Converting a Light-Driven Proton Pump into a Light-Gated Proton Channel
Converting a Light-Driven Proton Pump into a Light-Gated Proton Channel
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DOI：
10.1021/ja511788f
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发表时间：
2015-03-11
影响因子：
15
通讯作者：
Sudo, Yuki
中科院分区：
文献类型：
--
作者：
Inoue, Keiichi;Tsukamoto, Takashi;Sudo, Yuki
在自然界中有两种类型的嵌入膜离子传输机制。离子泵通过能量耦合的主动离子传递产生电化学电位，而离子通道通过刺激依赖的被动离子传递产生动作电位。光驱动质子泵archaerhodopsin-3 （AR3）和光门控阳离子通道channelrhodopsin （ChR）的氨基酸残基虽然在结构上非常相似，但在80%的氨基酸残基上是不同的。因此，问题出现了：这些蛋白质如何发挥不同的功能？与ChRs相比，离子泵的吸收最大值红移了约30100 nm，表明视网膜结合腔的结构不同。为了修饰空腔，通过替换位于视网膜周围的三个残基（即M128A、G132V和A225T），产生了一种蓝移的AR3，命名为AR3- t。AR3-T显示向内的氢离子通量穿过膜，这增加了它作为向内氢离子泵或氢离子通道的可能性。电生理实验表明，AR3-T的反膜电位接近于零，表明AR3-T具有光门控离子通道活性。光谱表征显示AR3-T的光化学性质与一些ChRs相似，包括全反式视网膜构型，质子化视网膜希夫碱与其反离子之间的强氢键，以及缓慢的光循环。从这些结果中，我们得出结论，H+转运体的功能决定因素位于跨膜结构域的中心，而不是在细胞质和细胞外结构域。
There are two types of membrane-embedded ion transport machineries in nature. The ion pumps generate electrochemical potential by energy-coupled active ion transportation, while the ion channels produce action potential by stimulus-dependent passive ion transportation. About 80% of the amino acid residues of the light-driven proton pump archaerhodopsin-3 (AR3) and the light-gated cation channel channelrhodopsin (ChR) differ although they share the close similarity in architecture. Therefore, the question arises: How can these proteins function differently? The absorption maxima of ion pumps are red-shifted about 30100 nm compared with ChRs, implying a structural difference in the retinal binding cavity. To modify the cavity, a blue-shifted AR3 named AR3-T was produced by replacing three residues located around the retinal (i.e., M128A, G132V, and A225T). AR3-T showed an inward H+ flux across the membrane, raising the possibility that it works as an inward H+ pump or an H+ channel. Electrophysiological experiments showed that the reverse membrane potential was nearly zero, indicating light-gated ion channeling activity of AR3-T. Spectroscopic characterization of AR3-T revealed similar photochemical properties to some of ChRs, including an all-trans retinal configuration, a strong hydrogen bond between the protonated retinal Schiff base and its counterion, and a slow photocycle. From these results, we concluded that the functional determinant in the H+ transporters is localized at the center of the membrane-spanning domain, but not in the cytoplasmic and extracellular domains.