| One of the central issues in understanding neuronal transmission is to define the functions of the numerous proteins that are localized within presynaptic terminals and are thought to participate in the regulation of the synaptic vesicle life cycle. Synapsins are a multigene family of neuron-specific phosphoproteins, and are the most abundant proteins on synaptic vesicles. Much evidence has indicated that synapsins regulate neurotransmitter release, but the underlying mechanism(s), and the specific domains of the synapsin molecule involved, are not completely understood.; The goal of this dissertation research has been to determine the function of synapsins in neurotransmitter release, using an electrophysiological approach at the squid giant synapse preparation. To establish the primary structure of the endogeneous proteins, two squid synapsin isoforms were cloned (s-syn-short and s-syn-long). In both cases, three of the domains (domains A, C and E) were homologous to domains in vertebrate synapsins. Synthetic peptides corresponding to regions spanning all the domains of both squid synapsin isoforms were injected into the squid giant synapse in an attempt to interfere with endogeneous protein function. Peptides derived from the conserved domains A, C and E reversibly inhibited neurotransmitter release, while peptides derived from variable domains B and V were ineffective.; Peptides derived from domain E were investigated in more detail, since in vertebrate synapsins, this domain is present only in the a-type splice variants. Injection of domain E peptide greatly reduced the number of synaptic vesicles in the periphery of the active zone, and increased the rate and extent of synaptic depression, suggesting that domain E is essential for synapsins to regulate a reserve pool of synaptic vesicles. Domain E peptide had no effect on the number of docked synaptic vesicles, yet reversibly inhibited and slowed the kinetics of neurotransmitter release, indicating a second role for synapsins that is more intimately associated with the release process itself. Together, these data suggest that synapsin domain E is involved in at least two distinct reactions that are crucial for exocytosis in presynaptic terminals. |
