α-SNAP rescues the block imposed by Munc18-1.
A, SLO-permeabilized spermatozoa were loaded with 4 nm Munc18-1 for 10 min at 37 °C and subsequently challenged with 0.5 mm CaCl2 for 10 min at 37 °C. We then added 100 nm wild type α-SNAP, 100 nm α-SNAP(160–295), or 100 nm α-SNAP-L294A and incubated at 37 °C for 15 min (black bars). B, SLO-permeabilized spermatozoa were loaded with 4 nm Munc18-1 for 10 min at 37 °C and subsequently challenged with 0.5 mm CaCl2 for 10 min at 37 °C. After this incubation, we added 100 nm wild type TeTx and incubated for 10 min at 37 °C. We stopped toxin activity with 2.5 μm TPEN for 10 min. At the end of the incubation, we released the block with 100 nm wild type α-SNAP for 10 min at 37 °C (black bar). Controls in A and B (gray bars) included the following: background acrosomal exocytosis in the absence of any stimulation (control); acrosomal exocytosis stimulated by 0.5 mm CaCl2 (calcium); inhibitory effect of 4 nm Munc18-1 or 100 nm wild type TeTx, and lack of effect of 100 nm α-SNAPs. Cells were fixed; acrosomal exocytosis was evaluated using PSA-FITC, and data were normalized as described under “Experimental Procedures.” The values represent mean ± S.E. of at least three independent experiments.