PIP₂ dependence of ATP-activated MEND. PIP2 mediates ATP-dependent MEND and induces MEND without involvement of G protein cycling. Composite results are expressed as percentage of peak Cm after Ca influx (n = 7–15). (A) Ca-activated MEND with high (8 mM) ATP is blocked by cytoplasmic GTPγS (0.5 mM). (B) Blockade of ATP-dependent MEND by GTPγS, as in A, is relieved by inclusion of the PLC inhibitor, U73122 (10 µM), in the pipette solution. (C) Pipette perfusion of 50 µM PIP₂ substitutes for ATP in the activation of MEND after a Ca transient. Bar graphs give normalized Cm in BHK cells before and after perfusion of 40 µM PIP₂ for 4 min. Before PIP₂ perfusion, cells were perfused with ATP/GTP-free solution for 4 min, followed by NCX1-mediated Ca influx for 6 s. (D) The ability of PIP₂ to induce MEND is unaffected by 0.5 mM GTPγS when PIP2 is introduced in Ca-free solution (3 mM EGTA). (E) PIP₂ is required for nucleotide reperfusion-induced MEND. Average Cm responses in BHK-NCX1 cells perfused with ATP/GTP-free solution for 4 min, followed by NCX1-mediated Ca influx for 6 s, and then perfusion of 2 mM ATP for 3 min. Inclusion of 0.1 mg/ml IPP5c in cytoplasmic solutions reduced ATP-activated MEND by >80%.