Macular degeneration is a major cause of visual impairment in the United States and is associated with the death of cone photoreceptors. The pathogenesis and the cellular events responsible for the distortion of central vision in macular degeneration are not understood. Circadian oscillators in retina photoreceptors are known to play important roles in regulating photoreceptor function and physiology, and disruption of these circadian oscillators leads to the death of photoreceptors and could contribute to the pathogenesis of macular degeneration. The objective of the proposed research is to elucidate the signaling pathways from the circadian oscillators leading to the regulation of L-type voltage-dependent calcium channels (VDCCs) in cone photoreceptors, since the L-type VDCCs are essential in regulating neurotransmitter release in the retina. For example, mutation of the L-type VDCC a1f subunit gene causes incomplete congenital stationary night blindness in humans, with a defect in neurotransmission between photoreceptors and second-order neurons. An integrative approach with multiple techniques including electrophysiological patch-clamp recordings, Western immunoblotting, immunocytochemistry with confocal imaging, quantitative real-time RT- PCR and gene transfection, will be used in this application to achieve the following four specific aims:
Specific Aim 1. Characterize the circadian rhythmicity of VDCCs in chick cone photoreceptors. The circadian rhythmicity of L-type VDCCs at multiple time points throughout the entire circadian day will be characterized in detail.
Specific Aim 2. Identify the cellular signaling pathways underlying the circadian regulation of VDCCs. The roles of PKA, MAP kinase, CaMKII and PI3 kinase as output pathways leading to the circadian regulation of VDCCs will be determined.
Specific Aim 3. Elucidate the roles of actin cytoskeleton rearrangement and protein trafficking on circadian regulation of VDCCs.
Specific Aim 4. Define the cellular mechanisms of the phase-dependent modulation of VDCCs by somatostatin. While somatostatin serves as a VDCC inhibitor in other neurons, it shows both phase-dependent enhancement and inhibition of L-type VDCCs in cones. This project will be among the very first studies to define the circadian regulation of L-type VDCCs and their molecular mechanisms in retina photoreceptors. Understanding the molecular signaling of circadian regulation of retina function may ultimately provide knowledge for developing new strategies to treat retinal diseases, especially age-related macular degeneration, and preventing blindness in the future, which are the long-term goals of the PI. ? ? ?
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