Regulation of cyclic GMP synthesis in photoreceptors

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The purpose of the proposed study is to define new processes that regulate phototransduction and adaptation in visual photoreceptors. The long-term goal is to determine the molecular mechanisms that regulate synthesis of a second messenger of phototransduction, cyclic GMP (cGMP), by retinal membrane guanylyl cyclases. The proposal is based on the finding that photoreceptor-specific calcium sensor proteins, GCAPs, that regulate guanylyl cyclases in response to the change in intracellular free calcium concentrations are essential for normal photoresponses and control photoreceptor viability. Recent evidence demonstrate that GCAPs are calcium/magnesium-binding proteins, and magnesium binding is essential for adjusting calcium sensitivity of cGMP synthesis to the physiological range of intracellular calcium. Therefore, the first aim of the proposal is to identify, by using directed mutagenesis, the function of the calcium- and magnesium-binding domains in GCAPs and to determine their contribution to activation and inhibition of guanylyl cyclase. Mutations in GCAP and guanylyl cyclase linked to congenital blindness alter calcium sensitivity of guanylyl cyclase. Recent evidence demonstrate that mutation in GCAP elevates intracellular free cGMP and calcium and results in photoreceptor degeneration in transgenic mice. Therefore, the second aim of this proposal is to replicate in transgenic mouse models the mutations in GCAP and guanylyl cyclase associated with human rod-cone and cone degenerations, to determine their physiological effects, and to determine the possibility of suppressing retinal degeneration caused by abnormal synthesis of cGMP by introducing mutations that elevate cGMP hydrolysis. Recent findings demonstrate that two different isoforms of GCAPs can accelerate recovery when introduced in mice lacking both GCAPs. Therefore, the third aim of this proposal is to determine the exact role for each of GCAP isoform in the recovery and light adaptation by using their individual gene disruption. The experiments proposed here are relevant to the understanding of the mechanisms that control photoreceptor activity and cause inherited retinal diseases in human patients.