Cholesterol enrichment enables fast Ca-activated MEND without polyamines. All results use BHK cells with ATP-, GTP-, and polyamine-free cytoplasmic solution. Composite Cm changes from multiple experiments (n = 4–6) are plotted by normalizing Cm after an intervention (open squares with standard errors) to Cm values before the intervention (filled squares). (A) Treatment of BHK cells with 10 mM HPCD for 5 min does not affect in an evident manner NCX1 currents or membrane fusion responses evoked by outward NCX1 current. Cm decreases by <10% during HPCD treatment. (B) Treatment of BHK cells with cholesterol-loaded HPCD for 5 min causes an average increase of Cm of 12%. Thereafter, the activation of outward NCX1 current causes an average 36% fall of Cm within 5 s, often with no evident preceding membrane fusion response. (C) Calcium transients force cells into a MEND-permissive state for many minutes. Although treatment of cells with HPCD complexes does not cause endocytic responses in control cells (see B), cholesterol enrichment causes profound MEND over 2–4 min when treatment is initialized after an NCX1-mediated Ca transient.