1. Presynaptic or simultaneous pre‐ and postsynaptic voltage‐clamp protocols were implemented in the squid giant synapse in order to determine the magnitude and time course of the presynaptic calcium current (ICa) and its relation to transmitter release before and after presynaptic injection of proteins. These included several forms of synapsin I, calcium‐calmodulin‐dependent protein kinase II (CaM kinase II) and avidin. 2. The quantities and location of these proteins were monitored by fluorescence video‐enhanced microscopy during the electrophysiological measurements. 3. Presynaptic injection of dephosphorylated synapsin I inhibited synaptic transmission with a time course consistent with diffusion of the protein through the terminal and action at the active release zone. A mathematical model relating the diffusion of synapsin I into the terminal with transmitter release was developed to aid in the interpretation of these results. 4. Synapsin I inhibition of transmitter release was reversible. 5. The action of synapsin I was highly specific, as phosphorylation of the tail region only or head and tail regions prevented synapsin I from inhibiting release. 6. Injections of heat‐treated synapsin I or of avidin, a protein with a size and isoelectric point similar to those of synapsin I, had no effect on transmitter release. 7. CaM kinase II injected presynaptically was found to facilitate transmitter release. This facilitation, which could be as large as 700% of the control response, was related to the level of penetration of the enzyme along the length of the preterminal A mathematical model of this facilitation indicates a reasonable fit between the distribution of CaM kinase II within the terminal and the degree of facilitation. 8. The overall shape of the postsynaptic response was not modified by either synapsin I or CaM kinase II injection. 9. The data suggest that, in addition to releasing transmitter, calcium also penetrates the presynaptic cytosol and activates CaM kinase II. When activated, CaM kinase II phosphorylates synapsin I, which reduces its binding to vesicles and/or cytoskeletal structures, enabling more vesicles to be released during a presynaptic depolarization. The amplitude of the postsynaptic response will then be both directly and indirectly regulated by depolarization induced Ca2+ influx. This model provides a molecular mechanism for synaptic potentiation.

The molecular events that control synaptic vesicle availability in chemical synaptic junctions have not been fully clarified. Among the protein molecules specifically located in presynaptic terminals, synapsin I and calcium/calmodulin-dependent protein kinase II (CaM kinase II) have been shown to modulate evoked transmitter release in the squid giant synapse. In the present study, analysis of synaptic noise in this chemical junction was used to determine whether these proteins also play a role in the control of spontaneous and enhanced spontaneous transmitter release. Injections of dephosphorylated synapsin I into the presynaptic terminal reduced the rate of spontaneous and enhanced quantal release, whereas injection of phosphorylated synapsin I did not modify such release. By contrast CaM kinase II injection increased enhanced miniature release without affecting spontaneous miniature frequency. These results support the view that dephosphorylated synapsin I "cages" synaptic vesicles while CaM kinase II, by phosphorylating synapsin I, "decages" these organelles and increases their availability for release without affecting the release mechanism itself.

Synapsin I, a neuron-specific, synaptic vesicle-associated phosphoprotein, is thought to play an important role in synaptic vesicle function. Recent microinjection studies have shown that synapsin I inhibits neurotransmitter release at the squid giant synapse and that the inhibitory effect is abolished by phosphorylation of the synapsin I molecule (Llinas et al., 1985). We have considered the possibility that synapsin I might modulate release by regulating the ability of synaptic vesicles to move to, or fuse with, the plasma membrane. Since it is not yet possible to examine these mechanisms in the intact nerve terminal, we have used video-enhanced microscopy to study synaptic vesicle mobility in axoplasm extruded from the squid giant axon. We report here that the dephosphorylated form of synapsin I inhibits organelle movement along microtubules within the interior of extruded axoplasm and that phosphorylation of synapsin I on sites 2 and 3 by calcium/calmodulin-dependent protein kinase II removes this inhibitory effect. Phosphorylation of synapsin I on site 1 by the catalytic subunit of cAMP-dependent protein kinase only partially reduces the inhibitory effect. In contrast to the inhibition of movement along microtubules seen within the interior of the axoplasm, movement along isolated microtubules protruding from the edges of the axoplasm is unaffected by dephospho-synapsin I, despite the fact that the synapsin I concentration is higher there. Thus, synapsin I does not appear to inhibit the fast axonal transport mechanism itself. Rather, these results are consistent with the possibility that dephospho- synapsin I acts by a crosslinking mechanism involving some component(s) of the cytoskeleton, such as F-actin, to create a dense network that restricts organelle movement. The relevance of the present observations to regulation of neurotransmitter release is discussed.

Protein phosphorylation is involved in the regulation of a wide variety of physiological processes in the nervous system. Studies in which purified protein kinases or kinase inhibitors have been microinjected into defined cells while a specific response is monitored have demonstrated that protein phosphorylation is both necessary and sufficient to mediate responses of excitable cells to extracellular signals. The precise molecular mechanisms involved in neuronal signal transduction processes can be further elucidated by identification and characterization of the substrate proteins for the various protein kinases. The roles of three such substrate proteins in signal transduction are described in this article: 1) synapsin I, whose phosphorylation increases neurotransmitter release and thereby modulates synaptic transmission presynaptically; 2) the nicotinic acetylcholine receptor, whose phosphorylation increases its rate of desensitization and thereby modulates synaptic transmission postsynaptically; and 3) DARPP-32, whose phosphorylation converts it to a protein phosphatase inhibitor and which thereby may mediate interactions between dopamine and other neurotransmitter systems. The characterization of the large number of additional phosphoproteins that have been found in the nervous system should elucidate many additional molecular mechanisms involved in signal transduction in neurons.

The data presented here provide evidence that the study of neuronal phosphoproteins can lead to the identification of previously unknown proteins and that these proteins may play important roles in neuronal communication. Specifically, in the case of synapsin I, direct evidence has been obtained that this phosphoprotein is involved in regulating neurotransmitter release. A tentative explanation of the results obtained in the micro-injection studies is as follows: synapsin I, in the dephosphostate, is bound to the cytoplasmic surface of synaptic vesicles and inhibits the ability of the vesicle to interact with the plasma membrane; increases in intracellular calcium activate calmodulin kinase II which in turn phosphorylates synapsin I and the phosphorylated synapsin I dissociates from the synaptic vesicle thus removing a constraint on the release of neurotransmitter. Clearly, more studies need to be done to rigorously test this hypothesis. Nevertheless these studies of synapsin I suggest that the study of previously unknown phosphoproteins will lead to the elucidation of previously unknown regulatory processes in neurons.

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.

Ca2+/calmodulin-dependent protein kinase II, an abundant brain protein proposed to mediate a number of Ca2+-regulated processes in neuronal tissue, is composed of autophosphorylatable subunits of Mr 50,000 and 60,000/58,000. A recent study (McGuinness, T. L., Lai, Y., Greengard, P., Woodgett, J.R., and Cohen, P. (1983) FEBS Lett. 163, 329-334) suggested that this kinase exists as isozymes which vary in the relative ratio of these subunits in different tissues or species. Other studies (Walaas, S. I., Nairn, A. C., and Greengard, P. (1983) J. Neurosci. 3, 291-301, 302-311) provided evidence which suggested that the ratio of these phosphopeptides might vary in different brain regions. In the present investigation, we have tested this possibility by comparing Ca2+/calmodulin-dependent protein kinase II purified from rat forebrain and cerebellum. The two kinases had similar purification characteristics, subunit compositions, physical properties, and substrate specificities. Gel filtration and sucrose density gradient centrifugation provided an estimated molecular weight of 550,000 for the forebrain kinase and 615,000 for the cerebellar kinase. The kinases from the two regions clearly differed in the relative proportions of the Mr 50,000 and 60,000/58,000 subunits. Three independent methods indicated that the forebrain kinase contained the Mr 50,000/(60,000/58,000) subunits in approximately a 3:1 ratio, while the cerebellar kinase contained the Mr 50,000/(60,000/58,000) subunits in approximately a 1:4 ratio. The forebrain kinase subunits were shown to be identical to the corresponding subunits of the cerebellar kinase by several criteria. The data are consistent with the existence in various brain regions of isozymic forms of Ca2+/calmodulin-dependent protein kinase II which differ in their relative subunit ratios.

Calcium/calmodulin-dependent protein kinase II (CaM kinase II) is a prominent enzyme in mammalian brain capable of phosphorylating a variety of substrate proteins. In the present investigation, the subcellular and regional distribution of CaM kinase II has been studied by light and electron microscopic immunocytochemistry using an antibody that recognizes the Mr 50,000 and 60,000/58,000 subunits of the enzyme. Light microscopy demonstrates strong immunoreactivity in neuronal somata and dendrites and weak immunoreactivity in axons. Electron microscopy, in addition to confirming light microscopic observations, reveals moderate immunoreactivity in spines and weak immunoreactivity in nerve terminals. An accumulation of immunoreaction product is also present on postsynaptic densities. The presence of CaM kinase II in diverse structures throughout the neuron supports the view that this enzyme may be involved in mediating a variety of calcium-dependent physiological processes. CaM kinase II immunoreactivity is present in neurons throughout the brain, but a marked regional variation in the strength of the immunoreactivity exists. Overall, there is a gradient of staining intensity with the strongest immunoreactivity in the telencephalon and the weakest in the myelencephalon. The most heavily labeled regions of the telencephalon are the hippocampal formation, lateral septum, cortical regions, neostriatum, and amygdaloid complex.

Polypeptides of Mr 50,000 and 60,000 in isolated synaptic junctions have been compared to polypeptides of corresponding molecular weight in Ca2+/calmodulin-dependent protein kinase II. The polypeptides of corresponding molecular weight from the two preparations were shown by several criteria to be indistinguishable. These criteria included 125I-labeled tryptic/chymotryptic peptide patterns, 32P-labeled proteolytic peptide maps, and crossreactivity on immunoblots using polyclonal and monoclonal antibodies. Furthermore, studies examining the phosphorylation of substrate proteins, by the endogenous synaptic junction kinase and by Ca2+/calmodulin-dependent protein kinase II, indicated that the two enzymes have similar substrate specificities. Since the Mr 50,000 polypeptide present in synaptic junctions is known to be the major postsynaptic density protein, the present results indicate that the major postsynaptic density protein is a component of Ca2+/calmodulin-dependent protein kinase II.

Calmodulin-dependent glycogen synthase kinase isolated from skeletal muscle and synapsin I kinase II isolated from brain have several properties that are very similar. These properties include: substrate and site-specificities, immunological cross-reactivity, and phosphopeptide maps following limited proteolysis. Both enzymes phosphorylate a wide variety of substrate proteins. The two kinases may represent different isozymes of a multifunctional calmodulin-dependent protein kinase that mediates many of the actions of Ca2+ in various tissues. Therefore, we propose the name 'calmodulin-dependent multi-protein kinase' for this broad specificity enzyme.