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含有两种感光细胞，杆状细胞和c状细胞，它们分别负责黄昏和白天的视觉。杆状和锥状视色素在光吸收前后表现出不同的分子特性。即锥体色素在吸收光后迅速形成和衰减G蛋白激活状态，而在黑暗中对羟胺表现出高度敏感性。导致前一种差异的氨基酸残基已被确定；我们已经确定了该位点，并使用敲入小鼠证明了该位点的效果。然而，由于棒状色素和锥状色素之间氨基酸序列的巨大差异，后者差异的残基尚未确定。我们克隆并表达了灵长类动物视紫红质，并将其与牛视紫红质对羟胺的敏感性进行了比较。结果表明，灵长类动物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动物和牛视紫红质的位点定向突变体，以确定造成这种差异的氨基酸残基。一些跨膜区疏水残基发生改变的突变体表现出对羟胺的易感性改变。这些结果表明，对羟胺的敏感性受跨膜区疏水相互作用离子的调节。由于视锥色素中的残基与牛视紫红质不同，这些发现将有助于阐明视杆色素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.