Reversal of TX100-induced MEND in BHK cells. (A) TX100-induced MEND reverses completely when the cytoplasmic solution contains 8 mM ATP, 0.2 mM GTP, no lithium, and a low free cytoplasmic Ca concentration (1 mM EGTA with 0.2 mM total Ca; free Ca calculated to be 0.15 µM; >20 observations). In this recording, MEND is induced with 200 µM TX100, and it reverses with a progressively longer halftime of 5–16 min over five repetitions. (B) MEND does not recover in the absence of ATP and the presence of a nonhydrolyzable ATP analogue (2 mM; AMP-PNP). (C) MEND recovery is completely blocked by 0.5 mM NEM added to both extracellular and cytoplasmic solutions (five similar observations). (D) MEND recovery is strongly inhibited by cytoplasmic application of 16 µM of the NO donor, nitroprusside (four similar observations), whereas it is unaffected by 0.3 mM cGMP (five similar observations). (E) When MEND recovery has progressed partially for 3 min, a Ca transient can induce complete recovery of membrane area in seconds. In this experiment, a Ca transient was evoked before MEND to determine the immediately available membrane pool for exocytosis. Thereafter, MEND amounts to a loss of 66% of the cell surface, and after 3 min, a Ca transient causes exocytosis of a three times greater amount of membrane than was initially available.