Figure 1.

SNAP-25 cleavage by BontA perturbs action potential-evoked neurotransmitter release. (A) Simultaneous pre- and postsynaptic whole-cell patch-clamp recordings obtained from cultured hippocampal neurons. Lower traces represent the action potential (in current clamp) whereas upper traces represent γ-aminobutyric acid-activated, chloride-mediated IPSCs. At control synapses, evoked synaptic currents were detected readily at an external Ca²⁺ concentration of 2 mM (Left). At BontA-treated synapses, no IPSCs were detectable in 2 mM Ca²⁺, but these became detectable as Ca²⁺ was increased gradually to higher concentrations (Right). (Calibration = 150 pA or 55 mV, 60 ms.) (B) The relative amplitude of IPSCs are plotted on a log–log plot for control synapses (at 1, 2, and 3 mM Ca²⁺) and BontA-treated synapses (at 3, 6, and 9 mM Ca²⁺). The data are plotted relative to the amplitude of IPSCs measured in 5 mM Ca²⁺ in control cultures and 15 mM Ca²⁺ in BontA-treated cultures. A shift to the right is observed without a notable change in slope (n). (C) Protein extracts from control and BontA-treated cultures were separated by SDS/PAGE. Blots were probed with anti-SNAP-25 and anti-syntaxin antibodies. All detectable SNAP-25 was cleaved by BontA and was shifted to a lower molecular weight compatible with complete cleavage whereas syntaxin was unaffected. (D) Synaptic delay measurements from five experiments were pooled for both control and BontA-treated inhibitory synapses. The relative distribution of synaptic delays in BontA-treated cultures (in 6 mM Ca²⁺; represented by black columns) and in control cultures (at 5 mM Ca²⁺; represented by white columns) is illustrated. Delays are longer and more variable after BontA. (E) These increased synaptic delays also are observed in excitatory synapses in which the variability in synaptic delay is made more striking by the faster rise times and decay times of these events. Paired recordings from 12 successive action potential-evoked excitatory synaptic currents in a cell pair are shown from a BontA-treated culture. (Calibration = 25 pA or 40 mV, 10 ms.)