Localized Ca2+ entry preferentially effects protein dephosphorylation, phosphorylation, and glutamate release.

The release of neurotransmitter glutamate from isolated nerve terminals (synaptosomes) was found to be tightly coupled to the entry of Ca2+ through voltage-dependent Ca2+ channels, but is relatively unresponsive to "bulk" increases in cytosolic Ca2+ concentrations ([Ca2+]c) effected by Ca2+ ionophore. Under the same conditions, this dependence on Ca2+ influx, specifically through Ca2+ channels, was also seen for the dephosphorylation of a 96-kDa protein, (P96), present in the nerve terminals, as well as the phosphorylation of proteins migrating at 75 kDa (P75), corresponding to the synapsins, a group of well characterized synaptic vesicle-associated proteins. P96 dephosphorylation, following Ca2+ influx, was persistent and insensitive to the phosphatase inhibitor okadaic acid, suggesting a phosphatase other than protein phosphatase 1 and 2A as being responsible. Perhaps through the same phosphatase activity the increase in P75 phosphorylation was rapidly reversed with a time course similar to P96 dephosphorylation. When release, P96 dephosphorylation, and P75 phosphorylation were considered as functions of the [Ca2+]c increases achieved by depolarization and Ca2+ ionophore, there was no correlation of any of these with the overall concentration of Ca2+ in the cytosol. Since the fura-2 method used to measure [Ca2+] gives an averaged [Ca2+]c, these results imply that the release and protein dephosphorylation events are functionally coupled to local [Ca2+]c, in the immediate vicinity of Ca2+ channels. The reported clustering of the latter at the active zone area of the synapse and the parallelism between synaptic vesicle exocytosis and the phosphorylation of synaptic vesicle-associated proteins (p75:synapsins Ia/Ib), suggests that P96 may be similarly localized at the active zone area and, therefore, may be of significance in a modulatory role in glutamate release.     

Ca2+ and Calmodulin-Dependent Phosphorylation of Endogenous Synaptic Vesicle Tubulin by a Vesicle-Bound Calmodulin Kinase System

Endogenous synaptic vesicle alpha- and beta-tubulin were shown to be the major substrates for a Ca2+-calmodulin-regulated protein kinase system in enriched synaptic vesicle preparations from rat cortex as determined by two-dimensional gel electrophoresis and peptide mapping. The activation of this endogenous tubulin kinase system was dependent on Ca2+ and the Ca2+ binding protein, calmodulin. Under maximally stimulated conditions, approximately 40% of the tubulin present in enriched synaptic vesicles was phosphorylated within less than 50 s by the vesicle Ca2+-calmodulin kinase. Evidence is presented indicating that the Ca2+-calmodulin tubulin kinase is an enzyme system distinct from previously described cyclic AMP protein kinases. alpha-Tubulin and beta-tubulin were identified as major components of previously designated vesicle phosphorylation bands DPH-L and DPH-M. The Ca2+-calmodulin tubulin kinase is very labile and specialized isolation procedures were necessary to retain activity. Ca2+-activated synaptic vesicle tubulin phosphorylation correlated with vesicle neurotransmitter release. Depolarization-dependent Ca2+ uptake in intact synaptosomes simultaneously stimulated the release of neurotransmitters and the phosphorylation of synaptic vesicle alpha- and beta-tubulin. The results indicate that regulation of the synaptic vesicle tubulin kinase by Ca2+ and calmodulin may play a role in the functional utilization of synaptic vesicle tubulin and may mediate some of the effects of Ca2+ on vesicle function and neurosecretion.     

Depolarization-induced phosphorylation of specific proteins, mediated by calcium ion influx, in rat brain synaptosomes.

Agents known to inphorylation of specific endogenous proteins in intact synaptosomes from rat brain. Synaptosome preparations, preincubated in vitro with 32Pi, incorporated 32P into a variety of specific proteins. Veratridine and high (60 mM) K+, which increase Ca2+ transport across membranes, through a mechanism involving membrane depolarization, as well as the calcium ionophore A23187, each markedly stimulated the incorporation of 32P into two specific proteins (80,000 and 86,000 daltons) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. All three agents failed to stimulate protein phosphorylation in calcium-free medium containing ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). Moreover, the Ca2+-dependent protein phosphorylation could be reversed by the addition of sufficient EGTA to chelate all free extracellular Ca2+. Veratridine, high K+, and A23187 also stimulated 45Ca2+ accumulation by synaptosomes. Tetrodotoxin blocked the stimulation both of protein phosphorylation and of 45Ca2+ accumulation by veratridine but not by high K+ or A23187. Cyclic nucleotides and several putative neurotransmitters were without effect on protein phosphorylation in these intact synaptosome preparations. The absence of any endogenous protein phosphorylation in osmotically shocked synaptosome preparations incubated with 32Pi, and the inability of added [gamma-32P]ATP to serve as a substrate for veratridine-stimulated protein phosphorylation in intact preparations, indicated that the Ca2+-dependent protein phosphorylation occurred within intact subcellular organelles. Fractionation of a crude synaptosome preparation on a discontinuous Ficoll/sucrose flotation gradient indicated that these organelles were synaptosomes rather than mitochondria. The data suggest that conditions which cause an accumulation of calcium by synaptosomes lead to a calcium-dependent increase in phosphorylation of specific endogenous proteins. These phosphoproteins may be involved in the regulation of certain calcium-dependent nerve terminal functions such as neurotransmitter synthesis and release.     

Cholesterol as a key player in the balance of evoked and spontaneous glutamate release in rat brain cortical synaptosomes

Membrane rafts are domains enriched in sphingolipids, glycolipids and cholesterol that are able to compartmentalize cellular processes. Noteworthy, many proteins have been assigned to membrane rafts including those related to the control of the synaptic vesicle release machinery, which is a important step for neurotransmission between synapses. In this work, we have investigated the role of cholesterol in key steps of glutamate release in isolated nerve terminals (synaptosomes) from rat brain cortices. Incubation of synaptosomes with methyl-β-cyclodextrin (MβCD) induced glutamate release in a dose-dependent fashion. HγCD, a cyclodextrin with low affinity for cholesterol, had no significant effect on spontaneous glutamate release. When we evaluated the effects of MβCD on glutamate release induced by depolarizing stimuli, we observed that MβCD treatment inhibited the KCl-evoked glutamate release. The glutamate release induced by MβCD was not altered by treatment with EGTA nor with EGTA-AM. The KCl-evoked glutamate release was no further inhibited when synaptosomes were incubated with MβCD in the absence of calcium. We therefore investigated whether the cholesterol removal by MβCD changes intrasynaptosomal sodium and calcium levels. Our results suggested that the cholesterol removal effect on spontaneous and evoked glutamate release might be upstream to sodium and calcium entry through voltage-activated channels. We therefore tested if MβCD would have a direct effect on synaptic vesicle exocytosis and we showed that cholesterol removal by MβCD induced spontaneous exocytosis and inhibited synaptic vesicle exocytosis evoked by depolarizing stimuli. Lastly, we investigated the effect of protein kinase inhibitors on the spontaneous exocytosis evoked by MβCD and we observed a statistically significant reduction of synaptic vesicles exocytosis. In conclusion, our work shows that cholesterol removal facilitates protein kinase activation that favors spontaneous synaptic vesicles and consequently glutamate release in isolated nerve terminals.     

Calmodulin, synchronous and asynchronous release of neurotransmitter

Evidence collected from studies on a wide range of secretory cells suggests that calmodulin may play an important role in stimulus-secretion coupling. Work on synaptosomes, central synaptic preparations and chromaffin cell preparations indicates that calmodulin probably also acts as the intracellular Ca2+-receptor for secretion in neuronal cells, Ca2+-binding resulting in activation of protein kinases and phosphorylation of certain secretory vesicle proteins. Studies on the effects of calmodulin-binding drugs at peripheral synapses have given surprising results, particularly the finding that evoked (synchronous) transmitter release is not suppressed by calmodulin inhibition, though asynchronous release can be markedly inhibited. It is suggested that the insensitivity of synchronous release to drug treatment is due to the fact that only vesicle-bound calmodulin is involved in this form of transmitter secretion. Asynchronous release, however, involves recruitment of cytosolic calmodulin and can therefore be inhibited by calmodulin-binding drugs.     

Ca2+-dependent protein phosphorylation of purely cholinergic Torpedo synaptosomes.

Preincubation of intact, purely cholinergic Torpedo synaptosomes with [32P]Pi results in the incorporation of 32P into about 10 specific proteins. Depolarizing the Torpedo synaptosomes by a high K+ buffer or treatment with the Ca2+ ionophore A23187 result in Ca2+ uptake, in acetylcholine (ACh) release, and in a marked increase of 32P incorporation into a specific protein band with an apparent subunit molecular weight of 100,000 (band alpha). The kinetics of synaptosomal 45Ca2+ uptake, of 32P incorporation into band alpha, and of ACh release is similar and reach maximal values about 45 s after the synaptosomes have been treated. Sr2+ and Ba2+ can replace Ca2+ in evoking both K+ depolarization-dependent ACh release and 32P incorporation into band alpha. The effectiveness of these ions (Ca2+ greater than Sr2+ greater than Ba2+) is similar in both cases. The data presented suggest that Ca2+ accumulation by Torpedo synaptosomes leads to an increase in the phosphorylation of a specific protein and to ACh release. This phosphoprotein may be involved in the regulation of presynaptic processes which underly ACh release.     

Vesicle dynamics: how synaptic proteins regulate different modes of neurotransmission

Central synapses operate neurotransmission in several modes: synchronous/fast neurotransmission (neurotransmitters release is tightly coupled to action potentials and fast), asynchronous neurotransmission (neurotransmitter release is slower and longer lasting), and spontaneous neurotransmission (where small amounts of neurotransmitter are released without being evoked by an action potential). A substantial body of evidence from the past two decades suggests that seemingly identical synaptic vesicles possess distinct propensities to fuse, thus selectively serving different modes of neurotransmission. In efforts to better understand the mechanism(s) underlying the different modes of synaptic transmission, many research groups found that synaptic vesicles used in different modes of neurotransmission differ by a number of synaptic proteins. Synchronous transmission with higher temporal fidelity to stimulation seems to require synaptotagmin 1 and complexin for its Ca²⁺ sensitivity, RIM proteins for closer location of synaptic vesicles (SV) to the voltage operated calcium channels (VGCC), and dynamin for SV retrieval. Asynchronous release does not seem to require functional synaptotagmin 1 as a calcium sensor or complexins, but the activity of dynamin is indispensible for its maintenance. On the other hand, the control of spontaneous neurotransmission remains less clear as deleting a number of essential synaptic proteins does not abolish this type of synaptic vesicle fusion. VGCC distance from the SV seems to have little control on spontaneous transmission, while there is an involvement of functional synaptic proteins including synaptotagmins and complexin. Recently, presynaptic deficits have been proposed to contribute to a number of pathological conditions including cognitive and mental disorders. In this review, we evaluate recent advances in understanding the regulatory mechanisms of synaptic vesicle dynamics and in understanding how different molecular substrates maintain selective modes of neurotransmission. We also highlight the implications of these studies in understanding pathological conditions.     

Calcium-dependent dissociation of synaptotagmin from synaptic SNARE complexes

The formation of the synaptic core (SNARE) complex constitutes a crucial step in synaptic vesicle fusion at the nerve terminal. The interaction of synaptotagmin I with this complex potentially provides a means of conferring Ca2+-dependent regulation of exocytosis. However, the subcellular compartments in which interactions occur and their modulation by Ca2+ influx remain obscure. Sodium dodecyl sulfate (SDS)-resistant core complexes, associated with synaptotagmin I, were enriched in rat brain fractions containing plasma membranes and docked synaptic vesicles. Depolarization of synaptosomes triggered [3H]GABA release and Ca2+-dependent dissociation of synaptotagmin from the core complex. In perforated synaptosomes, synaptotagmin dissociation was induced by Ca2+ (30-300 microM) but not Sr2+ (1 mM); it apparently required intact membrane bilayers but did not result in disassembly of trimeric SNARE complexes. Synaptotagmin was not associated with unstable v-SNARE/t-SNARE complexes, present in fractions containing synaptic vesicles and cytoplasm. These complexes acquired SDS resistance when N-ethylmaleimide-sensitive fusion protein (NSF) was inhibited with N-ethylmaleimide or adenosine 5'-O-(3-thiotriphosphate), suggesting that constitutive SNARE complex disassembly occurs in undocked synaptic vesicles. Our findings are consistent with models in which the Ca2+ triggered release of synaptotagmin precedes vesicle fusion. NSF may then dissociate ternary core complexes captured by endocytosis and recycle/prime individual SNARE proteins.     

Ca2+-dependent dephosphorylation of kinesin heavy chain on beta-granules in pancreatic beta-cells. Implications for regulated beta-granule transport and insulin exocytosis

The specific biochemical steps required for glucose-regulated insulin exocytosis from beta-cells are not well defined. Elevation of glucose leads to increases in cytosolic [Ca2+]i and biphasic release of insulin from both a readily releasable and a storage pool of beta-granules. The effect of elevated [Ca2+]i on phosphorylation of isolated beta-granule membrane proteins was evaluated, and the phosphorylation of four proteins was found to be altered by [Ca2+]i. One (a 18/20-kDa doublet) was a Ca2+-dependent increase in phosphorylation, and, surprisingly, three others (138, 42, and 36 kDa) were Ca2+-dependent dephosphorylations. The 138-kDa beta-granule phosphoprotein was found to be kinesin heavy chain (KHC). At low levels of [Ca2+]i KHC was phosphorylated by casein kinase 2, but KHC was rapidly dephosphorylated by protein phosphatase 2B beta (PP2Bbeta) as [Ca2+]i increased. Inhibitors of PP2B specifically reduced the second, microtubule-dependent, phase of insulin secretion, suggesting that dephosphorylation of KHC was required for transport of beta-granules from the storage pool to replenish the readily releasable pool of beta-granules. This is distinct from synaptic vesicle exocytosis, because neurotransmitter release from synaptosomes did not require a Ca2+-dependent KHC dephosphorylation. These results suggest a novel mechanism for regulating KHC function and beta-granule transport in beta-cells that is mediated by casein kinase 2 and PP2B. They also implicate a novel regulatory role for PP2B/calcineurin in the control of insulin secretion downstream of a rise in [Ca2+]i.     

Inhibition of noradrenaline release from PC12 cells by the long-term treatment with cholera toxin

Guanine nucleotide-binding (G) proteins are required for intracellular vesicular transport and endocytosis. In this study, we investigated the effects of short-term (2 h) and long-term (24 h) treatment with cholera toxin (CTX), which ADP-ribosylates proteins having arginine residues such as the alpha subunit of Gs (G(s alpha)), on exocytosis from the neurosecretory rat pheochromocytoma PC 12 cell line. Short-term treatment with CTX stimulated the accumulation of cyclic AMP, and synergistically enhanced both extracellular Ca2+-dependent [3H]noradrenaline (NA) releases (induced by high K+ and ATP) and Ca2+-independent release (induced by mastoparan, a peptide in wasp venom). Long-term treatment with CTX for 24h inhibited Ca2+-dependent and -independent stimulated [3H]NA release. The inhibitory effect of long-term CTX treatment was not derived from a cyclic AMP-dependent system, because (1) H-89, an inhibitor of protein kinase A, had no effect on the inhibition induced by CTX, (2) the long-term treatment with forskolin did not show an inhibitory effect. [32P]ADP-ribosylation of G(s alpha) and its immunoreactivity with anti-G(s alpha) antiserum in the crude membrane fraction was inhibited in the long-term CTX-treated cells, but not in the long-term forskolin-treated cells. [32P]ADP-ribosylation of G(s alpha) in the membrane fraction of short-term CTX-treated cells was approximately 90% of the level in the control cells. These findings suggest that CTX stimulates [3H]NA release via a cyclic AMP-dependent system in the short-term, and that long-term CTX treatment inhibited its release, maybe via ADP-ribosylation of CTX-sensitive proteins such as G(s alpha).     

Neurotransmitter release from viable purely cholinergic Torpedo synaptosomes

Viable synaptosomes from the electric organ of Torpedo have been prepared and partially purified. The synaptosomes contain about 100 fold more acetylcholine (Ach) than do mammalian synaptosomes, synaptic vesicles and mitochondria. The Torpedo synaptosomes release Ach by K depolarization in the presence of Ca ions, and manifest an ionophore-mediated Ca-dependent Ach release. These results demonstrate that the synaptosomes contain the neurosecretion apparatus in a functional viable state. Since this preparation uniquely contains only one neurotransmission system (cholinergic), it is most suitable for structural and functional investigations of neuro transmission.     

Antigen- and ionophore-induced signal transduction in rat basophilic leukemia cells involves protein tyrosine phosphorylation

Treatment of rat basophilic leukemia cells (RBL-2H3) with antigen or ionophore leads to an increase in cellular protein tyrosine phosphorylation. Three major proteins of molecular mass of 72, 92, and 110 kDa are targeted by antigen and a 110-kDa species by ionophore, A23187. The antigen- and ionophore-induced tyrosine phosphorylation responses are dose-dependent and correlate with increases in serotonin release from activated cells. The presence of extracellular Ca2+ is required to sustain the antigen- and ionophore-stimulated tyrosine phosphorylation as well as mediator release. A protein tyrosine kinase inhibitor, RG 50864, differentially inhibits the antigen-stimulated tyrosine phosphorylation in the decreasing order of 72, 91, and 110-kDa proteins. The compound inhibition of the 72-kDa protein tyrosine phosphorylation correlates with that of serotonin release. In ionophore-stimulated cells, the inhibition of the 110-kDa protein tyrosine phosphorylation and serotonin release by RG 50864 occurs in parallel. These results suggest that the 72- and 110-kDa phosphoproteins may represent the respective regulators of serotonin release in antigen- and ionophore-activated cells. The 110-kDa tyrosine phosphorylated proteins from antigen- and ionophore-stimulated cells exhibit identical electrophoretic mobility and V8 protease-generated phosphopeptide maps, suggesting that these two proteins may be the same. These results provide new evidence that both the stimulatory actions of antigen and ionophore on mediator release are mediated through enhanced protein tyrosine phosphorylation in RBL-2H3 cells. Significantly, the present study suggests the presence of multiple tyrosine phosphorylation signaling pathways in RBL cells and that their selective utility may be determined by the nature of the stimulus.     

Aluminium-induced impairment of Ca2+ modulatory action on GABA transport in brain cortex nerve terminals

The gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in vertebrate CNS. At GABAergic synapses, a high-affinity transporter exists, which is responsible for GABA reuptake and release during neurotransmission. GABA transporter activity depends on the phosphorylation/dephosphorylation state, being modulated by Ca(2+)/calmodulin-dependent protein phosphatase 2B (calcineurin). Aluminium is known to interfere with the Ca(2+)/calmodulin signalling pathway. In this work, we investigate the action of aluminium on GABA translocation mediated by the high-affinity transporter, using synaptic plasma membrane (SPM) vesicles and synaptosomes isolated from brain cortex. Aluminium completely relieved Ca(2+) downregulation of GABA transporter, when mediating uptake or release. Accordingly, aluminium inhibited Ca(2+)/calmodulin-dependent calcineurin activity present in SPM, in a concentration-dependent manner. The deleterious action of aluminium on the modulation of GABA transport was ascertained by comparative analysis of the aluminium effect on GABA uptake and release, under conditions favouring SPM dephosphorylation (presence of intracellular micromolar Ca(2+)) or phosphorylation (absence of Ca(2+) and/or presence of W-7, a selective calmodulin antagonist). In conclusion, aluminium-induced relief of Ca(2+) modulatory action on GABA transporter may contribute significantly to modify GABAergic signalling during neurotoxic events in response to aluminium exposure.     

Added ATP influences some responses of rat synaptosomes to glutamate.

ATP added externally to rat synaptosomes activated uptake of both Ca2+ and glutamate which was partially accounted for by the uptake phenomena of synaptic vesicles and mitochondria, as shown by using specific inhibitors of the latter. Increasing concentrations of glutamate stimulated Ca2+ entry linearly, as shown by using 45Ca or a Ca-specific electrode. The processes of glutamate and Ca2+ uptake shared some common features and their ATP-dependence may be correlated with an ouabain-insensitive synaptosomal ectonucleotidase activity measured by a 31P-NMR or a luminometric technique. The ATP hydrolysis catalysed by the synaptosomes was activated by both Ca2+ and glutamate. The present synaptosomal activities may represent a model for studying the modulatory effects of ATP on the glutamatergic neurotransmission.     

Non-genomic effect of L-triiodothyronine on calmodulin-dependent synaptosomal protein phosphorylation in adult rat cerebral cortex

The present study was undertaken to examine calmodulin-dependent effect of thyroid hormones (THs) on synaptosomal protein phosphorylation in mature rat brain. Effect of L-triiodothyronine (L-T3) on in vitro protein phosphorylation was measured in a hypotonic lysate of synaptosomes prepared from adult male rat cerebral cortex, incubated in presence and absence of calcium ion (Ca2+) and calmodulin. L-T3 significantly enhanced incorporation of 32P into synaptosomal proteins as compared to basal level of phosphorylation in the presence of Ca2+ and calmodulin. Under these conditions, increase in protein phosphorylation was 47, 74 and 52% for 10 nM, 100 nM and 1 microM L-T3, respectively. Chelation of Ca2+ using ethylene glycol-bis (2-aminoethylether)-N, N, N', N'-tetraacetic acid (EGTA) inhibited the effects of Ca2+/calmodulin on TH-stimulated protein phosphorylation levels. This study suggests that a high proportion of L-T3-stimulated protein phosphorylation involves Ca2+/calmodulin-dependent pathways in adult rat cerebrocortical synaptosomes.     

Src family tyrosine kinases differentially modulate exocytosis from rat brain nerve terminals

We have studied the role of src family tyrosine kinases in regulating synaptic transmitter release from rat brain synaptosomes by using two assays that measure different aspects of synaptic vesicle exocytosis: glutamate release (that directly measures exocytosis of vesicle contents) and release of FM 2-10 styryl dye (that is proportional to the time the synaptic vesicle is fused to the plasma membrane). Depolarisation was induced by KCl (30 mM) or 4-aminopyridine (4AP: 0.3mM) to induce release by full fusion (FF) exocytosis, or by 1 mM 4AP to induce release by both FF and kiss-and-run (KR)-like exocytosis. The src family selective inhibitor, PP1 (10 microM), increased KCl and 0.3 mM 4AP-evoked Ca2+ -dependent release of glutamate, but had little effect upon exocytosis evoked by 1mM 4AP. PP1 did not affect the release of FM 2-10 under any of the depolarisation conditions used. PP1 also had no effect on overall intracellular calcium levels, as measured by FURA2, suggesting that the effects of the inhibitor are downstream of calcium entry. At the same concentration the inactive analogue of this compound, PP3, had no effect on any measure. Immunoblotting with an antibody to phosphotyrosine revealed that phosphorylation of many synaptosomal proteins was reduced by PP1. The immunoreactivity of three protein bands increased upon depolarisation and this increase was blocked by PP1. Phosphorylation of src at tyrosine-416 was reduced by PP1 but changes in its phosphorylation did not correlate with the effects of PP1 on release. These results suggest one or more members of the src family of tyrosine kinases is a negative regulator of the KR mode of exocytosis in synaptosomes, perhaps by tonically inhibiting KR under normal stimulation conditions.     

The platelet activation induced by wheat germ agglutinin

In human platelets, wheat germ agglutinin (WGA) induced serotonin release without cell agglutination. WGA induced the phosphorylation of both 40-kDa and 20-kDa proteins in a parallel manner, and at least, the phosphorylation of 40-kDa protein was preceded by transient formation of endogenous diacylglycerol (DG) accompanied by a decrease in phosphatidylinositol (PI). Both phosphorylation of these two proteins and serotonin release were inhibited by prior treatment of platelets with dibutyryl cyclic AMP, W-7, or TMB-8. These results suggest that both phosphatidylinositol turnover and Ca2+ mobilization play an essential role in WGA-induced platelet activation.     

The calmodulin hypothesis of neurotransmission

Ca²⁺ plays a major role in neurotransmission and synaptic modulation. Evidence is presented to support the calmodulin hypothesis of neurotransmission developed in this laboratory stating that calmodulin, a major Ca²⁺ binding protein in brain, mediates the effects of Ca²⁺ on neurotransmission. Calmodulin was isolated from highly enriched preparations of synaptic vesicles and nerve terminal cytoplasm. Ca²⁺ and calmodulin were shown to regulate several synaptic processes in isolated and intact preparations, including endogenous synaptic Ca²⁺-calmodulin protein kinase activity, neurotransmitter release, and synaptic vesicle and synaptic membrane interactions. Ca²⁺ and calmodulin were shown to activate a synaptic tubulin kinase system which was shown to be a distinct enzyme system from the cyclic AMP protein kinase. Ca²⁺ and calmodulin stimulated phosphorylation of tubulin altered the properties of tubulin, forming insoluble tubulin fibrils. Evidence for the role of Ca²⁺-calmodulin kinase activity, especially the calmodulin-tubulin kinase, in neurotransmission are presented. The effects of several neuroactive drugs on the synaptic calmodulin system are presented. The results support the hypothesis that calmodulin mediates many of calcium's actions at the synapse, and that the effects of Ca²⁺ on synaptic protein phosphorylation, especially synaptic tubulin, may provide a biochemical mechanism for converting the Ca²⁺ signal into a motor force in the process of neurotransmission.     

A possible role of the phosphorylation of synaptic membrane proteins in the control of calcium ion permeability

Incubation of synaptosomes under conditions which result in complete phosphorylation of membrane bound accepter proteins does not affect the permeability to Na⁺ or K⁺ as measured by a spectrophotometric method. This technique was not, however, sensitive enough to determine permeability to Ca²⁺ which was thus estimated using ⁴⁵Ca²⁺. It was found that although phosphorylation did not affect the equilibrium binding of ⁴⁵Ca it did lower the rate of both Ca²⁺ uptake and efflux. The most likely interpretation of these results is that phosphorylation of proteins in the synaptic membrane lowers the permeability of the membrane to Ca²⁺. This could have a role in the regulation of synaptic transmission.