Regulation of cyclic GMP synthesis in photoreceptors

这项研究的目的是定义调节视觉光感受器中光传导和适应的新过程。长期目标是确定调节视网膜膜鸟苷酸环化酶合成第二光信号转导信使环GMP(CGMP)的分子机制。这一建议是基于这样的发现：光感受器特异的钙感受器蛋白(GCAPs)调节鸟苷酸环化酶以响应细胞内游离钙浓度的变化，对于正常的光反应和控制光感受器的活性是必不可少的。最近的证据表明，GCAP是钙/镁结合蛋白，而镁结合是调节cGMP合成对细胞内钙的生理范围的钙敏感性所必需的。因此，该提案的第一个目的是通过定向突变，确定GCAPs中钙和镁结合结构域的功能，并确定它们对鸟苷酸环化酶的激活和抑制的作用。与先天性失明相关的GCAP和鸟苷酸环化酶突变改变了鸟苷酸环化酶对钙的敏感性。最近的证据表明，GCAP基因突变使转基因小鼠细胞内游离cGMP和钙升高，导致光感受器变性。因此，这项建议的第二个目的是在转基因小鼠模型中复制与人类视杆细胞和视锥细胞退变相关的GCAP和鸟苷酸环化酶突变，以确定它们的生理效应，并确定通过引入提高cGMP水解率的突变来抑制由cGMP异常合成引起的视网膜变性的可能性。最近的发现表明，当两种不同的GCAP亚型被引入缺乏这两种GCAP的小鼠中时，可以加速恢复。因此，这项建议的第三个目的是通过利用各自的基因破坏来确定每一种GCAP亚型在恢复和光适应中的确切作用。这里提出的实验与理解控制光感受器活性和导致人类患者遗传性视网膜疾病的机制有关。

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.