Activation of MEND by the extracellular application of 1 U/ml Bacillus cereus SMase in BHK fibroblasts with Na-free solutions. (A) BHK cell perfused for >2 min with Na-, ATP-, and GTP-free cytoplasmic solution. The application of 1 mM Ca from outside has no effect on Cm, whereas the application of Ca with SMase causes a 54% drop of Cm at a peak rate of 12% per second. Membrane current (I_m) shows no response, access resistance (R_a) increases by 10%, and membrane conductance increases transiently by 0.8 nS in parallel with the first derivative of Cm (−dC_m/dt). (B) Cytoplasmic application of SMases does not cause MEND. Perfusion of purified SMase into the cytoplasm of a BHK cell causes at most 12% decline of Cm at the same concentration that causes >50% decline of Cm from outside within seconds. (C) Reversal of SMase-induced MEND. SMase application as in A caused on average a 53% fall of Cm within 15 s. With 6 mM ATP in the cytoplasmic solution (left), Cm recovered toward baseline by 60% over 5 min, whereas Cm did not recover in the absence of nucleotides (right).