Ca2+/phospholipid-binding and syntaxin-binding of native synaptotagmin I.

Synaptotagmin, a synaptic vesicle protein endowed with multiple properties, is the putative calcium sensor in neuroexocytosis. Ca2+/phospholipid binding and syntaxin binding activity of synaptotagmin were previously investigated using recombinant fusion proteins. In phospholipid binding the EC50 for calcium obtained was different when fusion proteins containing one (C2A) or both (C2A+C2B) binding domains were used. It was alternatively proposed that one or both synaptotagmin binding domains are important for calcium-sensing and triggering of transmitter release. In this study the binding activity of native full-length synaptotagmin, immobilized on beads, was investigated. We found the kinetic parameters of Ca2+/phospholipid binding to be compatible with the role of calcium sensor for synaptotagmin (EC50 for calcium = 72 +/- 7 microM), with the two C2 domains supporting separate and complementary calcium sensing properties. The binding of native syntaxin to synaptotagmin was measurable in the absence of calcium, but was markedly stimulated (2.2-fold) in the presence of mM calcium. It may be speculated that the two domains have a synergistic action in fast synchronous transmitter release, whereas C2B domain alone may support slow asynchronous release, working as a high affinity calcium sensor.

Long-term blockade of serotonin reuptake affects synaptotagmin phosphorylation in the hippocampus.

Synaptic vesicle trafficking and transmitter release from presynaptic terminals are precisely regulated by a complex array of protein/protein interactions. Several of these proteins are substrates of endogenous protein kinases present in presynaptic terminals. The activity of Ca2+/calmodulin-dependent protein kinase II(CaMKII), one of the kinases involved in the modulation of transmitter release, was previously shown to increase in the hippocampus after long-term blockade of 5-hydroxytryptamine (5-HT) reuptake (a treatment known to elicit an increase in 5-HT release in this area). To investigate the changes induced in presynaptic protein phosphorylation by 5-HT reuptake blockade and concomitant CaMKII up-regulation, we analyzed two major CaMKII presynaptic substrates (synapsin I and synaptotagmin). All 5-HT reuptake blockers that we used, which induce an increase in CaMKII activity and autophosphorylation, also caused a large (2-3-fold) increase in the Ca2+/calmodulin-dependent post hoc phosphorylation of synaptotagmin. Conversely, the phosphorylation of synapsin I is much less affected. The change in synaptotagmin phosphorylation, as determined through immunoprecipitation and quantitative immunoblot analysis after fluvoxamine treatment, is due exclusively to increased phosphate incorporation (presumably caused by the increased kinase activity) and not to a change in the level of substrate protein after the treatment. Thus, drugs known to induce an increase in 5-HT release simultaneously induce an increase in the activity of presynaptic CaMKII and in the phosphate incorporation (post hoc) by a major CaMKII substrate in synaptic vesicles (synaptotagmin). This finding establishes a link between the facilitation of transmitter release induced by antidepressant drugs and the phosphorylation of synaptotagmin by CaMKII.