FIGURE 6.

Recombinant Munc18-1 inhibits acrosomal exocytosis by sequestering monomeric syntaxin. A, SLO-permeabilized human sperm were treated for 15 min at 37 °C with increasing concentrations of syntaxin 1(1–262, white circles), syntaxin 1(25–262, black circles), or syntaxin 1-I233A (gray circles). Next, we added 0.5 mm CaCl₂ and incubated for an additional 15 min to stimulate exocytosis. B, SLO-permeabilized sperm were treated for 15 min at 37 °C with 4 nm Munc18-1 pre-mixed with 100 nm syntaxin 1(1–262), 100 nm syntaxin 1(25–262), or 100 nm syntaxin 1-I233A. Next, we added 0.5 mm CaCl₂ and incubated for an additional 15 min to stimulate exocytosis (black bars). Several controls were included (gray bars) as follows: background exocytosis in the absence of any stimulation (control); exocytosis stimulated by 0.5 mm CaCl₂ (calcium); Munc18-1 effect on calcium-stimulated exocytosis; lack of effect of low concentration of syntaxin 1(1–262), syntaxin 1(25–262), and syntaxin 1-I233A. A and B, 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.