The Li Yulong laboratory of the School of Life Sciences developed a new GRAB fluorescent probe for detecting the spatiotemporal dynamic changes of extracellular ATP

Purine molecules such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine (Ado) are widely present inside and outside the cell
.

Intracellular purine molecules are mainly responsible for regulating cell energy metabolism and other processes; while extracellular purine molecules are used as signaling molecules (called "purine transmitters"), which regulate breathing and taste by acting on their corresponding receptors.

On December 22, 2021, Peking University’s Li Yulong Laboratory published an online research paper entitled " A sensitive GRAB sensor for detecting extracellular ATP in vitro and in vivo " in Neuron magazine , reporting a new gene-encoded ATP probe GRAB _{ATP1 .}

0
development and application in vitro and in live animals .

Screenshot of the paper

In this work, Li Yulong’s laboratory used its previously designed GRAB probe strategy (GPCR Activation-Based sensor) to develop the ATP probe GRAB _ATP1 based on the human ATP receptor P2Y ₁ and the cyclically rearranged green fluorescent protein _cpEGFP.

.
0 (referred to as ATP1.

Figure 1: The performance of ATP1.
0 on HEK293T cells, astrocytes and neurons

Can the ATP1.
0 probe be used to detect endogenously released ATP? The author started with primary cultured hippocampal cells and found that ATP1.
0 can detect ATP release caused by mechanical stimulation and low osmotic pressure stimulation.
Pharmacological experiments and mutant probe experiments further verified the specificity of ATP1.
0 detection signal ( Figure 2)
.

Interestingly, when no additional stimulation is given, ATP1.

Figure 2: ATP1.
0 detects the release of endogenous ATP in primary cultured hippocampal cells

Can the ATP1.
0 probe be used in live animals? Past studies have found that when cells are damaged, the intracellular millimolar level of ATP is released outside the cell, which is sensed by the surrounding glial cells as a "danger signal", thereby activating microglia to release chemokines, etc.
, and produce immunity Response
.

The purine receptors expressed on glial cells play an important role in the process of microglial cell activation, migration and secretion of signal factors

Figure 3: ATP1.
0 reports the extracellular ATP signal in the brain when zebrafish is locally injured and when the mouse undergoes an immune response

When the brain is in a disease state, what kind of changes will the release of ATP show? As mentioned above, purinergic signals play an important role in immunity
.

In order to detect the changes in the ATP signal in the brain during the immune response, the author triggered a systemic immune response in mice by intraperitoneal injection of lipopolysaccharide (LPS), and at the same time expressed ATP1.

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Video: Changes in ATP signal of cerebral cortex in mouse inflammation model of intraperitoneal injection of LPS, pay attention to the signal of local ATP release

In addition to the development of a highly sensitive ATP1.
0 probe, the author also developed an ATP probe ATP1.
0-L with faster reaction kinetics and lower affinity
.

The affinity (EC ₅₀ ) of ATP1.

In summary, in this work, the author developed a new genetically-encoded ATP fluorescent probe, which achieved high spatio-temporal resolution recording of extracellular ATP
.

Prior to this, Li Yulong's research team also developed another GRAB fluorescent probe for the purine transmitter adenosine in 2020, and helped the Xu Min team at the Center for Excellence in Brain Science and Intelligent Technology of the Chinese Academy of Sciences in the study of sleep regulation

Postdoctoral student Wu Zhaofa of the School of Life Sciences of Peking University is the first author of this article.
Professor Li Yulong and Dr.
Wu Zhaofa are the co-corresponding authors.
Undergraduate graduate He Kaikai, doctoral students Pan Sunlei, Li Bohan, Deng Fei and Wang Huan have made important contributions to the article
.
The Peking University research team and the Jing Miao team (Chen Yue, Xi Fengxue) of the Beijing Brain Science and Brain-like Research Center and the Du Jiulin team (Li Hongyu, Liu Tingting) of the Center for Excellence in Brain Science and Intelligent Technology of the Chinese Academy of Sciences (Li Hongyu, Liu Tingting), etc.
have launched a full cooperation.
The University’s State Key Laboratory of Membrane Biology, Peking University-Tsinghua Life Sciences Joint Center, National Natural Science Foundation of China, Beijing Municipal Commission of Science and Technology, Feng Foundation and Boehringer Ingelheim Postdoctoral Scholarship and other institutions and funding support
.