Reconstitution of phototransduction channel from frog rod outer segments in planar lipid bilayers

脊椎动物光感受器由于外节膜上cGMP依赖性阳离子通道的关闭而对光刺激显示出超孔化反应。本课题的目的是鉴定和纯化cGMP依赖性离子通道蛋白。用[^3H]cGMP标记蛙视杆外节的完整膜蛋白组分，鉴定出分子量分别为250K、100K、92K和66K的四种cGMP结合蛋白。的incoropration的馏分成平面脂质双层导致至少三种通道活动的外观，其中两个是有关的cGMP。由于100K和92K是磷酸二酯酶，因此250K和66K蛋白强烈建议是cGMP依赖性通道蛋白。我们对250K蛋白进行了部分纯化，并成功地将其重组到脂质体中。用抗250K蛋白的多克隆抗体（IGG），通过免疫电镜观察了250K蛋白在细胞内的分布。250 K蛋白似乎是丰富的光盘膜，而不是质膜，这可能是在和谐与最近的报告表明，在光盘膜cGMP依赖性通道的富集。有趣的是，IgG级分与250K和66K蛋白反应，表明前者是后者的替代物。这些蛋白质也可能是不同类型的cGMP依赖性通道。这些问题将通过进一步纯化和重组这些蛋白质来解决。

Vertebrate photoreceptors show hyperporalizing responses to light stimulations due to the closure of cGMP-dependent cation channels on outer segment membranes. The aim of this project is to identifiy and purify the cGMP-dependent ion channel protein. Integral membrane protein fraction from frog rod outer segments was labeled with [^3H]cGMP and four cGMP binding proteins with molecular weights of 250K, 100K, 92K, and 66K were identified. The incoropration of the fraction into planar lipid bilayers resulted in the appearance of at least three kinds of channel activity; two of them were related to cGMP. Since the 100K and 92K were phosphodiesterase, the 250K and/ar 66K proteins were strongly suggested to be cGMP dependent channel proteins. We performed partial purification of the 250K protein and succeeded in reconstituting of it into liposomes. By using polyclonal antibody (IGG) against the 250K protein the distribution of the protein was investagted by means of immuno-electronmicroscopy. The 250K protein seemed to be enriched on disc membranes rather than plasma membranes, which may be in harmony with recent reports indicating an enrichment of cGMP-dependent channels in disc membranes. Interestingly the IgG fraction reacts with both 250K and 66K proteins suggesting that the former is a precurser of the latter. It may also be possible these proteins are different types of cGMP-dependent channels. These problems would be solved by the further purification and reconstitution of these proteins.