Calcium (Ca2+) is a ubiquitous signaling molecule that controls the function and survival of neurons. The disrupted Ca2+ homeostasis in a wide range of photoreceptor mutations is believed to cause cell death, retinal degeneration and blindness. In vertebrate photoreceptors, Ca2+ changes also modulate the shutoff of the phototransduction cascade to accelerate light response recovery and background adaptation. It is thought that the concentration of Ca2+ in the outer segments of vertebrate photoreceptors is controlled by a dynamic balance between influx via the cGMP-gated (CNG) channels and extrusion via cell-specific Na+/Ca2+, K+ exchangers (NCKX), NCKX1 in rods and NCKX2 in cones. However, the extent to which these exchangers control the Ca2+ homeostasis in mammalian photoreceptors and modulate phototransduction and cell survival has not been determined. In addition, it is not known whether other active or passive mechanisms for extruding Ca2+ are at play in the outer segments of mammalian rods and cones. We will perform experiments to establish the role of CNG and NCKX1 in regulating the Ca2+ homeostasis in mammalian rods and their effect on long-term rod survival and degeneration. We will also test the hypothesis that abnormal photoreceptor Ca2+ homeostasis mediates photoreceptor degeneration in a variety of blinding diseases and will determine the therapeutic potential of restoring the Ca2+ flux balance in photoreceptor channelopathies. We have identified NCKX4 as a second Na+/Ca2+, K+ exchanger expressed in mammalian cones. We will perform experiments to analyze the expression profile, morphology, and functional properties of NCKX2- and NCKX4- deficient mouse cones. These experiments will establish the molecular mechanisms for the efficient extrusion of Ca2+ from mammalian cone photoreceptors critical for the fast response kinetics and background adaptation of cones as our daytime photoreceptors as well as their effect on cone long-term survival and degeneration. Collectively, our experiments will establish the molecular mechanisms that mediate the extrusion of Ca2+ from mammalian photoreceptors. They will also help us understand the link between abnormal Ca2+ homeostasis and photoreceptor degeneration and might potentially lead to the development of treatments for channelopaties.
The experiments outlined in this proposal seek to identify the molecular mechanisms for achieving efficient extrusion of calcium from mammalian rod and cone photoreceptors, critical for the fast response kinetics and background adaptation of photoreceptors. These studies will also determine whether abnormal photoreceptor calcium homeostasis mediates photoreceptor degeneration in a variety of blinding diseases and will determine the therapeutic potential of restoring the calcium flux balance in photoreceptor channelopathies.
