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 VGCCa1C and VGCCa1D subunits, we found that the immunofluorescence intensities of both VGCCa1C and VGCCa1D in photoreceptors are higher during the subjective night. However, the mRNA levels of VGCCa1D, but not VGCCa1C, 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, voltagegated 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 lightdark (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 cG...

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 oscillators in chicken cone photoreceptors regulate the gating properties of cGMP-gated cationic channels (CNGCs) such that they have a higher apparent affinity for cGMP during the subjective night. Here we show that cAMP, acting through protein kinase A (PKA), Ras, and Erk, is part of the circadian output pathway controlling CNGCs. Endogenous and exogenous cAMP cause activation of Erk and Ras, which are more active at night in cones, and increase the apparent affinity of CNGCs for cGMP. The Ras farnesyl transferase inhibitor manumycin-A, and a dominant-negative form of Ras (RasN17) block the circadian rhythms in CNGC gating, as well as the effects of cAMP. A dominant-negative form of the MEK kinase B-Raf also blocks circadian and cAMP modulation of CNGCs. The circadian output pathway modulating CNGC channels is comprised in part of cAMP 3 PKA 3 Ras 3 B-Raf 3 MEK 3 Erk 3 3 CNGCs. cAMP activation of Ras and Erk occur within minutes, whereas modulation of CNGCs requires Ͼ1 hr. However, cAMP protagonists do not alter rhythms in cPer2 mRNA, and their effects on CNGCs cannot be attributed to clock phase-shifting.

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.

cGMP-gated channels are essential for phototransduction in the vertebrate retina. Here we show that the affinity of these channels for cGMP in chick cones is substantially higher during the subjective night than during the subjective day. This effect persists in constant environmental conditions after entrainment to 12:12 hr light-dark cycles in vitro or in ovo. Circadian modulation of ligand affinity is a posttranslational effect and is driven by rhythms in the activities of two protein kinases: Erk and Ca2+/calmodulin-dependent protein kinase II (CaMKII). Erk is maximally active during the subjective night, whereas CaMKII is maximally active during the subjective day. Acute inhibition of these signaling pathways causes phase-dependent changes in the affinity of the channels for cGMP.