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，视杆细胞和视锥细胞，它们分别负责黄昏和日光视觉。棒状和锥状视觉颜料在光吸收前后表现出不同的分子性质。也就是说，锥体色素在吸收光线后表现出快速的G蛋白激活状态的形成和衰退，而它们在黑暗中表现出对羟胺的高度敏感性。导致前者差异的氨基酸残基已经确定；我们已经确定了该位点，并使用敲入小鼠演示了该位点的作用。然而，由于杆状和锥状色素氨基酸序列的巨大差异，导致后一种差异的残基(S)尚未确定。我们克隆并表达了灵长类视紫红质，并比较了其对羟胺和牛视紫质的敏感性。结果，灵长类视紫红质的敏感性显著高于牛视紫红质。我们还设计并表达了灵长类和牛视紫质的定点突变体，以确定导致差异的氨基酸残基(S)。一些突变体的跨膜区疏水残基发生了改变，表现出对羟胺的敏感性改变。这些结果表明，对羟胺的敏感性是由跨膜区的疏水相互作用离子调节的。由于视锥色素中的残留与牛视紫质不同，这些发现将有助于阐明视锥视色素与视锥色素对羟胺敏感性不同的机制。

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.