Margaret E. Graham

Neuronal Ca2+ Sensor 1, the Mammalian Homologue of Frequenin, Is Expressed in Chromaffin and PC12 Cells and Regulates Neurosecretion from Dense-core Granules

Neuronal Ca2+ sensor 1 (NCS-1) is the mammalian homologue of the Ca2+-binding protein frequenin previously implicated in regulation of neurotransmission inDrosophila (Pongs, O., Lindemeier, J., Zhu, X. R., Theil, T., Endelkamp, D., Krah-Jentgens, I., Lambrecht, H.-G., Koch, K. W., Schwemer, J., Rivosecchi, R., Mallart, A., Galceran, J., Canal, I., Barbas, J. A., and Ferrus, A. (1993) Neuron11, 15–28). NCS-1 has been considered to be expressed only in neurons, but we show that NCS-1 expression can be detected in bovine adrenal chromaffin and PC12 cells, two widely studied model neuroendocrine cells. NCS-1 was present in both cytosolic and membrane fractions including purified chromaffin granules, and in immunofluorescence, its distribution overlapped with peripheral punctate staining seen with the synaptic-like microvesicle marker synaptophysin in PC12 cells. The possible functional role of NCS-1 in exocytosis of dense-core granules was tested using transient transfection in PC12 cells and assay of co-transfected growth hormone (GH) release. Overexpression of NCS-1 increased evoked GH release in intact cells in response to ATP. No effect of overexpression was seen on GH release because of Ca2+ in permeabilized cells suggesting that NCS-1 may have a regulatory but not direct role in neurosecretion.

Dynamin-dependent and dynamin-independent processes contribute to the regulation of single vesicle release kinetics and quantal size

Accumulating evidence suggests that the kinetics of release from single secretory vesicles can be regulated and that quantal size can be modified during fast kiss-and-run fusion. Multiple pathways for vesicle retrieval have been identified involving clathrin and dynamin. It has been unclear whether dynamin could participate in a fast kiss-and-run process to reclose a transient fusion pore and thereby limit vesicle release. We have disrupted dynamin function in adrenal chromaffin cells by expression of the amphiphysin Src-homology domain 3 (SH3) or by application of guanosine 5′-[γ-thio]triphosphate (GTPγS), and have monitored single vesicle release events, evoked by digitonin and Ca2+, by using carbon-fiber amperometry. Under both conditions, there was an increase in mean quantal size accompanying an increase in the half-width of amperometric spikes and a slowing of the fall time. These data suggest the existence of a dynamin-dependent process that can terminate vesicle release under basal conditions. Protein kinase C activation changed release kinetics and decreased quantal size by shortening the release period. The effects of phorbol ester treatment were not prevented by expression of the amphiphysin SH3 domain or by GTPγS suggesting the existence of alternative dynamin-independent process underlying fast kiss-and-run exocytosis.

Neurotrophic effects of NMDA receptor activation on developing cerebellar granule cells

SummaryGlutamate acting on N-methyl-D-aspartate (NMDA) receptors controls a variety of aspects of neuronal plasticity in the adult and developing brain. This review summarizes its effects on developing cerebellar granule cells. The glutamatergic mossy fibre input to cerebellar granule cells exerts a neurotrophic effect on these cells during development. The investigation of potential neurotrophic agents can be carried out using enriched granule cell cultures. Considerable evidence now indicates that glutamate acting on N-methyl-D-aspartate receptors is an important neurotrophic factor that regulates granule cell development. In culture, neurite growth, differentiation and cell survival are all stimulated by N-methyl-D-aspartate receptor activation. The intracellular pathways involved following Ca2+ entry through the N-methyl-D-aspartate receptor channel are beginning to be elucidated. The cerebellar granule cell culture system may provide an ideal model to investigate the molecular mechanisms involved in long term N-methyl-D-aspartate receptor-mediated changes in neuronal function.

Phosphorylation of cysteine string protein by protein kinase A. Implications for the modulation of exocytosis.

Cyclic AMP-dependent protein kinase (PKA) enhances regulated exocytosis in neurons and most other secretory cells. To explore the molecular basis of this effect, known exocytotic proteins were screened for PKA substrates. Both cysteine string protein (CSP) and soluble NSF attachment protein-α (α-SNAP) were phosphorylated by PKA in vitro, but immunoprecipitation of cellular α-SNAP failed to detect 32P incorporation. In contrast, endogenous CSP was phosphorylated in synaptosomes, PC12 cells, and chromaffin cells. In-gel kinase assays confirmed PKA to be a cellular CSP kinase, with phosphorylation occurring on Ser10. PKA phosphorylation of CSP reduced its binding to syntaxin by 10-fold but had little effect on its interaction with HSC70 or G-protein subunits. Furthermore, an in vivo role for Ser10 phosphorylation at a late stage of exocytosis is suggested by analysis of chromaffin cells transfected with wild type or non-phosphorylatable mutant CSP. We propose that PKA phosphorylation of CSP could modulate the exocytotic machinery, by selectively altering its availability for protein-protein interactions.

Cysteine residues of SNAP-25 are required for SNARE disassembly and exocytosis, but not for membrane targeting

The release of neurotransmitter at a synapse occurs via the regulated fusion of synaptic vesicles with the plasma membrane. The fusion of the two lipid bilayers is mediated by a protein complex that includes the plasma membrane target soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (t-SNAREs), syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25), and the vesicle SNARE (v-SNARE), vesicle-associated membrane protein (VAMP). Whereas syntaxin 1A and VAMP are tethered to the membrane by a C-terminal transmembrane domain, SNAP-25 has been suggested to be anchored to the membrane via four palmitoylated cysteine residues. We demonstrate that the cysteine residues of SNAP-25 are not required for membrane localization when syntaxin 1A is present. Analysis of the 7 S and 20 S complexes formed by mutants that lack cysteine residues demonstrates that the cysteines are required for efficient SNARE complex dissociation. Furthermore, these mutants are unable to support exocytosis, as demonstrated by a PC12 cell secretion assay. We hypothesize that syntaxin 1A serves to direct newly synthesized SNAP-25 through the Golgi transport pathway to the axons and synapses, and that palmitoylation of cysteine residues is not required for targeting, but to optimize interactions required for SNARE complex dissociation.

Measurement of exocytosis by amperometry in adrenal chromaffin cells: effects of clostridial neurotoxins and activation of protein kinase C on fusion pore kinetics.

We have used carbon-fibre amperometry to examine the kinetics of individual secretory granule fusion/release events in bovine adrenal chromaffin cells. Transfection with plasmids encoding the light chains of botulinum neurotoxins (BoNTs) was used to investigate the effects of cleavage of syntaxin or SNAP-25 on exocytosis. Expression of BoNT/C1 or BoNT/E inhibited the extent of exocytosis that was evoked by application of digitonin/Ca(2+) to permeabilise and stimulate single chromaffin cells. Following neurotoxin expression, the residual release events were no different from those of control cells in their magnitude and kinetics from analysis of the amperometric spikes. In contrast, activation of protein kinase C (PKC) resulted in a modification of the kinetics of single granule release events. Following phorbol ester treatment, the amperometric spikes showed a significant decrease in their total charge due to a decrease in their mean half-width with increases in the rate of the initial rise and also the fall to baseline of the spikes. These changes were prevented by pre-treatment with the PKC inhibitor bisindolylmaleimide. These results suggest that PKC regulates the rate of fusion pore expansion and also subsequent pore closure or granule retrieval. A PKC-mediated regulation of kiss-and-run fusion may, therefore, control the extent of catecholamine release from single secretory granules. The experimental approach used here may provide further information on the protein constituents and regulation of the fusion pore machinery.

A gain-of-function mutant of Munc18-1 stimulates secretory granule recruitment and exocytosis and reveals a direct interaction of Munc18-1 with Rab3

Munc18-1 plays a crucial role in regulated exocytosis in neurons and neuroendocrine cells through modulation of vesicle docking and membrane fusion. The molecular basis for Munc18 function is still unclear, as are the links with Rabs and SNARE [SNAP (soluble N-ethylmaleimide-sensitive factor-attachment protein) receptor] proteins that are also required. Munc18-1 can bind to SNAREs through at least three modes of interaction, including binding to the closed conformation of syntaxin 1. Using a gain-of-function mutant of Munc18-1 (E466K), which is based on a mutation in the related yeast protein Sly1p, we have identified a direct interaction of Munc18-1 with Rab3A, which is increased by the mutation. Expression of Munc18-1 with the E466K mutation increased exocytosis in adrenal chromaffin cells and PC12 cells (pheochromocytoma cells) and was found to increase the density of secretory granules at the periphery of PC12 cells, suggesting a stimulatory effect on granule recruitment through docking or tethering. Both the increase in exocytosis and changes in granule distribution appear to require Munc18-1 E466K binding to the closed form of syntaxin 1, suggesting a role for this interaction in bridging Rab- and SNARE-mediated events in exocytosis.

The functions of Munc18-1 in regulated exocytosis.

The activation of regulated exocytosis occurs by a rise in cytosolic Ca2+ concentration. Synaptotagmins act as the Ca2+ sensors, whereas the machinery that allows fusion of secretory vesicles with the plasma membrane consists of the soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) proteins, including syntaxin 1, SNAP‐25, and VAMP. Within the pathway leading to exocytosis, there is an essential requirement for a member of the conserved Sec1/Munc18 (SM) protein family, which in neurotransmitter and neurohormone release in mammalian cells is Munc18‐1. The exact role of Munc18‐1 and the steps within exocytosis in which it acts have been intensively investigated. Current evidence suggests that Munc18‐1 acts via distinct modes of interactions with syntaxin 1 and the other SNARE proteins and influences all of the steps leading to exocytosis, including vesicle recruitment, tethering, docking, priming, and membrane fusion.

S-nitrosylation of syntaxin 1 at Cys(145) is a regulatory switch controlling Munc18-1 binding

Exocytosis is regulated by NO in many cell types, including neurons. In the present study we show that syntaxin 1a is a substrate for S-nitrosylation and that NO disrupts the binding of Munc18-1 to the closed conformation of syntaxin 1a in vitro. In contrast, NO does not inhibit SNARE {SNAP [soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein] receptor} complex formation or binding of Munc18-1 to the SNARE complex. Cys(145) of syntaxin 1a is the target of NO, as a non-nitrosylatable C145S mutant is resistant to NO and novel nitrosomimetic Cys(145) mutants mimic the effect of NO on Munc18-1 binding in vitro. Furthermore, expression of nitrosomimetic syntaxin 1a in living cells affects Munc18-1 localization and alters exocytosis release kinetics and quantal size. Molecular dynamic simulations suggest that NO regulates the syntaxin-Munc18 interaction by local rearrangement of the syntaxin linker and H3c regions. Thus S-nitrosylation of Cys(145) may be a molecular switch to disrupt Munc18-1 binding to the closed conformation of syntaxin 1a, thereby facilitating its engagement with the membrane fusion machinery.

Activation of metabotropic glutamate receptors by L-AP4 stimulates survival of rat cerebellar granule cells in culture

The results presented here show that the metabotropic glutamate receptor agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4) is capable of markedly stimulating the survival of rat cerebellar granule cells in culture. This is the first demonstration of a neurotrophic role for metabotropic glutamate receptors. The survival promoting action of L-AP4 does not involve a large, rapid rise in [Ca2+]i which is seen with other neurotrophic agents in granule cells such as N-methyl-D-aspartate, ionomycin and high potassium. In addition, the survival-promoting effect of L-AP4 did not appear to be related to changes in cAMP levels. Survival due to L-AP4 was enhanced by pertussis toxin and by forskolin and was unaffected by inhibitors of cAMP-dependent protein kinase. Measurement of cAMP levels after long-term treatment with neurotrophic agents showed no clear relationship between cAMP concentration and granule cell survival. The mechanism of L-AP4 stimulated cell survival is unknown but seems unlikely to involve an acute rise in [Ca2+]i or modulation of cAMP levels. Survival induced by L-AP4 was not blocked by the antagonist (RS)-alpha-methyl-4-carboxyphenylglycine. Similarity in these properties with those of the mGLu7 receptor suggests that granule cell survival was stimulated by an mGlu7-like metabotropic receptor.