Functional characterization of the rod visual pigment of the echidna (Tachyglossus aculeatus), a basal mammal
Functional characterization of the rod visual pigment of the echidna (Tachyglossus aculeatus), a basal mammal
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DOI：
10.1017/s0952523812000223
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发表时间：
2012-07-01
影响因子：
1.9
通讯作者：
Chang, Belinda S. W.
中科院分区：
文献类型：
--
作者：
Bickelmann, Constanze;Morrow, James M.;Chang, Belinda S. W.
单孔类是最基本的产卵哺乳动物，由两个现存的属组成，主要是夜间活动的。视觉色素是眼睛光感受器感觉转导级联的第一步，已经在各种脊椎动物中进行了研究，但对单孔动物的视紫红质的研究工作很少。我们分离了夜间短喙针鹭(Tachyglossus Aculeatus)的视紫红质基因，并在体外进行了表达和功能鉴定。还表达并鉴定了三个突变体：N83D，一个重要的光谱调谐和变紫红质动力学位点，以及两个针鼠特有的氨基酸位点(T158A和F169A)。T158A(498.0+/-1.3 nm)和F169A(499.4+/-0.1 nm)的视紫红质的最大吸收波长(497.9+/-1.1 nm)在T158A(498.0+/-1.3 nm)和F169A(499.4+/-0.1 nm)中无显著差异，而在N83D(503.8+/-1.5 nm)中发生了红移。与其他哺乳动物视紫红质不同，针鼠视紫红质在接触羟胺时确实会发生反应，尽管速度不如视锥细胞视紫红质那么快。荧光光谱法测定的视紫红质的半衰期为9.5±2.6min(-1)，明显短于牛视紫质的半衰期。N83D突变体的半衰期为5.1+/-0.1min(-1)，甚至比野生型的半衰期短。我们的结果表明，在羟胺敏感性和视网膜释放方面，野生型针鼠视紫红质与所有以前描述的哺乳动物视紫红质有很大的不同，并且看起来更类似于其他非哺乳动物脊椎动物视紫红质，如鸡和阿诺尔。然而，我们的N83D突变结果表明，该位点可能通过增加光激活中间产物的稳定性来调节针鼠对昏暗光环境的适应。这项研究是第一次对最基本的哺乳动物的视紫红质进行表征，并表明哺乳动物的视紫红质可能存在比先前假设更多的功能变异。
Monotremes are the most basal egg-laying mammals comprised of two extant genera, which are largely nocturnal. Visual pigments, the first step in the sensory transduction cascade in photoreceptors of the eye, have been examined in a variety of vertebrates, but little work has been done to study the rhodopsin of monotremes. We isolated the rhodopsin gene of the nocturnal short-beaked echidna (Tachyglossus aculeatus) and expressed and functionally characterized the protein in vitro. Three mutants were also expressed and characterized: N83D, an important site for spectral tuning and metarhodopsin kinetics, and two sites with amino acids unique to the echidna (T158A and F169A). The lambda(max) of echidna rhodopsin (497.9 +/- 1.1 nm) did not vary significantly in either T158A (498.0 +/- 1.3 nm) or F169A (499.4 +/- 0.1 nm) but was redshifted in N83D (503.8 +/- 1.5 nm). Unlike other mammalian rhodopsins, echidna rhodopsin did react when exposed to hydroxylamine, although not as fast as cone opsins. The retinal release rate of light-activated echidna rhodopsin, as measured by fluorescence spectroscopy, had a half-life of 9.5 +/- 2.6 min(-1), which is significantly shorter than that of bovine rhodopsin. The half-life of the N83D mutant was 5.1 +/- 0.1 min(-1), even shorter than wild type. Our results show that with respect to hydroxylamine sensitivity and retinal release, the wild-type echidna rhodopsin displays major differences to all previously characterized mammalian rhodopsins and appears more similar to other nonmammalian vertebrate rhodopsins such as chicken and anole. However, our N83D mutagenesis results suggest that this site may mediate adaptation in the echidna to dim light environments, possibly via increased stability of light-activated intermediates. This study is the first characterization of a rhodopsin from a most basal mammal and indicates that there might be more functional variation in mammalian rhodopsins than previously assumed.