Phosphorylation of a synaptic vesicle-associated protein by an inositol hexakisphosphate-regulated protein kinase

Despite the fact that inositol hexakisphosphate (InsP(6)) is the most abundant inositol metabolite in cells, its cellular function has remained an enigma. In the present study, we present the first evidence of a protein kinase identified in rat cerebral cortex/hippocampus that is activated by InsP(6). The substrate for the InsP(6)-regulated protein kinase was found to be the synaptic vesicle-associated protein, pacsin/syndapin I. This brain-specific protein, which is highly enriched at nerve terminals, is proposed to act as a molecular link coupling components of the synaptic vesicle endocytic machinery to the cytoskeleton. We show here that the association between pacsin/syndapin I and dynamin I can be increased by InsP(6)-dependent phosphorylation of pacsin/syndapin I. These data provide a model by which InsP(6)-dependent phosphorylation regulates synaptic vesicle recycling by increasing the interaction between endocytic proteins at the synapse.     

A cerebellar Purkinje cell marker P400 protein is an inositol 1,4,5-trisphosphate (InsP3) receptor protein. Purification and characterization of InsP3 receptor complex.

P400 protein is a 250 kd glycoprotein, characteristic of the cerebellum, which is accumulated at the endoplasmic reticulum, at the plasma membrane and at the post-synaptic density of Purkinje cells. In this study, we purified inositol 1,4,5-trisphosphate (InsP3) receptor from mouse cerebellum and examined the possibility that P400 protein is identical with cerebellar InsP3 receptor protein. InsP3 receptor was solubilized with Triton X-100 from a post-nuclear fraction of ddY mouse cerebellum and was purified with high yield by sequential column chromatography on DE52, heparin-agarose, lentil lectin-Sepharose and hydroxylapatite. In these chromatographies, P400 protein co-migrated completely with the InsP3 binding activity. The purified receptor is a 250 kd protein with a Bmax of 2.1 pmol/microgram and a KD of 83 nM. It reacted with three different monoclonal antibodies against P400 protein, indicating that P400 protein is the same substance as the InsP3 receptor (P400/InsP3 receptor protein). Electron microscopy of the purified receptor showed a square shape with sides approximately 25 nm long. Binding assays of the cerebella of Purkinje cell-degeneration (pcd) mice with [3H]InsP3 demonstrated that the InsP3 binding sites in the cerebellum are distributed exclusively on the Purkinje cells. Immunohistochemical analysis indicated that P400/InsP3 receptor is present at the dendrites, cell bodies, axons and synaptic boutons of the Purkinje cells.     

Ryanodine and inositol trisphosphate receptors coexist in avian cerebellar Purkinje neurons

Two intracellular calcium-release channel proteins, the inositol trisphosphate (InsP3), and ryanodine receptors, have been identified in mammalian and avian cerebellar Purkinje neurons. In the present study, biochemical and immunological techniques were used to demonstrate that these proteins coexist in the same avian Purkinje neurons, where they have different intracellular distributions. Western analyses demonstrate that antibodies produced against the InsP3 and the ryanodine receptors do not cross-react. Based on their relative rates of sedimentation in continuous sucrose gradients and SDS-PAGE, the avian cerebellar InsP3 receptor has apparent native and subunit molecular weights of approximately 1,000 and 260 kD, while those of the ryanodine receptors are approximately 2,000 and 500 kD. Specific [3H]InsP3- and [3H]ryanodine-binding activities were localized in the sucrose gradient fractions enriched in the 260-kD and the approximately 500-kD polypeptides, respectively. Under equilibrium conditions, cerebellar microsomes bound [3H]InsP3 with a Kd of 16.8 nM and Bmax of 3.8 pmol/mg protein; whereas, [3H]ryanodine was bound with a Kd of 1.5 nM and a capacity of 0.08 pmol/mg protein. Immunolocalization techniques, applied at both the light and electron microscopic levels, revealed that the InsP3 and ryanodine receptors have overlapping, yet distinctive intracellular distributions in avian Purkinje neurons. Most notably the InsP3 receptor is localized in endomembranes of the dendritic tree, in both the shafts and spines. In contrast, the ryanodine receptor is observed in dendritic shafts, but not in the spines. Both receptors appear to be more abundant at main branch points of the dendritic arbor. In Purkinje neuron cell bodies, both the InsP3 and ryanodine receptors are present in smooth and rough ER, subsurface membrane cisternae and to a lesser extent in the nuclear envelope. In some cases the receptors coexist in the same membranes. Neither protein is observed at the plasma membrane, Golgi complex or mitochondrial membranes. Both the InsP3 and ryanodine receptors are associated with intracellular membrane systems in axonal processes, although they are less abundant there than in dendrites. These data demonstrate that InsP3 and ryanodine receptors exist as unique proteins in the same Purkinje neuron. These calcium-release channels appear to coexist in ER membranes in most regions of the Purkinje neurons, but importantly they are differentially distributed in dendritic processes, with the dendritic spines containing only InsP3 receptors.     

SV2 mediates entry of tetanus neurotoxin into central neurons

Tetanus neurotoxin causes the disease tetanus, which is characterized by rigid paralysis. The toxin acts by inhibiting the release of neurotransmitters from inhibitory neurons in the spinal cord that innervate motor neurons and is unique among the clostridial neurotoxins due to its ability to shuttle from the periphery to the central nervous system. Tetanus neurotoxin is thought to interact with a high affinity receptor complex that is composed of lipid and protein components; however, the identity of the protein receptor remains elusive. In the current study, we demonstrate that toxin binding, to dissociated hippocampal and spinal cord neurons, is greatly enhanced by driving synaptic vesicle exocytosis. Moreover, tetanus neurotoxin entry and subsequent cleavage of synaptobrevin II, the substrate for this toxin, was also dependent on synaptic vesicle recycling. Next, we identified the potential synaptic vesicle binding protein for the toxin and found that it corresponded to SV2; tetanus neurotoxin was unable to cleave synaptobrevin II in SV2 knockout neurons. Toxin entry into knockout neurons was rescued by infecting with viruses that express SV2A or SV2B. Tetanus toxin elicited the hyper excitability in dissociated spinal cord neurons - due to preferential loss of inhibitory transmission - that is characteristic of the disease. Surprisingly, in dissociated cortical cultures, low concentrations of the toxin preferentially acted on excitatory neurons. Further examination of the distribution of SV2A and SV2B in both spinal cord and cortical neurons revealed that SV2B is to a large extent localized to excitatory terminals, while SV2A is localized to inhibitory terminals. Therefore, the distinct effects of tetanus toxin on cortical and spinal cord neurons are not due to differential expression of SV2 isoforms. In summary, the findings reported here indicate that SV2A and SV2B mediate binding and entry of tetanus neurotoxin into central neurons.     

Immunocytochemistry of the olfactory marker protein.

The olfactory marker protein has been localized, by means of immunohistochemical techniques in the primary olfactory neurons of mice. The olfactory marker protein is not present in the staminal cells of the olfactory neuroepithelium, and the protein may be regarded as indicative of the functional stage of the neurons. Our data indicate that the olfactory marker protein is present in the synaptic terminals of the olfactory neurons at the level of the olfactory bulb glomeruli. The postsynaptic profiles of both mitral and periglomerular cells are negative.     

Localization of inositol 1,4,5-trisphosphate receptor-like protein in plasmalemmal caveolae

Activation of various receptors by extracellular ligands induces an influx of Ca2+ through the plasma membrane, but its molecular mechanism remains elusive and seems variable in different cell types. In the present study, we utilized mAbs generated against the cerebellar type I inositol 1,4,5-trisphosphate (InsP3) receptor and performed immunocytochemical and immunochemical experiments to examine its localization in several non-neuronal cells. By immunogold electron microscopy of ultrathin frozen sections as well as permeabilized tissue specimens, we found that a mAb to the type I InsP3 receptor (mAb 4C11) labels the plasma membrane of the endothelium, smooth muscle cell and keratinocyte in vivo. Interestingly, the labeling with the antibody was confined to caveolae, smooth vesicular inpocketings of the plasma membrane. The reactive protein, with an M(r) of 240,000 by SDS-PAGE, could be biotinylated with a membrane-impermeable reagent, sulfo-NHS-biotin, in intact cultured endothelial cells, and recovered by streptavidin-agarose beads, which result further confirmed its presence on the cell surface. The present findings indicate that a protein structurally homologous to the type I InsP3 receptor is localized in the caveolar structure of the plasma membrane and might be involved in the Ca2+ influx.     

Targeted disruption of Sept3, a heteromeric assembly partner of Sept5 and Sept7 in axons, has no effect on developing CNS neurons

The septins constitute a family of GTPase proteins that are involved in many cytological processes such as cytokinesis and exocytosis. Previous studies have indicated that mammalian Sept3 is a brain-specific protein that is abundant in synaptic terminals. Here, we further investigated the localization and function of Sept3 in the mouse brain. Sept3 is expressed in several types of post-mitotic neurons, including granule cells in the cerebellum and pyramidal neurons in the cerebral cortex and hippocampus. In primary cultures of hippocampal pyramidal neurons, Sept3 protein is enriched at the tips of growing neurites during differentiation. Sept3 directly binds to Sept5 and Sept7 and forms a heteromeric complex at nerve terminals adjacent to where a synaptic vesicle marker, synaptophysin, is expressed in mature neurons. When over-expressed in HEK293 cells, Sept3 forms filamentous structures that are dependent on the presence of its GTP- and phosphoinositide-binding domains. To investigate the physiological roles of Sept3, we generated Sept3 deficient mice. These mice show no apparent abnormalities in histogenesis nor neuronal differentiation in culture. Expression of synaptic proteins and other septins are unaltered, indicating that Sept3 is dispensable for normal neuronal development.     

Translational mobility of the type 3 inositol 1,4,5-trisphosphate receptor Ca2+ release channel in endoplasmic reticulum membrane

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an integral membrane protein in the endoplasmic reticulum (ER) which functions as a ligand-gated Ca2+ release channel. InsP3-mediated Ca2+ release modulates the cytoplasmic free Ca2+ concentration ([Ca2+]i), providing a ubiquitous intracellular signal with high temporal and spatial specificity. Precise localization of the InsP3R is believed to be important for providing local [Ca2+] regulation and for ensuring efficient functional coupling between Ca2+ release sites by enabling graded recruitment of channels with increasing stimulus strength in the face of the intrinsically unstable regenerative process of Ca2+-induced Ca2+ release. Highly localized Ca2+ release has been attributed to the ability of the InsP3R channels to cluster and to be localized to discrete areas, suggesting that mechanisms may exist to restrict their movement. Here, we examined the lateral mobility of the type 3 isoform of the InsP3R (InsP3R3) in the ER membrane by performing confocal fluorescence recovery after photobleaching of an InsP3R3 with green fluorescent protein fused to its N terminus. In Chinese hamster ovary and COS-7 cells, the diffusion coefficient D was approximately 4 x 10(-10) cm2/s at room temperature, a value similar to that determined for other ER-localized integral membrane proteins, with a high fraction (approximately 75%) of channels mobile. D was modestly increased at 37 degrees C, and it as well as the mobile fraction were reversibly reduced by ATP depletion. Although disruption of the actin cytoskeleton (latrunculin) was without effect, disruption of microtubules (nocodazole) reduced D by half without affecting the mobile fraction. We conclude that the entire ER is continuous in these cells, with the large majority of InsP3R3 channels free to diffuse throughout it, at rates that are comparable with those measured for other polytopic ER integral membrane proteins. The observed InsP3R3 mobility may be higher than its intrinsic diffusional mobility because of additional ATP- and microtubule-facilitated motility of the channel.     

Glutamate-induced declustering of post-synaptic adaptor protein Cupidin (Homer 2/vesl-2) in cultured cerebellar granule cells

Cupidin (Homer 2/vesl-2) is a post-synaptic adaptor protein that associates with glutamate receptor complexes and the actin cytoskeleton. We analyzed the developmental and activity-dependent localization of Cupidin in mouse cerebellar granule cells. Cupidin is predominantly localized to granule cell post-synapses connecting with mossy fiber terminals in developing post-natal cerebellum, but is diminished in adult cerebellum. In cultured granule cells 7 days in vitro, Cupidin was present as synaptic and extra-synaptic punctate clusters that largely co-localized with the actin-cytoskeletal binding partners F-actin and drebrin, as well as a post-synaptic scaffold protein PSD-95. Upon stimulation with glutamate, Cupidin clusters were rapidly dissociated without protein degradation, and by short-term but not sustained stimulation they were recovered after post-incubation without glutamate. The glutamate-induced declustering of Cupidin preceded that of F-actin and drebrin, was elicited by NMDA receptor-mediated Ca2+ influx, and was followed by a downstream pathway including MAPK/ERK and protein tyrosine kinase. Specific isoforms with post-translational modification were reduced depending on Ca2+-dependent protein phosphatase activity. In cultured hippocampal neurons, Homer family members Homer 1, Cupidin/Homer 2 and Homer 3 showed similar glutamate-induced declustering. We suggest that Cupidin acts as a mobile adaptor protein that changes the distribution states, clustered versus declustered, in response to synaptic activity.     

Inositol trisphosphate and ryanodine receptors share a common functional Ca2+ pool in cerebellar Purkinje neurons

Changes in the intracellular free calcium concentration ([Ca2+]i) control many important processes in excitable and nonexcitable cells. In cerebellar Purkinje neurons, increases in [Ca2+]i modulate excitability by turning on calcium-activated potassium and chloride conductances, and modifying the synaptic efficacy of inhibitory and excitatory inputs to the cell. Calcium release from the intracellular stores plays an important role in the regulation of [Ca2+]i. Purkinje neurons contain both inositol trisphosphate (InsP3) and ryanodine (Ry) receptors. With the exception of the dendritic spines, where only InsP3 receptors are found, InsP3 and Ry receptors are present in the entire cell. The distribution of the two calcium release channels, however, is not uniform, and it has been suggested that InsP3 and Ry receptors use separate Ca2+ pools. The functional properties of InsP3 and Ry Ca2+ pools were investigated by flash photolysis and single-cell microspectrofluorimetry. It was found that depletion of ryanodine-sensitive Ca2+ stores renders InsP3 incapable of releasing more Ca2+ from the stores. Abolishing calcium-induced calcium release by blocking ryanodine receptors with ruthenium red did not have a significant effect on InsP3-evoked Ca2+ release. It is concluded that InsP3 receptors use the same functional Ca2+ pool as that utilized by Ry receptors in Purkinje neurons.     

ARIA is concentrated in the synaptic basal lamina of the developing chick neuromuscular junction

ARIA is a member of a family of polypeptide growth and differentiation factors that also includes glial growth factor (GGF), neu differentiation factor, and heregulin. ARIA mRNA is expressed in all cholinergic neurons of the central nervous systems of rats and chicks, including spinal cord motor neurons. In vitro, ARIA elevates the rate of acetylcholine receptor incorporation into the plasma membrane of primary cultures of chick myotubes. To study whether ARIA may regulate the synthesis of junctional synaptic acetylcholine receptors in chick embryos, we have developed riboprobes and polyclonal antibody reagents that recognize isoforms of ARIA that include an amino-terminal immunoglobulin C2 domain and examined the expression and distribution of ARIA in motor neurons and at the neuromuscular junction. We detected significant ARIA mRNA expression in motor neurons as early as embryonic day 5, around the time that motor axons are making initial synaptic contacts with their target muscle cells. In older embryos and postnatal animals, we found ARIA protein concentrated in the synaptic cleft at neuromuscular junctions, consistent with transport down motor axons and release at nerve terminals. At high resolution using immunoelectron microscopy, we detected ARIA immunoreactivity exclusively in the synaptic basal lamina in a pattern consistent with binding to synapse specific components on the presynaptic side of the basal lamina. These results support a role for ARIA as a trophic factor released by motor neuron terminals that may regulate the formation of mature neuromuscular synapses.     

Glucose increases synaptic transmission from vagal afferent central nerve terminals via modulation of 5-HT3 receptors

Acute hyperglycemia has profound effects on vagally mediated gastrointestinal functions. We have reported recently that the release of glutamate from the central terminals of vagal afferent neurons is correlated directly with the extracellular glucose concentration. The present study was designed to test the hypothesis that 5-HT(3) receptors present on vagal afferent nerve terminals are involved in this glucose-dependent modulation of glutamatergic synaptic transmission. Whole-cell patch-clamp recordings were made from neurons of the nucleus tractus solitarius (NTS) in thin rat brainstem slices. Spontaneous and evoked glutamate release was decreased in a concentration-dependent manner by the 5-HT(3) receptor selective antagonist, ondansetron. Alterations in the extracellular glucose concentration induced parallel shifts in the ondansetron-mediated inhibition of glutamate release. The changes in excitatory synaptic transmission induced by extracellular glucose concentration were mimicked by the serotonin uptake inhibitor, fenfluramine. These data suggest that glucose alters excitatory synaptic transmission within the rat brainstem via actions on tonically active 5-HT(3) receptors, and the number of 5-HT(3) receptors on vagal afferent nerve terminals is positively correlated with the extracellular glucose concentration. These data indicate that the 5-HT(3) receptors present on synaptic connections between vagal afferent nerve terminals and NTS neurons are a strong candidate for consideration as one of the sites where glucose acts to modulate vagovagal reflexes.     

Synaptic plasticity and functionality at the cone terminal of the developing zebrafish retina

Previous studies have analyzed photoreceptor development, some inner retina cell types, and specific neurotransmitters in the zebrafish retina. However, only minor attention has been paid to the morphology of the synaptic connection between photoreceptors and second order neurons even though it represents the transition from the light sensitive receptor to the neuronal network of the visual system. Here, we describe the appearance and differentiation of pre- and postsynaptic elements at cone synapses in the developing zebrafish retina together with the maturation of the directly connecting second order neurons and a dopaminergic third order feedback-neuron from the inner retina. Zebrafish larvae were examined at developmental stages from 2 to 7dpf (days postfertilization) and in the adult. Synaptic maturation at the photoreceptor terminals was examined with antibodies against synapse associated proteins. The appearance of synaptic plasticity at the so-called spinule-type synapses between cones and horizontal cells was assessed by electron microscopy, and the maturation of photoreceptor downstream connection was identified by immunocytochemistry for GluR4 (AMPA-type glutamate receptor subunit), protein kinase beta(1) (mixed rod-cone bipolar cells), and tyrosine hydroxylase (dopaminergic interplexiform cells). We found that developing zebrafish retinas possess first synaptic structures at the cone terminal as early as 3.5dpf. Morphological maturation of these synapses at 3.5-4dpf, together with the presence of synapse associated proteins at 2.5dpf and the maturation of second order neurons by 5dpf, indicate functional synaptic connectivity and plasticity between the cones and their second order neurons already at 5dpf. However, the mere number of spinules and ribbons at 7dpf still remains below the adult values, indicating that synaptic functionality of the zebrafish retina is not entirely completed at this stage of development.     

Regulation of presynaptic phosphatidylinositol 4,5-biphosphate by neuronal activity

Phosphatidylinositol 4,5-biphosphate (PIP2) has been implicated in a variety of cellular processes, including synaptic vesicle recycling. However, little is known about the spatial distribution of this phospholipid in neurons and its dynamics. In this study, we have focused on these questions by transiently expressing the phospholipase C (PLC)-delta1 pleckstrin homology (PH) domain fused to green fluorescent protein (GFP) in cultured hippocampal neurons. This PH domain binds specifically and with high affinity to PIP2. Live confocal imaging revealed that in resting cells, PH-GFP is localized predominantly on the plasma membrane. Interestingly, no association of PH-GFP with synaptic vesicles in quiescent neurons was observed, indicating the absence of detectable PIP2 on mature synaptic vesicles. Electrical stimulation of hippocampal neurons resulted in a decrease of the PH-GFP signal at the plasma membrane, most probably due to a PLC-mediated hydrolysis of PIP2. This was accompanied in the majority of presynaptic terminals by a marked increase in the cytoplasmic PH-GFP signal, localized most probably on freshly endocytosed membranes. Further investigation revealed that the increase in PH-GFP signal was dependent on the activation of N-methyl-D-aspartate receptors and the consequent production of nitric oxide (NO). Thus, PIP2 in the presynaptic terminal appears to be regulated by postsynaptic activity via a retrograde action of NO.     

GRAB: a physiologic guanine nucleotide exchange factor for Rab3A, which interacts with inositol hexakisphosphate kinase

Diphosphoinositol-pentakisphosphate (InsP7) and bis-diphosphoinositol tetrakisphosphate (InsP8) possess pyrophosphate bonds. InsP7 is formed from inositol hexakisphosphate (InsP6) by recently identified InsP6 kinases designated InsP6K1 and InsP6K2. We now report the identification, cloning, and characterization of a novel protein, GRAB (guanine nucleotide exchange factor for Rab3A), which interacts with both InsP6K1 and Rab3A, a Ras-like GTPase that regulates synaptic vesicle exocytosis. GRAB is a physiologic GEF (guanine nucleotide exchange factor) for Rab3A. Consistent with a role of Rab3A in synaptic vesicle exocytosis, GRAB regulates depolarization-induced release of dopamine from PC12 cells and nicotinic agonist-induced hGH release from bovine adrenal chromaffin cells. The association of InsP6K1 with GRAB fits with a role for InsP7 in vesicle exocytosis.     

Cardiac type 2 inositol 1,4,5-trisphosphate receptor: interaction and modulation by calcium/calmodulin-dependent protein kinase II

The type 2 inositol 1,4,5-trisphosphate receptor (InsP(3)R2) was identified previously as the predominant isoform in cardiac ventricular myocytes. Here we reported the subcellular localization of InsP(3)R2 to the cardiomyocyte nuclear envelope (NE). The other major known endo/sarcoplasmic reticulum calcium-release channel (ryanodine receptor) was not localized to the NE, indicating functional segregation of these channels and possibly a unique role for InsP(3)R2 in regulating nuclear calcium dynamics. Immunoprecipitation experiments revealed that the NE InsP(3)R2 associates with Ca(2+)/calmodulin-dependent protein kinase IIdelta (CaMKIIdelta), the major isoform expressed in cardiac myocytes. Recombinant InsP(3)R2 and CaMKIIdelta(B) also co-immunoprecipitated after co-expression in COS-1 cells. Additionally, the amino-terminal 1078 amino acids of the InsP(3)R2 were sufficient for interaction with CaMKIIdelta(B) and associated upon mixing following separate expression. CaMKII can also phosphorylate InsP(3)R2, as demonstrated by (32)P labeling. Incorporation of CaMKII-treated InsP(3)R2 into planar lipid bilayers revealed that InsP(3)-mediated channel open probability is significantly reduced ( approximately 11 times) by phosphorylation via CaMKII. We concluded that the InsP(3)R2 and CaMKIIdelta likely represent two central components of a multiprotein signaling complex, and this raises the possibility that calcium release via InsP(3)R2 in the myocyte NE may activate local CaMKII signaling, which may feedback on InsP(3)R2 function.     

Mitochondrial malic enzyme activity is much higher in mitochondria from cortical synaptic terminals compared with mitochondria from primary cultures of cortical neurons or cerebellar granule cells

Most of the malic enzyme activity in the brain is found in the mitochondria. This isozyme may have a key role in the pyruvate recycling pathway which utilizes dicarboxylic acids and substrates such as glutamine to provide pyruvate to maintain TCA cycle activity when glucose and lactate are low. In the present study we determined the activity and kinetics of malic enzyme in two subfractions of mitochondria isolated from cortical synaptic terminals, as well as the activity and kinetics in mitochondria isolated from primary cultures of cortical neurons and cerebellar granule cells. The synaptic mitochondrial fractions had very high mitochondrial malic enzyme (mME) activity with a Km and a Vmax of 0.37 mM and 32.6 nmol/min/mg protein and 0.29 mM and 22.4 nmol/min mg protein, for the SM2 and SM1 fractions, respectively. The Km and Vmax for malic enzyme activity in mitochondria isolated from cortical neurons was 0.10 mM and 1.4 nmol/min/mg protein and from cerebellar granule cells was 0.16 mM and 5.2 nmol/min/mg protein. These data show that mME activity is highly enriched in cortical synaptic mitochondria compared to mitochondria from cultured cortical neurons. The activity of mME in cerebellar granule cells is of the same magnitude as astrocyte mitochondria. The extremely high activity of mME in synaptic mitochondria is consistent with a role for mME in the pyruvate recycling pathway, and a function in maintaining the intramitochondrial reduced glutathione in synaptic terminals.     

Mechanism of Ca2+ inhibition of inositol 1,4,5-trisphosphate (InsP3) binding to the cerebellar InsP3 receptor.

Ca2+ efficiently inhibits binding of inositol 1,4,5-trisphosphate (InsP3) to the InsP3 receptor in cerebellar membranes but not to the purified receptor. We have now investigated the mechanism of action by which Ca2+ inhibits InsP3 binding. Our results suggest that Ca2+ does not cause the stable association of a Ca(2+)-binding protein with the receptor. Instead, Ca2+ leads to the production of a soluble, heat-stable, low molecular weight substance from cerebellar membranes that competes with InsP3 for binding. This inhibitory substance probably represents endogenously generated InsP3 as judged by the fact that it co-purifies with InsP3 on anion-exchange chromatography, competes with [3H]InsP3 binding in a pattern similar to unlabeled InsP3, and is in itself capable of releasing 45Ca2+ from permeabilized cells. A potent Ca(2+)-activated phospholipase C activity producing InsP3 was found in cerebellar microsomes that exhibited a Ca2+ dependence identical to the Ca(2+)-dependent inhibition of InsP3 binding. Together these results suggest that the Ca(2+)-dependent inhibition of InsP3 binding to the cerebellar receptor is due to activation of a Ca(2+)-sensitive phospholipase C enriched in cerebellum. Nevertheless, Ca2+ probably also modulates the InsP3 receptor function by a direct interaction with the receptor that does not affect InsP3 binding but regulates InsP3-dependent channel gating.     

Recordings from neuron–HEK cell cocultures reveal the determinants of miniature excitatory postsynaptic currents

Spontaneous exocytosis of single synaptic vesicles generates miniature synaptic currents, which provide a window into the dynamic control of synaptic transmission. To resolve the impact of different factors on the dynamics and variability of synaptic transmission, we recorded miniature excitatory postsynaptic currents (mEPSCs) from cocultures of mouse hippocampal neurons with HEK cells expressing the postsynaptic proteins GluA2, neuroligin 1, PSD-95, and stargazin. Synapses between neurons and these heterologous cells have a molecularly defined postsynaptic apparatus, while the compact morphology of HEK cells eliminates the distorting effect of dendritic filtering. HEK cells in coculture produced mEPSCs with a higher frequency, larger amplitude, and more rapid rise and decay than neurons from the same culture. However, mEPSC area indicated that nerve terminals in synapses with both neurons and HEK cells release similar populations of vesicles. Modulation by the glutamate receptor ligand aniracetam revealed receptor contributions to mEPSC shape. Dendritic cable effects account for the slower mEPSC rise in neurons, whereas the slower decay also depends on other factors. Lastly, expression of synaptobrevin transmembrane domain mutants in neurons slowed the rise of HEK cell mEPSCs, thus revealing the impact of synaptic fusion pores. In summary, we show that cocultures of neurons with heterologous cells provide a geometrically simplified and molecularly defined system to investigate the time course of synaptic transmission and to resolve the contribution of vesicles, fusion pores, dendrites, and receptors to this process.