Ca-activated MEND in five different protocols. Electrical parameters are monitored via 0.2–0.5-kHz/20-mV square wave voltage oscillations. (A) MEND with high cytoplasmic ATP (8 mM). From top to bottom, solid records give the calculated cell Cm in pF, conductance (G_m) in nS, membrane current (I_m) in pA, and access resistance (R_a) in MΩ. The dotted record is a representative Cm response of a cell when ATP is omitted from the pipette solution. (B) MEND incurred by perfusion of ATP into cells after nucleotide depletion and the introduction of a Ca transient without nucleotides, as in Fig. 2. The ATP/GTP-free period was 3 min, NCX1 current was activated for 3 s, and 20 s later the pipette was perfused with solution containing 2 mM ATP and 0.2 mM GTP. (C and D) MEND induced by perfusion of Ca-buffered pipette solutions. Cm responses were monitored during pipette perfusion of solutions buffered to free Ca concentrations of 5–200 µM. The experimental record illustrates MEND generated by perfusion of solution with 200 µM of free Ca (10 mM NTA and 0.5 mM ATP plus 0.1 mM GTP). The cytoplasmic solution contains 80 mM Li, 40 mM Cs, and no Na. As shown in the graph for experiments with five different free Ca concentrations, the half-maximal Ca concentration was 9 µM. Standard errors were <15%. (E) Ca-activated MEND in the presence of 1 mM cytoplasmic spermidine. MEND occurs within seconds during Ca influx via NCX1 and stops rather quickly when Ca influx is terminated. (F) Immediate Ca-activated MEND response in a cholesterol-enriched BHK cell without spermidine. Patch pipettes were dipped in a hot (60°C) mineral oil–cholesterol solution (150 mg cholesterol/1 ml oil with 10% ethanol) before seal formation. Seals were highly stable, and MEND occurred very rapidly upon activating Ca influx, with almost no detectable exocytic response.