Intraterminal injection of synapsin I or calcium/calmodulin-dependent protein kinase II alters neurotransmitter release at the squid giant synapse.

Abstract

Synapsin I and calcium/calmodulin-dependent protein kinase II were pressure-injected into the preterminal digit of the squid giant synapse to test directly the possible regulation of neurotransmitter release by these substances. Neurotransmitter release was determined by measuring the amplitude, rate of rise, and latency of the postsynaptic potential generated in response to presynaptic depolarizing steps under voltage clamp conditions. Injection of dephosphosynapsin I decreased the amplitude and rate of rise of the postsynaptic potential, whereas injection of either phosphosynapsin I or heat-treated dephosphosynapsin I was without effect. Conversely, injection of calcium/calmodulin-dependent protein kinase II, which phosphorylates synapsin I on site II, increased the rate of rise and amplitude and decreased the latency of the postsynaptic potential. The effects of these proteins were observed without any detectable change in the initial phase of the presynaptic calcium current. A synapsin I-like protein and calcium/calmodulin-dependent protein kinase II were demonstrated by biochemical and immunochemical techniques to be present in squid nervous tissue. The data support the hypothesis that synapsin I regulates the availability of synaptic vesicles for release; we propose that calcium entry into the nerve terminal activates calcium/calmodulin-dependent protein kinase II, which phosphorylates synapsin I on site II, dissociating it from the vesicles and thereby removing a constraint in the release process.The coupling between presynaptic membrane depolarization and transmitter release in chemical synaptic transmission has been the subject of a large number of studies over several decades (cf. ref. 1). It is clear that this depolarization causes the opening of voltage-sensitive calcium channels, entry of calcium into the nerve terminal, and calcium-dependent release of neurotransmitter (cf. refs. 1 and 2). The present study represents a step towards elucidating the molecular mechanism(s) by which the increase in intracellular calcium regulates transmitter availability or release.Synapsin I is a neuron-specific phosphoprotein localized to presynaptic terminals, where it is associated with synaptic vesicles (3)(4)(5)(6) (3-6, 16, 17). In the present study the possible roles of synapsin I and of calmodulin kinase II in synaptic transmission were examined by injecting these substances into the presynaptic terminal of the squid giant synapse and measuring the effect on calcium entry and neurotransmitter release. For this purpose, well-established electrophysiological methods for studying the squid giant synapse were used (18-21). METHODS Intracellular Injection and Electrophysiological Recording.The experiments were performed on Loligo pealii at the Marine Biological Laboratory (Woods Hole, MA), and the techniques for the electrophysiological portion of the work were similar to those described previously (19). The stellate ganglion was held in a three-compartment chamber that was superfused with Tris-buffered a...

Synaptic vesicle recycling in synapsin I knock-out mice.

“…Synapsin I has been implicated in the regulation of neurotransmitter release in a variety of preparations (Llinas et al, 1985(Llinas et al, , 1991Lin et al, 1990;Lu et al, 1992;Pieribone et al, 1995). Numerous in vitro studies have shown that synapsin I acts to cross-link synaptic vesicles with actin filaments in a phosphorylation-dependent manner (B/ihler and Greengard, 1987;Petrucci and Morrow, 1987;Benfenati et al, 1993), a notion supported by electron microscopic observations of short bridging strands between synaptic vesicles and the cytoskeleton in nerve terminals (Landis et al, 1988;Hirokowa et al, 1989).…”

Presynaptic plasticity: The regulation of Ca2+-dependent transmitter release

“…The activity of these kinases, especially the Ca 2 +-dependent kinase, may thus be a mechanism to modulate transmitter release. Indeed, a number of studies have shown that presynaptic stimulation, Ca2+-calmodulin kinase activity and synapsin phosphorylation correlate with the transmitter output (Nestler and Greengard, 1984;Llinas et al, 1985;Nichols et al, 1990). Both synapsin and the kinase may not be primary requirements for regulated exocytosis to function properly since mice lacking synapsin I or Ca: +-calmodulin kinase II both exhibit no overt phenotype and show normal synaptic transmission, but the absence of these proteins has clear effects on certain aspects of synaptic plasticity (Silva et al, 1992;Rosahl et al, 1993).…”

Inhibition of Ca2+/calmodulin-dependent protein kinase II by arachidonic acid and its metabolites.

“…The Ca2+/calmodulin-independent form of CaM-kinase II was prepared by autophosphorylation, essentially as described (25). In brief, CaM-kinase II was incubated in a reaction mixture containing CaCl2 (0.5 mM), calmodulin (0.75 puM) and nonradioactive ATP (3 Proteolysis of CaM-Kinase II. CaM-kinase II was autophosphorylated for 5 min at 00C in standard kinase buffer containing 0.5 mM ATP (26).…”

“…In nerve terminals, CaM-kinase II catalyzes the phosphorylation of several proteins, including synapsin I, a protein associated with synaptic vesicles (2). Phosphorylation of synapsin I by CaM-kinase II is thought to participate in presynaptic modulation of neurotransmitter release (3).…”