ANATOMY AND PHYSIOLOGY OF VISION

视觉的解剖学和生理学

基本信息

批准号：
2159325
负责人：
JOEL E BROWN
金额：
$ 30.3万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-10-01 至 1999-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2159325
关键词：
3'5' cyclic nucleotide phosphodiesterase cyclic GMP enzyme activity horseshoe crabs immunocytochemistry light adaptations membrane channels photoactivation photostimulus squid visual photoreceptor visual phototransduction

项目摘要

The long-term objective of this project is a biophysical and molecular understanding of the processes of transduction in general, and in photoreceptor cells in particular. The details of the cascade of reactions that mediates excitation by linking the absorption of photons by photopigments to changes in conductance of the plasma membrane are not entirely understood. Moreover, the reactions that modulate the phototransduction cascade to produce "adaptation are also incompletely understood. Our specific aims are to investigate certain steps in the transduction cascade for excitation and adaptation in invertebrate photoreceptors. The candidacy of cGMP as the excitatory messenger that gates the light-induced channels in Limulus ventral photoreceptors will be investigated. (I) A light-induced increase in intracellular cGMP near the plasma membrane of these cells will be assayed by introducing exogenous channels that are gated by cGMP into the plasma membrane. mRNA that is specific for cGMP-gated channels from bovine rods or olfactory receptors will be injected intracellularly and allowed to express the channel proteins. Expressed channels will be identified by recording electrophysiological responses to intracellular injections of cGMP and identified by immunohistochemistry for the expressed proteins. (II) We will attempt to purify cGMP-gated channels from squid photoreceptors by affinity chromatography. To examine if cGMP participates in the transduction cascade in a way other than by gating channels, (III) activated bovine cGMP-specific phosphodiesterase will be injected intracellularly into Limulus photoreceptors to attempt to perturb the electrophysiological responses to light and (IV) the time course of light-induced changes in phosphodiesterase activity will be measured by a continuous fluorescence assay in broken cell preparations of squid photoreceptors.

该项目的长期目标是生物物理和分子理解一般的转导过程，特别是感光细胞。级联的细节通过连接光子的吸收来介导兴奋的反应通过对质膜电导变化的变化不是完全理解。而且，调节的反应产生适应的光转导级联也不完全理解。我们的具体目的是研究某些步骤无脊椎动物的激发和适应的转导级联感受器。 CGMP作为兴奋性使者的候选人资格 Limulus腹侧感光器中的光引起的通道将被调查。（i）在附近的细胞内CGMP的光引起的增加这些细胞的质膜将通过引入来测定由CGMP门控进入质膜的外源通道。 mRNA 这是针对牛棒或嗅觉的CGMP门控通道受体将被细胞内注射，并允许表达通道蛋白。表达的渠道将通过录制来确定对CGMP的细胞内注射的电生理反应通过免疫组织化学确定表达蛋白质。（ii）我们将尝试通过通过亲和色谱。检查CGMP是否参与通过门控通道以外的其他方式转导级联，（iii）活化的牛CGMP特异性磷酸二酯酶将被注入细胞内进入Limulus感光器，以尝试扰动电生理对光的反应和（iv）时间过程光引起的磷酸二酯酶活性的变化将通过鱿鱼破裂的细胞制剂中的连续荧光测定感受器。