Modulation of an early step in the secretory machinery in hippocampal nerve terminals

In hippocampal neurons, neurotransmitter release can be regulated by protein kinase A (PKA) through a direct action on the secretory machinery. To identify the site of PKA modulation, we have taken advantage of the ability of the neurotoxin Botulinum A to cleave the synaptic protein SNAP-25. Cleavage of this protein decreases the Ca(2+) responsiveness of the secretory machinery by partially uncoupling Ca(2+)-sensing from fusion per se. This is expressed as a shift toward higher Ca(2+) levels of the Ca(2+) to neurotransmitter release relationship and as a perturbation of synaptic delay under conditions where secretion induced by the Ca(2+)-independent secretagogue ruthenium red is unimpaired. We find that SNAP-25 cleavage also perturbs PKA-dependent modulation of secretion; facilitation of ruthenium red-evoked neurotransmitter release by the adenylyl cyclase activator forskolin is blocked completely after Botulinum toxin A action. Together with our observation that forskolin modifies the Ca(2+) to neurotransmitter release relationship, our results suggest that SNAP-25 acts as a functional linker between Ca(2+) detection and fusion and that PKA modulates an early step in the secretory machinery related to calcium sensing to facilitate synaptic transmission.