This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Though CNG channel in rods has been well characterized, our understanding on the cone channel is quite limited, which is primarily due to the difficulty of investigating cone system in mammalian retinas. Cones play an essential role in central vision and are responsible for color vision. To date, over 80 mutations in cone CNG channel have been linked to various forms of achromatopsia and progressive cone dystrophy in humans. Thus, it is overt that elucidating the pathogenesis of cone dystrophy requires understanding of the channel normal structure and function. The recently characterized cone-dominant retinas of Nrl-/- mice and cone-derived cell line, the 661W cells, are excellent models to study cone-specific proteins. The main goal of this proposal is to establish the biochemical and functional characteristics of cone CNG channel using the cone models. The first specific aim is to identify the cone CNG channel interacting partners. The potential associations between the cone channel and those proteins known to interact with the rod channel will be investigated using co-immunoprecipitation and GST pull-down assay. Yeast two-hybrid screening of a mouse retinal cDNA library and mass spectrometry will be used to further identify the cone channel interacting partners. The second specific aim is to characterize the biochemical properties of the cone CNG channel complex and the subunit association. Cone CNG channel complex in the Nrl-/- retinas will be analyzed using blue native and SDS gel electrophoresis and chemical cross-linking. Subunit association of the channel complex will be determined through co-immunoprecipitation and GST pull-down assay. The third specific aim is to evaluate the role of Ca2+/calmodulin (CaM) and phosphorylation in regulating the cone CNG channel activity. The channel subunits will be heterologously co-expressed in 661W cells and the channel function will be studied using electrophysiological recording techniques. The dynamic binding of CaM to the cone channel subunits will be evaluated in the light-treated Nrl-/- retinas using co-immunoprecipitation and immunostaining and in situ phosphorylation of the channel subunits will be examined in the Nrl-/- retinas. Findings from the proposed studies will add substantially to the field of cone biology and pathogenesis and the insights will facilitate the development of therapeutic strategies for human cone diseases.
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