Figure 3. Channel Activity in Membranes of Liposome–Fused Mitochondria.

Panel A. Isolated, liposome–fused mitochondria from cultured HeLa cells were studied in planar lipid bilayers as described in Methods. Mitochondria were added to the cis chamber of a planar lipid bilayer cuvette with a 450 to 150 mM KCl from the cis to trans chambers. After 3–7 minutes current levels stabilized and the cis solution was perfused to 150 mM KCl. Using voltage clamp configuration command ramps (lower panel) were applied to the bilayer after fusion events were observed. Panel B. Using buffer containing 100 µM CaCl₂ and 100 µM EGTA the stable voltage dependence of fM was studied. Data were obtained by holding indicated the membrane potential for 10 seconds and using the the average current of the final two seconds. EGTA to 100 µM was added to the cis chamber and after 5 minutes the voltage dependence was determined again. The high calcium perfusion, voltage dependence, EGTA addition and voltage dependence re–determination was repeated until the bilayer membrane collapsed, 3 cycles. These were averaged for this panel and the s.d. at each point shown in the figure. Panel C. Bilayer conductance was calculated from voltage ramps (±60mV) collected at 1.67 kHz with a 1.2 second duration for each ramp. The bilayers were formed and Ca²⁺ concentration adjusted as described in Panel A. The currents were normalized to a peak conductance of 1 for comparison.