Fig. 5. Membrane-depolarization-induced effects on SNARE complexes in PC12 cells that were stimulated with high levels of K⁺ require elevations of intracellular Ca²⁺. (A) Effects of membrane depolarization and repolarization on SNARE complexes in cells at different extracellular Ca²⁺ concentrations or in the presence of 1 mM Cd²⁺. 230 kDa and 100 kDa SNAP-25-immunoreactive bands from PC12 cell membrane extracts from cells treated for 5 minutes in the following way: (lane C) incubation in physiological buffer containing 1.5 mM Ca²⁺ (control), (lane D) stimulation with 80 mM K⁺ buffer containing 1.5 mM Ca²⁺, (lane D/R) stimulation with 80 mM K⁺ buffer containing 1.5 mM Ca²⁺, followed by a 45 minute incubation at 37°C in physiological buffer containing 0.1 mM Ca²⁺, (lane D 0.1) stimulation with 80 mM K⁺ buffer containing 0.1 mM Ca²⁺, (lane D Cd²⁺) stimulation with 80 mM K⁺ buffer containing 1.5 mM Ca²⁺ and 1 mM Cd²⁺. (B) The effect of membrane depolarization in PC12 cells with 110 mM K⁺ on syntaxin (clone 78.3)-immunoreactive protein bands (intact cells, left panel) is shown together with a comparison of the intensities of protein bands recognized by the same antibody in PC12 cell membrane extracts from permeabilized cells exposed to 0.1 µM free Ca²⁺ and 250 µM free Ca²⁺ (right panel). Immunoblots were performed on heat-treated nitrocellulose membrane. Stimulation of intact cells with 110 mM K⁺ for 10 minutes led to a decrease in the intensity of the 230 kDa band (62% of the band intensity obtained from an extract of non-depolarized control cells, left column) and to an increase in the intensity of the 100 kDa band (157% of control). Exposure of permeabilized cells to 250 µM free Ca²⁺ showed a reduced intensity of the 230 kDa band (to 63%) when compared with the intensity of the 230 kDa band when 100 nM free Ca²⁺ were present. The intensities of the 100 kDa band after stimulation of cells exposed to 0.1 µM and 250 µM free Ca²⁺ showed no difference (intensity in 250 µM Ca²⁺ was 99.6% of intensity in 0.1 µM Ca²⁺). Notice that the 100 kDa and 230 kDa bands are from films exposed to the blotting membrane for different durations. (C) Effect of depolarization, repolarization and digitonin (digi)-permeabilization of PC12 cells. SNAP-25-immunoreactive protein bands obtained from membrane extracts from PC12 cells exposed for 10 minutes to the indicated conditions are compared with those of the control cells (4 mM K⁺ and 10 mM Ca²⁺) (bands on left side). Repolarization was for 45 minutes at 37°C (bands on right side).