నైరూప్య

Immobilization of firefly luciferase on the cell plasma membrane as a quantitative biosensor for measurement of ATP in the pericellular space in live mammalian cells

Khue Vu Nguyen, Kefeng Li, Robert K Naviaux

The purinergic signaling system consists of transporters, enzymes and receptors responsible for the synthesis, release, action and extracellular inactivation of adenosine-5’-triphosphate (ATP) and its extracellular breakdown product adenosine. Up to date, the full appreciation of the role of ATP as an extracellular signal has been hampered by lack of proper biosensors for accurate local real-time measurement of extracellular ATP concentration in the pericellular space from individual cells under physiological and pathological conditions. We describe herein the development of simple, sensitive, and reliable dual-function biosensor for the local real-time measurement of extracellular ATP concentration in the pericellular space in live HEK 293 cells by performing an immobilization of firefly luciferase (Fluc) coupled with the green fluorescent protein (GFP) on the plasma membrane of HEK 293 cells via a glycosyl-phosphatidylinositol, GPI, anchor derived from the human folate receptor 1 (FOLR1) protein: pmeLUC. Our pmeLUC2 dual-function reporter construct was shown to be fluorescent and bioluminescent and could detect pericellular ATP at concentrations under physiological conditions: <5 μM and the apparent KM of immobilized Fluc for ATP are to be 2-3 × 10-6 M, values much lower than the 51 × 10-6 M found for the free Fluc. In addition, there was no loss of immobilized Fluc activity in pmeLUC2 after more than 15 ATP measurements followed by 90 days stored at +4°C in PBS. The method used for the construction of our pmeLUC2 probe may pave the way for new strategies applicable to rational pmeLUC design. Its use in live cells and organisms, especially for identifying a new pathway for ATP secretion as a signaling molecule, promise to further expand its utility.