Once in the pre-synaptic terminal, calcium triggers a series of events that lead to neurotransmitter release. The entire process, from the opening of Ca2+ channels to neurotransmitter release, may occur quickly within 200 micro-seconds or take up to 50 milli-seconds. Time variations are most likely attributed to the location of pre-synaptic vesicles when Ca2+ enters the neuron. For rapid release it is assumed that the synaptic vesicles involved and the site of exocytosis are located at or very close to the Ca2+ channels. In delayed reactions, synaptic vesicles must be recruited and guided to the active zones before neurotransmitter release can occur.
The first step in the Ca2+ dependent process of neurotransmitter release is to free synaptic vesicles from actin and other cytoskeletal elements. At rest, the synaptic vesicle protein synapsin I is found in a dephosphorylated state where it binds to elements of the cytoskeleton including actin filaments, microtubules, and spectrin as well as exhibiting a high affinity toward synaptic vesicles. Synapsin I, by binding to both the cytoskeleton and the vesicle, immobilizes vesicles.
Synapsin I, however, when phosphorylated, loses its binding affinity for synaptic vesicles. Phosphorylation, a process where phosphate (PO4-) is transferred from adenosine triphosphate (ATP) to a protein, is a mechanism for changing the state or shape of a protein, and thus affecting its function.
Synapsin I is phosphorylated by the Ca2+ dependent protein, Ca2+ /calmodulin-dependent protein kinase II (CaM kinase II). With Ca2+ influx, Ca2+ /calmodulin is formed, which then binds to CaM kinase II. In its inactive state, CaM kinase II has an auto-inhibitory sequence which overlaps with the Ca2+ /calmodulin binding domain. Binding of Ca2+ /calmodulin changes the conformation of the kinase, relieves the inhibitory block, and activates the kinase. When activated, CaM kinase II phosphorylates synapsin I. Consequently, synapsin I loses its affinity for synaptic vesicles and the vesicles are released from the cytoskeleton. It has been found that most synaptic vesicles within 30 nm of the terminal membrane do not have synapsin I (Hirokawa et al 1989). Thus, it seems dephosphorylated synapsin I holds vesicles in place at a distance from the synaptic terminal. Vesicles released by phosphorylated synapsin I, however, move to the active zone where they are ready for exocytosis.