investigate the circadian regulation and acute illumination effects on the expression and secretion of retinoschisin from vertebrate retinas. METHODS Retinas were studied on the second day of constant darkness (DD) after several days of entrainment to 12-hour light/12-hour dark (LD) cycles in ovo or in vitro. Quantitative real-time PCR and Western immunoblotting were used to examine the mRNA and protein expressions of retinoschisin at different circadian time points. Pharmacologic treatments in whole retina and dissociated retinal cell cultures were used to investigate the cellular mechanisms underlying the circadian regulation of retinoschisin content and secretion. Different illumination conditions were given to examine changes in retinoschisin content in association with acute light/dark adaptation. RESULTS The mRNA level, protein expression, and secretion of retinoschisin were under circadian control, all of which were higher at night and lower during the day. The Ras, MAP kinase Erk, CaMKII pathway served as part of the circadian output regulating the rhythmicity of retinoschisin. Blockage of L-type VGCCs dampened the retinoschisin rhythm, but inhibition of L-type VGCCs did not completely abolish the secretion of retinoschisin. The protein expression of retinoschisin also responded to acute illumination changes. CONCLUSIONS The mRNA and protein expression, as well as retinoschisin secretion, are under circadian control. L-type VGCCs play a role in the circadian regulation of retinoschisin, but the molecular mechanism underlying retinoschisin secretion does not depend on L-type VGCCs. Protein expression of retinoschisin in response to acute illumination changes depends on previous light exposure experience.