IMAGING OF PHOTOTRANSDUCTION PROCESS IN RENTINAL CELLS

这项与韦恩州立大学克雷格眼科研究所Akio山崎博士的国际合作研究的目标是揭示脊椎动物光感受器细胞中光传导过程的形态对应。特别是，我们的目标是通过对磷酸二酯酶(PDE)亚单位的功能和结构变化的表征，来探讨光信号转导中的关键成分之一--磷酸二酯酶(PDE)的调控，并阐明PDE在视觉信号转导机制中的新作用。与理解光信号转导中的信号流和每个组分在信号流中的作用的重大进展相比，这些组分的机制和结构之间的关系仍然不清楚。尤其是由于对完整P-αβ的纯化和表达的不同，以及对P-αβ-P-γ相互作用的调控缺乏信息，因此PDE亚基之间的关系尚不清楚。然而，在这项研究中，P-αβ-P-γ的相互作用被部分阐明，关于P-γ修饰机制的新发现和关于PDE在杆状外节胞质cGMP水平增加中的作用的意外发现。我们最近在牛P-αβ的分离纯化中加入或不加入P-γ的研究进展也使得在电子显微镜下观察其分子成为可能。所有类型的PDE(αβγγ，αβγ，αβ)基本上由两条具有特征形状的扭曲的链组成，排列成环状。在大约30-60℃时，这两个组分相对于弯曲或扭转彼此相对。遗憾的是，γ亚基太小，不能用这种技术准确地检测出来。然而，P-γ从完整的PDE错位引起的可能的结构变化被阻止为分子的三维位置移动。无论如何，了解P-αβ功能结构的分子基础为理解光信号转导机制提供了新的视角。

The objective in this international cooperative research with Dr Akio Yamazaki, Kresge Eye Institute, Wayne State University, was to reveal morphological counterpart of the photo-transduction process in the vertebrate photoreceptor cells. In paticular, we aim to explore regulation of phosphodiesterase (PDE), one of the key components in phototransduction, by characterizing both functional and structural changes of PDE subunits and to elucidate new roles of PDE in visual signal transduction mechanism. In contrast to significant progress in understanding the flow of signals in phototransduction and the roles of each component in the flow of signals, the relationship between mechanism and structure of these components still remain unclear. Especially, the relationship in PDE subunits is unknown because of diffeculty of purification and expression of intact Pαβand lack of information about regulation of Pαβ-Pγ interactions. However, in this study, Pαβ-Pγ interactions were partially elucidated with new findings on the machanism of Pγ modifications and unexpected finding on the PDE role in the increase of cytoplasmic cGMP level in rod outer segments. Our recent development in the isolation and purification of bovine Pαβ with or without Pγ also made possible to observe the molecules under electron microscope. All types of PDE (αβγγ,αβγ,αβ) consisted basically of two crooked strands with characteristic shape that were arranged as making a ring. Theses two components faced each other relative to bending or twisting at about 30 - 60℃. Unfortunately, γ subunit was too small to be dected accurately by this technique. Nevertheless, suggestible structural changes induced by dislocation of Pγ from complete PDE were arrested as three dimentional shift of molecules in position.At any rate, understanding molecular basis of Pαβ functional structure provided new insight into phototransduction mechanism.