Novel iGluSnFR Variants Optimised for Rapid Glutamate Imaging
Novel iGluSnFR Variants Optimised for Rapid Glutamate Imaging
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针对快速谷氨酸成像而优化的新型 iGluSnFR 变体
DOI：
10.1016/j.bpj.2017.11.858
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发表时间：
2018
影响因子：
3.4
通讯作者：
Helassa N
中科院分区：
文献类型：
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作者：
Helassa N
基于强度的谷氨酸传感荧光报告基因iGlu-“嗅探器”（iGluSnFR）是神经科学的有用工具，它能够检测单个突触前末梢的谷氨酸释放。然而，探针的荧光上升和衰减动力学似乎太慢，无法准确读出高频率爆发期间突触处的谷氨酸动力学。因此，我们产生了新的变种更快的谷氨酸结合动力学的氨基酸残基的突变协调谷氨酸在结合位点。快速变体iGlu f和iGlu u具有与iGluSnFR相当的亮度和荧光动态范围。用平衡结合滴定法在20 ℃下测定谷氨酸的Kd值，从33 μM（iGluSnFR）分别增加到137 μM和600 μM（iGluf和iGluu）。在34 ℃下，通过停流荧光法测量的体外解离速率从iGluSnFR的233 s− 1（τ off= 4.3 ms）增加到iGlu u的1481 s− 1（τ off= 0.7 ms），使iGlu u成为迄今为止最快的谷氨酸荧光报告基因。在海马切片培养物中以100 Hz刺激的单个突触前末梢处，iGlu u具有比iGluSnFR快5倍的“关闭”速率（τ off= 2.6 ms），其中信号在每次刺激之间返回到基线，揭示了在高频释放事件之间突触谷氨酸的完全清除。谷氨酸神经传递在高频率爆发期间表现出明显的抑制，这可以归因于突触前资源的耗尽或突触后受体的脱敏。通过比较iGlu u信号和AMPA受体电流，我们发现在100 Hz训练过程中突触抑制完全是由于谷氨酸释放减少，而500 ms后的恢复具有突触后成分。德国研究基金会（SPP 1665，SFB 936，FOR 2419）和JSW（SPP 1926，FOR 2419）;欧洲研究理事会（ERC-2016-StG 714762）。
Intensity-based glutamate-sensing fluorescent reporter iGlu-‘sniffer’(iGluSnFR) is a useful tool for neuroscience that has enabled detection of glutamate release from single presynaptic terminals. However the probe's fluorescence rise and decay kinetics appeared too slow to give an accurate readout of glutamate dynamics at the synapse during high frequency bursts. We thus generated novel variants with faster glutamate binding kinetics by mutation of amino acid residues coordinating glutamate at the binding site. Fast variants iGlu f and iGlu u have comparable brightness and fluorescence dynamic range to iGluSnFR. The K d for glutamate measured by equilibrium binding titration at 20 C is increased from 33 μM (iGluSnFR) to 137 μM and 600 μM (iGlu f and iGlu u, respectively). At 34 C, in vitro dissociation rate measured by stopped-flow fluorimetry are increased up to 6-fold from 233 s− 1 for iGluSnFR (τ off= 4.3 ms) to 1481 s− 1 for iGlu u (τ off= 0.7 ms), making iGlu u the fastest glutamate fluorescent reporter to-date. At single presynaptic terminals stimulated at 100 Hz in hippocampal slice culture, iGlu u has 5-fold faster “off” rate (τ off= 2.6 ms) than iGluSnFR, with the signal returning to baseline between each stimulus, revealing complete clearing of synaptic glutamate between high frequency release events. Glutamate neurotransmission shows pronounced depression during high frequency bursts that can be attributed to a depletion of presynaptic resources or desensitization of postsynaptic receptors. By comparing iGlu u signals and AMPA receptor currents, we show that synaptic depression during 100 Hz trains is entirely due to reduced glutamate release while the recovery after 500 ms has a postsynaptic component.This work was funded by the Wellcome Trust 094385/Z/10/Z and BBSRC BB/M02556X/1 to KT; German Research Foundation to TGO (SPP 1665, SFB 936, FOR 2419) and JSW (SPP 1926, FOR 2419); European Research Council to JSW (ERC-2016-StG 714762).