KINETIC-ANALYSIS OF THE PHOSPHORYLATION-DEPENDENT INTERACTIONS OF SYNAPSIN-I WITH RAT-BRAIN SYNAPTIC VESICLES

1. Synapsin I, a major synaptic vesicle (SV)-associated phosphoprotein , is involved in the regulation of neurotransmitter release and synaps e formation. By binding to both phospholipid and protein components of SV with high affinity and in a phosphorylation-dependent fashion, syn apsin I is believed to cluster SV and to attach them to the actin-base d cytoskeleton of the nerve terminal. 2. In the present study we have investigated tile kinetic aspects of synapsin I-SV interactions and th e mechanisms of their modulation by ionic strength and site-specific p hosphorylation, using fluorescence resonance energy transfer between s uitable fluorophores linked to synapsin I and to the membrane bilayer. 3. The binding of synapsin I to the phospholipid and protein component s of SV has fast kinetics: mean time constants ranged between 1 and 4 s for association and 9 and Ils for ionic strength-induced dissociatio n at 20 degrees C. The interaction with the phospholipid component con sists predominantly of a hydrophobic binding with the core of the memb rane which may account for the membrane stabilizing effect of synapsin I. 4. Phosphorylation of synapsin I by either SV-associated or purifi ed exogenous Ca2+/calmodulin-dependent protein kinase II (CaMPKII) inh ibited the association rate and the binding to SV at steady state by a cting on the ionic strength-sensitive component of the binding. When d ephosphorylated synapsin I was previously bound to SV, exposure of SV to Ca2+/calmodulin in the presence of ATP triggered a prompt dissociat ion of synapsin I with a time constant similar to that of ionic streng th-induced dissociation. 5. In conclusion, the reversible interactions between synapsin I and SV are highly regulated by site-specific phosp horylation and have kinetics of the same order of magnitude as the kin etics of SV recycling determined in mammalian neurons under comparable temperature conditions. These findings are consistent with the hypoth esis that synapsin I associates with, and dissociates from, SV during the exo-endocytotic cycle. The on-vesicle phosphorylation of synapsin I by the SV-associated CaMPKII, and the subsequent dissociation of the protein from the vesicle membrane, though not involved. in mediating exocytosis of primed vesicles evoked by a single stimulus, may represe nt a prompt and efficient mechanism for the modulation of neurotransmi tter release and presynaptic plasticity.