Circadian Regulation of L-Type Voltage-Gated Calcium Channels in Avian Retina

Abstract

The circadian clock is an endogenous time-keeping mechanism that allows an organism to synchronize itself with external time cues and prepares the organism to anticipate upcoming environmental changes on a daily basis. The retina is a light-sensitive neuronal tissue located in the back of the eye. The circadian clocks in the retina enable the retina to anticipate daily ambient illumination over at least twelve orders of magnitude and initiate the adaptive processes with visual system throughout the course of a day. The retinal photoreceptors are responsible for phototransduction and transmitting the visual information into the brain. Unlike most neurons, photoreceptors do not fire action potentials, and they release neurotransmitter in a sustained manner, which is governed by the L-type voltage-gated calcium channels (L-VGCCs). The mRNA and protein expression of the α1 pore forming subunit of L-VGCCs are under circadian control, in which the protein expression of L-VGCCα1 with a corresponding increase in the L-VGCC current density is higher at night than during the day. Using the chicken embryo as a model system, an integrative strategy was used through combining biochemical, molecular, morphological, and electrophysiological analyses to investigate cellular mechanisms of the circadian regulation of L-VGCCs in the photoreceptors.

Three important cell signaling molecules and their pathways were investigated in this dissertation: calcineurin, mechanistic/mammalian target of rapamycin complex 1 (mTORC1), and AMP-activated protein kinase (AMPK). The activities of the protein phosphatase calcineurin, as well as the protein kinase mTORC1 exhibited circadian oscillation with their activities higher at night than during the day, while the activities of AMPK are greater during the day compared to the activities at night. Inhibition of calcineurin and mTORC1 dampened the current densities and protein expression of L-VGCCs at night, while activation of AMPK decreased L-VGCC currents at night. These signaling molecules interacted with cAMP-Ras-MAPK and cAMP-Ras-PI3K-AKT signaling pathways to modulate the L-VGCC trafficking from the cytosol onto the plasma membrane in a circadian phase-dependent manner. The results demonstrated that the complex of cellular signaling pathways participated in the circadian regulation of L-VGCCs in the photoreceptors. Understanding the molecular mechanism underlying the circadian regulation of L-VGCCs in cone photoreceptors will provide important knowledge on how circadian clocks regulate retinal physiology and function in healthy states. Changes in L-VGCCs and these cell-signaling molecules might be indicators or biomarkers for age-related macular degeneration or other retinal degenerative diseases.