Funding Source: NIH R01GM134380 – Extracellular ATP and ADP are mediators of purinergic signaling in cell-to-cell communication in virtually every tissue. Autocrine and paracrine purinergic signaling contributes to normal cellular functions such as immune cell chemotaxis and cell volume regulation, and purinergic signaling also contributes to pathology in injury, infection, sepsis, and a number of diseases. Notably, extracellular ATP and ADP mediate purinergic signaling between cells with both short-term and long-term effects. However, it has been a challenge to study the cellular and molecular mechanisms by which ATP and ADP control distinct signaling events because their levels are spatially heterogeneous and purinergic signaling can vary significantly from cell-to-cell.

Clearly, direct measurements of extracellular nucleotides are needed, but current methods are not well adapted to measuring fluctuations in the low extracellular nucleotide concentrations expected in live specimens. To overcome this barrier, we will use protein engineering to develop high affinity cell-surface sensors that can be used to resolve the complex spatial and temporal dynamics of ATP and ADP release, clearance, and purinergic receptor activation. To demonstrate the application of our sensors and also guide their optimization, we will study neuron-glia purinergic signaling in the brain. In particular, we will use our sensors to learn how metabolic state affects the function of microglia, the resident immune cells in the brain, in regulating the regional purinergic dynamics that impact local neuronal activity.

The proposed series of multiplexed imaging studies that pair our purinergic sensors with sensors of downstream signaling should directly visualize core elements of neuron-glia purinergic communication in an integrated manner. This should provide valuable insight into novel mechanisms responsible for the coupling between brain energy metabolism and excitability. Furthermore, the genetically-encoded tools developed through this proposal should be broadly applicable to deeper study of any purinergic system.