自 2006 年以来,光敏离子通道已成为神经科学中不可或缺的光遗传学工具。使用基因工程技术在特定神经元上强制表达光敏离子通道之后,可以用特定波长的光照射这些细胞以激发或抑制每个靶神经元。这种技术的时空准确性是药理学或电学等常规方法所不具备的特点。
另外,信号因子,例如由环核苷酸 cAMP 和 cGMP 介导的细胞内信号转导通路,是对各种重要的生物功能起至关重要作用的因子。激活腺苷酸环化酶或抑制磷酸二酯酶会增加细胞内的 cAMP 浓度,这会刺激环核苷酸门控 (CNG) 通道、蛋白激酶 A (PKA) 和交换蛋白直接被 cAMP 激活 (Epac)。这些过程可诱发各种细胞反应,例如受体电位的产生、神经元轴突分支或延长、心率增加和糖原分解。
我们将推出一种光遗传学工具“PAC”,它可以控制 cAMP 的产生,并诱发这些由不同强度的蓝光所触发的细胞反应。
PAC 最初在光合鞭毛虫 Euglena glacilis 中被鉴定为通过光照诱导细胞内 cAMP 生成的光敏分子(参考文献 1)。最近,在几种原核生物中广泛发现了 PAC 的同源蛋白。
PAC 对 cAMP 信号的控制很容易处理,因为该过程独立于 G 蛋白偶联受体 GPCR 介导的信号级联。由于 PAC 仅通过光照产生 cAMP,因此 cAMP 的产生可以在时间和空间上进行高精度的调节。因此,该方法被寄予厚望,能够仅通过光照来直接控制细胞和组织内的各种生理功能(参考文献 2)。
摘自 Scientific Reports 5,19679,doi:10.1038/srep19679 (2016)
在迄今为止发现的 PAC 中,来自蓝藻细菌的 OaPAC 和来自硫细菌的 bPAC 的结构已被确定。这两种 PAC 在 N 端都有一个光敏 BLUF 结构域,在 C 端都有一个腺苷酸环化酶 (AC) 结构域,并且这些结构域彼此相邻排列。虽然 OaPAC 和 bPAC 的序列和结构相似,但它们的光敏性相差一百倍(参考文献 3)。这种差异应该为修改 PAC 设计提供线索,使其成为一种适用于多种变体的光遗传学工具。在与新光子学产业创建研究生院 (GPI) 的合作中,我们在 C 端构建了许多缺失突变体,并比较了它们的酶活性。我们发现,C 端区域归属的时间越长,酶活性越低,其光活性的变化超过五十倍(参考文献 4)。
OaPAC 的氨基酸序列来自 Oscillatoria acuminata (NCBI, WP_015149803.1) 及其缺失变体 (Oa-363, Oa-360, Oa-357, Oa-354, Oa-351, Oa-348),bPAC 来自土壤细菌 Beggiatoa (GenBank: GU461306.2),PACα 来自 Euglena gracilis (GenBank: BAB85619.1) C 端的氨基酸序列。黄色高亮:保守氨基酸。
摘自 Scientific Reports 9, 20262 (2019)
野生型 OaPAC 的低感光灵敏度一直是实际应用的瓶颈,因为它很容易因强光照射而出现光毒性。我们已经解决了这个问题,并可以将 OaPAC 用于各种应用。两种具有不同感光灵敏度的用户友好型 OaPAC 突变体,即 OaPAC360 和 OaPAC348,现已作为我们的产品上市。
※这两种产品已委托给 COSMO BIO CO., LTD 进行销售。有关产品的咨询,请访问下面的链接。
摘自 Scientific Reports 9, 20262 (2019)
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