High-throughput development of a hybrid-type fluorescent glutamate sensor for analysis of synaptic transmission.
High-throughput development of a hybrid-type fluorescent glutamate sensor for analysis of synaptic transmission.
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DOI：
10.1002/anie.201407181
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发表时间：
2014-12
影响因子：
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通讯作者：
Kenji Takikawa;D. Asanuma;S. Namiki;H. Sakamoto;Tetsuro Ariyoshi;Naoya Kimpara;K. Hirose
中科院分区：
文献类型：
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作者：
Kenji Takikawa;D. Asanuma;S. Namiki;H. Sakamoto;Tetsuro Ariyoshi;Naoya Kimpara;K. Hirose
荧光传感器是可视化细胞分子动力学的强大工具。我们提出了一种高通量筛选系统，称为混合型荧光指示剂开发（HyFInD），用于识别由配体结合蛋白突变体与小分子荧光团的组合组成的混合型传感器中的最佳位置特异性荧光团标记。我们在通过四种半胱氨酸反应性荧光探针与AMPA型谷氨酸受体GluA2亚基的274个氨基酸S1S2结构域的一组半胱氨酸扫描突变体反应获得的杂合分子中筛选谷氨酸传感器。 HyFInD 确定了一种谷氨酸响应探针（增强型谷氨酸光学传感器：eEOS），其动态范围 >2400%、良好的光稳定性和高选择性。当 eEOS 被特定地束缚在神经元表面时，它可靠地可视化单个突触谷氨酸释放的时空动态，揭示突触与突触的短期可塑性异质性。
Fluorescent sensors are powerful tools for visualizing cellular molecular dynamics. We present a high-throughput screening system, designated hybrid-type fluorescence indicator development (HyFInD), to identify optimal position-specific fluorophore labeling in hybrid-type sensors consisting of combinations of ligand-binding protein mutants with small molecular fluorophores. We screened sensors for glutamate among hybrid molecules obtained by the reaction of four cysteine-reactive fluorescence probes with a set of cysteine-scanning mutants of the 274 amino acid S1S2 domain of AMPA-type glutamate receptor GluA2 subunit. HyFInD identified a glutamate-responsive probe (enhanced glutamate optical sensor: eEOS) with a dynamic range >2400 %, good photostability, and high selectivity. When eEOS was specifically tethered to neuronal surfaces, it reliably visualized the spatiotemporal dynamics of glutamate release at single synapses, revealing synapse-to-synapse heterogeneity of short-term plasticity.