Understanding of the photoreceptor cell function in vision by molecular replacement (knock-in) methods

大多数脊椎动物的视网膜包含两种类型的感光细胞，分别负责微光和日光视觉。视杆细胞和视锥细胞的视色素在光吸收前后表现出不同的分子特性。也就是说，锥状色素在吸收光时显示G蛋白激活状态的快速形成和衰减，而它们在黑暗中显示对羟胺的高敏感性。负责前一个差异的氨基酸残基已被确定;我们已经确定了该网站，并证明了该网站使用敲入小鼠的效果。然而，由于视杆和视锥色素之间氨基酸序列的巨大差异，导致后一差异的残基尚未被鉴定。我们克隆并表达了灵长类视紫红质，并比较了其与牛视紫红质对羟胺的敏感性。结果表明，灵长类视紫红质的敏感性明显高于牛视紫红质。我们还设计并表达了灵长类和牛视紫红质的定点突变体，以鉴定导致差异的氨基酸残基。一些突变体中的跨膜区的疏水残基发生变化，表现出改变的敏感性羟胺。这些结果表明，羟胺的敏感性是由跨膜区的疏水相互作用离子调节。由于视锥色素中的残留物与牛视紫红质中的残留物不同，这些发现将有助于阐明视杆和视锥色素对羟胺敏感性差异的机制。

Most vertebrate retinas contain two types of photoreceptor cells, rods and c ones, which are responsible for twilight and daylight vision, respectively. The rod and cone visual pigments exhibit different molecular properties before and after light absorption. That is, cone pigments show rapid formation and decay of G protein-activating states upon absorption of light, while they show high susceptibility to hydroxylamine in the dark. The amino acid residues responsible for the former difference have been identified ; We have identified the site and demonstrated the effect of the site using knock-in mice. However, the residue(s) for the latter difference have not been identified due to the vast divergence of amino acid sequence between rod and cone pigments. We have cloned and expressed primate rhodopsin and compared its susceptibility to hydroxylamine with bovine rhodopsin. As a result, primate rhodopsin showed significantly higher susceptibility than bovine rhodopsin. We also designed and expressed site directed mutants of primate and bovine rhodopsin to identify the amino acid residue(s) responsible for the difference. Some mutants in which the hydrophobic residues in the transmembrane regions are changed showed altered susceptibility to hydroxylamine. These results suggest that the susceptibility to hydroxylamine is regulated by the hydrophobic interact ions in transmembrane regions. Because the residues in cone pigments are different from bovine rhodopsin, these findings would give a due to elucidating the mechanism for the difference in the susceptibility to hydroxylamine between rod and cone visual pigments.