Sakata Souhei, Jinno Yuka, Kawanabe Akira, Okamura Yasushi
Laboratory of Integrative Physiology, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan; Institute for Academic Initiatives, Osaka University, Suita, Osaka 565-0871, Japan
Laboratory of Integrative Physiology, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan;
Proc Natl Acad Sci U S A. 2016 Jul 5;113(27):7521-6. doi: 10.1073/pnas.1604218113. Epub 2016 Jun 21.
The cytoplasmic region of voltage-sensing phosphatase (VSP) derives the voltage dependence of its catalytic activity from coupling to a voltage sensor homologous to that of voltage-gated ion channels. To assess the conformational changes in the cytoplasmic region upon activation of the voltage sensor, we genetically incorporated a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), into the catalytic region of Ciona intestinalis VSP (Ci-VSP). Measurements of Anap fluorescence under voltage clamp in Xenopus oocytes revealed that the catalytic region assumes distinct conformations dependent on the degree of voltage-sensor activation. FRET analysis showed that the catalytic region remains situated beneath the plasma membrane, irrespective of the voltage level. Moreover, Anap fluorescence from a membrane-facing loop in the C2 domain showed a pattern reflecting substrate turnover. These results indicate that the voltage sensor regulates Ci-VSP catalytic activity by causing conformational changes in the entire catalytic region, without changing their distance from the plasma membrane.
电压感应磷酸酶（VSP）的胞质区域通过与电压门控离子通道的电压传感器同源物偶联，获得其催化活性的电压依赖性。为了评估电压传感器激活后胞质区域的构象变化，我们通过基因工程将一种荧光非天然氨基酸3-（6-乙酰萘-2-基氨基）-2-氨基丙酸（Anap）引入到玻璃海鞘VSP（Ci-VSP）的催化区域。在非洲爪蟾卵母细胞中进行电压钳制时对Anap荧光的测量表明，催化区域根据电压传感器的激活程度呈现出不同的构象。荧光共振能量转移（FRET）分析表明，无论电压水平如何，催化区域都位于质膜下方。此外，来自C2结构域中面向膜的环的Anap荧光显示出反映底物周转的模式。这些结果表明，电压传感器通过引起整个催化区域的构象变化来调节Ci-VSP的催化活性，而不改变它们与质膜的距离。
