The long-term objectives of this research program are to understand cGMP-gated cation channel protein structure/function relationships in rod photoreceptors and to translate this understanding to the treatment of patients with retinitis pigmentosa (RP) and related disorders. We will focus our studies on the channel b- subunit and the associated soluble glutamic acid rich protein (GARP) GARP2, both encoded by the Cngb1 locus, examining the structural and functional roles of the GARP region in three specific aims. We previously generated a homozygous Cngb1 photoreceptor null (X1 KO) mouse resulting not only in partial loss of channel function but also severe structural perturbations establishing in vivo that these proteins are necessary for normal rod outer segment ROS disk morphogenesis and structural integrity. We hypothesized that the GARP2 sequence on the b-subunit is required for plasma membrane/disc membrane interaction. To test this we created transgenic mice expressing an N-terminally truncated b-subunit (Tb) devoid of all GARP2 sequence on the X1 KO background. Despite the absence of soluble GARP2, the entire b-subunit GARP2 region and only one glutamate rich segment remaining on Tb, there is significant but not complete structural and functional rescue. To determine the basis for the observed rescue (A.) we will analyze these mice structurally using histology, immunocytochemistry and ultrastructure analysis by transmission and Cryo-EM and functionally using ERG, single cell physiology and a now established retina punch preparation. In the X1 KO and X26 mice that do not express the b-subunit and in X1 KO also missing soluble GARPs the photoresponse is attenuated. In WT mice overexpressing GARP2 a significant increase in phototransduction gain was observed demonstrating a previously unknown role for GARP2 in modulating phototransduction. (B.) We hypothesize that GARP2 regulates rod dark noise and also contributes to novel slow Burnsian adaptation. To test this we will use our established physiologic tools to compare phototransduction parameters in WT, GARP2 overexpressing, Tb transgenic mice and mice expressing full length b-subunit during activation, recovery and adaptation to further define the role of GARP2 and the b-subunit in modulating the photoresponse. Using novel zinc finger nuclease technology we have established GARP2-specific knockout mice deleted for the last unique GARP2 exon and a potential hypomorph that is missing the GARP2 3'-UT region. (C.) We will use these mice to test the hypothesis that GARP2 is required for structure and function in the rods. We will perform structural analysis to determine effects on disk morphogenesis and overall outer segment structure and functional analysis to complement studies proposed in Aims A. and B. and to directly examine the role of GARP2 in rod function. The proposed studies document a comprehensive plan to define the structural importance of the GARP region and to define the role of GARP2 in modulating the rod photoresponse. The studies may also yield new targets for intervention treatments for certain forms of hereditary retinal degeneration.
The proposed studies are focused on a novel gene in the retina that is required for normal vision. Defects in the gene cause some forms of the disease retinitis pigmentosa, a progressive hereditary form of blindness. The disease affects a significant portion of the US and world populations and currently there is no established treatment. This application seeks to identify fundamental properties of the genes encoded proteins that will provide essential information needed to develop a treatment for this blinding disorder.
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