Membrane targeting calcium sensors in vision

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The overall objectives are to develop nuclear magnetic resonance (NMR) techniques and use them in concert with experimental functional approaches to elucidate the molecular structure and physiologic functions of selected membrane-targeting proteins involved in phototransduction in vision and other signal transduction processes. During the next five years, we will use nuclear magnetic resonance (NMR), fluorescence, microcalorimetry, spin-label EPR, xray crystallography, and computational analysis to delineate the structure, dynamics and mechanisms of a family of neuronal calcium sensor proteins (calcium-myristoyl switches) that serve as membrane- targeting regulators in calcium signaling and are linked to retinal and neurological diseases. Our studies will determine the structural basis of: (1) retinal guanylyl cyclase (RetGC) regulation by GCAP1 and retinal degeneration 3 (RD3) protein, and their role in autosomal dominant cone dystrophy and Leber Congenital Amaurosis; (2) Ca2+-dependent inactivation of photoreceptor cyclic nucleotide gated (CNG) channels controlled by calmodulin (CaM); and (3) Ca2+-dependent activation of retinal L-type Ca2+ channels (CaV1.4) mediated by calcium binding protein-4 (CaBP4), and implicated in congenital stationary night blindness. By continuing our intensive structural analysis of retinal Ca2+ sensor proteins and by broadening the scope to encompass membrane trafficking regulators (RD3) and protein targets (RetGCs and ion channels), we hope to gain an atomic-level understanding of how retinal calcium sensor proteins regulate their membrane-bound target proteins in retinal disease processes. The specific aims are 3-fold: (1) Determine atomic-level structures of GCAP1 and RD3 each bound to RetGC to elucidate activation mechanism of RetGCs and thus provide a structural basis for understanding mechanisms of visual recovery and retinal degenerative diseases; (2) Determine structures of CaM bound to CNG channels to understand molecular mechanisms of light-adaptation in rod and cone photoreceptors; (3) Determine atomic-level structures of the retinal calcium sensor protein (CaBP4) bound to the retinal L-type voltage-gated Ca2+ channel (CaV1.4) at the rod synapse to understand the Ca2+- dependent regulatory mechanism of ion channels linked to congenital stationary night blindness.