Munc13-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion

Munc13-4 is a widely expressed member of the CAPS/Munc13 protein family proposed to function in priming secretory granules for exocytosis. Munc13-4 contains N- and C-terminal C2 domains (C2A and C2B) predicted to bind Ca(2+), but Ca(2+)-dependent regulation of Munc13-4 activity has not been described. The C2 domains bracket a predicted SNARE-binding domain, but whether Munc13-4 interacts with SNARE proteins is unknown. We report that Munc13-4 bound Ca(2+) and restored Ca(2+)-dependent granule exocytosis to permeable cells (platelets, mast, and neuroendocrine cells) dependent on putative Ca(2+)-binding residues in C2A and C2B. Munc13-4 exhibited Ca(2+)-stimulated SNARE interactions dependent on C2A and Ca(2+)-dependent membrane binding dependent on C2B. In an apparent coupling of membrane and SNARE binding, Munc13-4 stimulated SNARE-dependent liposome fusion dependent on putative Ca(2+)-binding residues in both C2A and C2B domains. Munc13-4 is the first priming factor shown to promote Ca(2+)-dependent SNARE complex formation and SNARE-mediated liposome fusion. These properties of Munc13-4 suggest its function as a Ca(2+) sensor at rate-limiting priming steps in granule exocytosis.     

CAPS1 regulates catecholamine loading of large dense-core vesicles

CAPS1 is thought to play an essential role in mediating exocytosis from large dense-core vesicles (LDCVs). We generated CAPS1-deficient (KO) mice and studied exocytosis in a model system for Ca2+-dependent LDCV secretion, the adrenal chromaffin cell. Adult heterozygous CAPS1 KO cells display a gene dosage-dependent decrease of CAPS1 expression and a concomitant reduction in the number of docked vesicles and secretion. Embryonic homozygous CAPS1 KO cells show a strong reduction in the frequency of amperometrically detectable release events of transmitter-filled vesicles, while the total number of fusing vesicles, as judged by capacitance recordings or total internal reflection microscopy, remains unchanged. We conclude that CAPS1 is required for an essential step in the uptake or storage of catecholamines in LDCVs.     

CAPS in search of a lost function

Ca2+-dependent activator protein for secretion (CAPS) is an evolutionarily conserved secretory protein that was previously thought to mediate Ca2+-triggered fusion of dense-core vesicles. In an elegant study of CAPS1-deficient mice, Speidel et al. (this issue of Neuron) now show that CAPS function may have been misunderstood. CAPS appears to act upstream of fusion in the biogenesis or maintenance of mature secretory vesicles, raising the possibility of a completely new type of function for an essential component of the secretory machinery.     

Tissue distribution of Ca2+-dependent activator protein for secretion family members CAPS1 and CAPS2 in mice.

The family of Ca2+-dependent activator proteins for secretion (CAPS) is involved in dense-core vesicle exocytosis. CAPS1/CADPS1 and CAPS2/CADPS2 have been identified in mammals. CAPS1 regulates catecholamine release from neuroendocrine cells, whereas CAPS2 is involved in the release of brain-derived neurotrophic factor and neurotrophin-3 from cerebellar granule cells. CAPS1 and CAPS2 are predominantly expressed in brain. Here we show the immunohistochemical localization of the CAPS family proteins in various mouse tissues. In the pituitary gland, CAPS1 and CAPS2 were localized to the pars nervosa and the pars intermedia, respectively. In non-neural tissues, CAPS1 was observed in the islets of Langerhans, minor cell types of the spleen and stomach, and medullary cells of the adrenal gland, whereas CAPS2 was present in bronchial epithelial cells, thyroid parafollicular cells, chief cells of the stomach, ductal epithelium of the salivary gland, kidney proximal tubules, and minor cell types of the thymus, spleen, and colon. These results suggest that secretion from distinct cell types in various tissues involves either or both members of the CAPS family.     

Ca2+-dependent activator protein for secretion 1 is critical for constitutive and regulated exocytosis but not for loading of transmitters into dense core vesicles

Although CAPS1 was originally identified as a soluble factor that reconstitutes Ca(2+)-dependent secretion from permeabilized neuroendocrine cells, its exact function in intact mammalian cells remains controversial. Here we investigate the role for CAPS1 by generating stable cell lines in which CAPS1 is strongly down-regulated. In these cells, Ca(2+)-dependent secretion was strongly reduced not only of catecholamine but also of a transfected neuropeptide. These secretion defects were rescued by infusion of CAPS1-containing brain cytosol or by transfection-mediated expression of CAPS1. Whole cell patch clamp recording revealed significant reductions in slow burst and sustained release components of exocytosis in the knockdown cells. Unexpectedly, they also accumulated higher amounts of endogenous and exogenous transmitters, which were attributable to reductions in constitutive secretion. Electron microscopy did not reveal abnormalities in the number or docking of dense core vesicles. Our results indicate that CAPS1 plays critical roles not only in Ca(2+)-dependent, regulated exocytosis but also in constitutive exocytosis downstream of vesicle docking. However, they do not support the role for CAPS1 in loading transmitters into dense core vesicles.     

Munc13 homology domain-1 in CAPS/UNC31 mediates SNARE binding required for priming vesicle exocytosis

Neuropeptide and peptide hormone secretion from neural and endocrine cells occurs by Ca(2+)-triggered dense-core vesicle exocytosis. The membrane fusion machinery consisting of vesicle and plasma membrane SNARE proteins needs to be assembled for Ca(2+)-triggered vesicle exocytosis. The related Munc13 and CAPS/UNC31 proteins that prime vesicle exocytosis are proposed to promote SNARE complex assembly. CAPS binds SNARE proteins and stimulates SNARE complex formation on liposomes, but the relevance of SNARE binding to CAPS function in cells had not been determined. Here we identify a core SNARE-binding domain in CAPS as corresponding to Munc13 homology domain-1 (MHD1). CAPS lacking a single helix in MHD1 was unable to bind SNARE proteins or to support the Ca(2+)-triggered exocytosis of either docked or newly arrived dense-core vesicles. The results show that MHD1 is a SNARE-binding domain and that SNARE protein binding is essential for CAPS function in dense-core vesicle exocytosis.     

CAPS Activity in Priming Vesicle Exocytosis Requires CK2 Phosphorylation

CAPS (Ca²⁺-dependent activator protein for secretion) functions in priming Ca²⁺-dependent vesicle exocytosis, but the regulation of CAPS activity has not been characterized. Here we show that phosphorylation by protein kinase CK2 is required for CAPS activity. Dephosphorylation eliminated CAPS activity in reconstituting Ca²⁺-dependent vesicle exocytosis in permeable and intact PC12 cells. Ser-5, -6, and -7 and Ser-1281 were identified by mass spectrometry as the major phosphorylation sites in the 1289 residue protein. Ser-5, -6, and -7 but not Ser-1281 to Ala substitutions abolished CAPS activity. Protein kinase CK2 phosphorylated CAPS in vitro at these sites and restored the activity of dephosphorylated CAPS. CK2 is the likely in vivo CAPS protein kinase based on inhibition of phosphorylation by tetrabromo-2-benzotriazole in PC12 cells and by the identity of in vivo and in vitro phosphorylation sites. CAPS phosphorylation by CK2 was constitutive, but the elevation of Ca²⁺ in synaptosomes increased CAPS Ser-5 and -6 dephosphorylation, which terminates CAPS activity. These results identify a functionally important N-terminal phosphorylation site that regulates CAPS activity in priming vesicle exocytosis.Regulated neurotransmitter secretion is central to intercellular communication in the nervous system. Two types of secretory vesicles mediate neurotransmitter release; that is, synaptic vesicles that release transmitters such as glutamate at synapses and dense-core vesicles that release modulatory transmitters and neuropeptides at non-synaptic sites. Both types of secretory vesicles are recruited to docking sites on the plasma membrane where they are primed to a ready release state to undergo fusion in response to Ca²⁺ elevations. Many of the proteins that mediate the targeting, docking, priming, and Ca²⁺-dependent fusion of vesicles with the plasma membrane function in both synaptic vesicle and dense-core vesicle pathways (1). CAPS-1² (also known as Cadps1) is a 1289-residue protein that reconstitutes Ca²⁺-triggered dense-core vesicle exocytosis in permeable neuroendocrine cells at a priming step (2–4). CAPS is required for secretion of a subset of transmitters in Caenorhabditis elegans (5) and Drosophila melanogaster (6) and for priming dense-core vesicle exocytosis in neuroendocrine cells (7) and synaptic vesicle exocytosis in neurons (8). Vesicle priming reactions are extensively modulated during physiological demand (9), but mechanisms that regulate CAPS function remain to be identified.Reversible protein phosphorylation is a major mechanism for the regulation of cellular processes including vesicle exocytosis. Many proteins that function in evoked vesicle exocytosis are phosphoproteins (10, 11). The neuronal SNARE proteins syntaxin 1A, VAMP-2, and SNAP-25 are phosphorylated by several protein kinases in vitro (12–14). Protein kinase C and protein kinase A sites on SNAP-25 affect refilling rates and size, respectively, of the primed pool of vesicles in chromaffin cells (15, 16). Several SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-binding proteins such as munc18, RIM1, and rabphilin undergo regulated phosphorylation, but it is not known whether phosphorylation affects function (10, 11, 17).Because the function of CAPS at a priming step in vesicle exocytosis may be regulated, we determined whether CAPS is phosphorylated. We show that CAPS is a phosphoprotein with functionally essential N-terminal phosphorylated Ser residues. Ser-5, -6, and -7 in CAPS were substrates for protein kinase CK2 in vitro and in vivo as well as for a Ca²⁺-dependent dephosphorylation mechanism. The results indicate that phosphorylation by protein kinase CK2 is necessary for CAPS activity in priming vesicle exocytosis and that regulated dephosphorylation may constitute a mechanism for terminating CAPS activity.     

The Anx7(+/-) knockout mutation alters electrical and secretory responses to Ca(2+)-mobilizing agents in pancreatic β-cells

Insulin secretion from the pancreatic β-cell is controlled by changes in membrane potential and intracellular Ca(2+). The contribution of intracellular Ca(2+) stores to this process is poorly understood. We have previously shown that β-cells of mice lacking one copy of the Annexin 7 gene (Anx7(+/-)) express reduced levels of IP(3) receptors and defects in IP(3)-dependent Ca(2+) signaling. To further elucidate the effect of the Anx7(+/-) mutation on signaling related to intracellular Ca(2+) stores in the β-cell, we measured the effects of Ca(2+) mobilizing agents on electrical activity, intracellular Ca(2+) and insulin secretion in control and mutant β-cells. We found that the muscarinic agonist carbachol and the ryanodine receptor agonists caffeine and 4-chloro-m-cresol had more potent depolarizing effects on Anx7(+/-) β-cells compared to controls. Accordingly, glucose-induced insulin secretion was augmented to a greater extent by caffeine in mutant islets. Surprisingly, ryanodine receptor-mediated Ca(2+) mobilization was not affected by the Anx7(+/-) mutation, suggesting that the mechanism underlying the observed differences in electrical and secretory responsiveness does not involve intracellular Ca(2+) stores. Our results provide evidence that both IP3 receptors and ryanodine receptors play important roles in regulating β-cell membrane potential and insulin secretion, and that the Anx7(+/-) mutation is associated with alterations in the signaling pathways related to these receptors.     

PIKfyve negatively regulates exocytosis in neurosecretory cells

Regulated secretion depends upon a highly coordinated series of protein-protein and protein-lipid interactions. Two phosphoinositides, phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3-phosphate, are important for the ATP-dependent priming of the secretory apparatus prior to Ca(2+)-dependent exocytosis. Mechanisms that control phosphoinositide levels are likely to play an important role in priming fine tuning. Here we have investigated the involvement of PIKfyve, a phosphoinositide 5-kinase that can phosphorylate phosphatidylinositol 3-phosphate to produce phosphatidylinositol 3,5-bisphosphate on large dense core vesicle exocytosis from neuroendocrine cells. PIKfyve localizes to a subpopulation of secretory granules in chromaffin and PC12 cells. Nicotine stimulation promoted recruitment of PIKfyve-EGFP onto secretory vesicles in PC12 cells. YM-201636, a selective inhibitor of PIKfyve activity, and PIKfyve knockdown by small interfering RNA potentiated secretory granule exocytosis. Overexpression of PIKfyve or its yeast orthologue Fab1p inhibited regulated secretion in PC12 cells, whereas a catalytically inactive PIKfyve mutant had no effect. These results demonstrate a novel inhibitory role for PIKfyve catalytic activity in regulated secretion and provide further evidence for a fine tuning of exocytosis by 3-phosphorylated phosphoinositides.     

Ca2+-secretion coupling is impaired in diabetic Goto Kakizaki rats

The Goto Kakizaki (GK) rat is a widely used animal model to study defective glucose-stimulated insulin release in type-2 diabetes (T2D). As in T2D patients, the expression of several proteins involved in Ca(2+)-dependent exocytosis of insulin-containing large dense-core vesicles is dysregulated in this model. So far, a defect in late steps of insulin secretion could not be demonstrated. To resolve this apparent contradiction, we studied Ca(2+)-secretion coupling of healthy and GK rat beta cells in acute pancreatic tissue slices by assessing exocytosis with high time-resolution membrane capacitance measurements. We found that beta cells of GK rats respond to glucose stimulation with a normal increase in the cytosolic Ca(2+) concentration. During trains of depolarizing pulses, the secretory activity from GK rat beta cells was defective in spite of upregulated cell size and doubled voltage-activated Ca(2+) currents. In GK rat beta cells, evoked Ca(2+) entry was significantly less efficient in triggering release than in nondiabetic controls. This impairment was neither due to a decrease of functional vesicle pool sizes nor due to different kinetics of pool refilling. Strong stimulation with two successive trains of depolarizing pulses led to a prominent activity-dependent facilitation of release in GK rat beta cells, whereas secretion in controls was unaffected. Broad-spectrum inhibition of PKC sensitized Ca(2+)-dependent exocytosis, whereas it prevented the activity-dependent facilitation in GK rat beta cells. We conclude that a decrease in the sensitivity of the GK rat beta-cell to depolarization-evoked Ca(2+) influx is involved in defective glucose-stimulated insulin secretion. Furthermore, we discuss a role for constitutively increased activity of one or more PKC isoenzymes in diabetic rat beta cells.     

Annexin 7 mobilizes calcium from endoplasmic reticulum stores in brain

Mobilization of intracellular calcium from inositol-1,4,5-triphosphate (IP3)-sensitive endoplasmic reticulum (ER) stores plays a prominent role in brain function. Mice heterozygous for the annexin A7 (Anx7) gene have a profound reduction in IP3 receptor function in pancreatic islets along with defective insulin secretion. We examined IP3-sensitive calcium pools in the brains of Anx7 (+/-) mice by utilizing ATP/Mg(2+)-dependent (45)Ca(2+) uptake into brain membrane preparations and tissue sections. Although the Anx7 (+/-) mouse brain displayed similar levels of IP3 binding sites and thapsigargin-sensitive (45)Ca(2+) uptake as that seen in wild-type mouse brain, the Anx7 (+/-) mouse brain Ca(2+) pools showed markedly reduced sensitivity to IP3. A potent and saturable Ca(2+)-releasing effect of recombinant ANX7 protein was demonstrated in mouse and rat brain membrane preparations, which was additive with that of IP3. We propose that ANX7 mobilizes Ca(2+) from an endoplasmic reticulum-like pool, which can be recruited to enhance IP3-mediated Ca(2+) release.     

v-SNAREs control exocytosis of vesicles from priming to fusion

SNARE proteins (soluble NSF-attachment protein receptors) are thought to be central components of the exocytotic mechanism in neurosecretory cells, but their precise function remained unclear. Here, we show that each of the vesicle-associated SNARE proteins (v-SNARE) of a chromaffin granule, synaptobrevin II or cellubrevin, is sufficient to support Ca(2+)-dependent exocytosis and to establish a pool of primed, readily releasable vesicles. In the absence of both proteins, secretion is abolished, without affecting biogenesis or docking of granules indicating that v-SNAREs are absolutely required for granule exocytosis. We find that synaptobrevin II and cellubrevin differentially control the pool of readily releasable vesicles and show that the v-SNARE's amino terminus regulates the vesicle's primed state. We demonstrate that dynamics of fusion pore dilation are regulated by v-SNAREs, indicating their action throughout exocytosis from priming to fusion of vesicles.     

Doc2 alpha and Munc13-4 regulate Ca(2+) -dependent secretory lysosome exocytosis in mast cells

The Doc2 family comprises the brain-specific Doc2alpha and the ubiquitous Doc2beta and Doc2gamma. With the exception of Doc2gamma, these proteins exhibit Ca(2+)-dependent phospholipid-binding activity in their Ca(2+)-binding C2A domain and are thought to be important for Ca(2+)-dependent regulated exocytosis. In excitatory neurons, Doc2alpha interacts with Munc13-1, a member of the Munc13 family, through its N-terminal Munc13-1-interacting domain and the Doc2alpha-Munc13-1 system is implicated in Ca(2+)-dependent synaptic vesicle exocytosis. The Munc13 family comprises the brain-specific Munc13-1, Munc13-2, and Munc13-3, and the non-neuronal Munc13-4. We previously showed that Munc13-4 is involved in Ca(2+)-dependent secretory lysosome exocytosis in mast cells, but the involvement of Doc2 in this process is not determined. In the present study, we found that Doc2alpha but not Doc2beta was endogenously expressed in the RBL-2H3 mast cell line. Doc2alpha colocalized with Munc13-4 on secretory lysosomes, and interacted with Munc13-4 through its two regions, the N terminus containing the Munc13-1-interacting domain and the C terminus containing the Ca(2+)-binding C2B domain. In RBL-2H3 cells, Ca(2+)-dependent secretory lysosome exocytosis was inhibited by expression of the Doc2alpha mutant lacking either of the Munc13-4-binding regions and the inhibition was suppressed by coexpression of Munc13-4. Knockdown of endogenous Doc2alpha also reduced Ca(2+)-dependent secretory lysosome exocytosis, which was rescued by re-expression of human Doc2alpha but not by its mutant that could not bind to Munc13-4. Moreover, Ca(2+)-dependent secretory lysosome exocytosis was severely reduced in bone marrow-derived mast cells from Doc2alpha knockout mice. These results suggest that the Doc2alpha-Muunc13-4 system regulates Ca(2+)-dependent secretory lysosome exocytosis in mast cells.     

Inhibition of calpain blocks pancreatic beta-cell spreading and insulin secretion

In addition to promoting insulin secretion, an increase in cytosolic Ca(2+) triggered by glucose has been shown to be crucial for spreading of beta-cells attached on extracellular matrix (804G matrix). Calpains are Ca(2+)-dependent cysteine proteases involved in an extended spectrum of cellular responses, including cytoskeletal rearrangements and vesicular trafficking. The present work aimed to assess whether calpain is also implicated in the process of Ca(2+)-induced insulin secretion and spreading of rat pancreatic beta-cells. The results indicate calpain dependency of beta-cell spreading on 804G matrix. Indeed, treatment with three distinct calpain inhibitors (N-Ac-Leu-Leu-norleucinal, calpeptin, and ethyl(+)-(2S,3S)-3-[(S)-3-methyl-1-(3-methylbutylcarbamoyl)butyl-carbamoyl]-2-ox-iranecarboxylate) inhibited cell spreading induced by glucose and KCl, whereas cell attachment was not significantly modified. Calpain inhibitors also suppressed glucose- and KCl-stimulated insulin secretion without affecting insulin synthesis. Washing the inhibitor out of the cell culture restored spreading on 804G matrix and insulin secretory response after 24 h. In addition, incubation with calpeptin did not affect insulin secretory response to mastoparan that acts on exocytosis downstream of intracellular calcium [Ca(2+)]i. Finally, calpeptin was shown to affect the [Ca(2+)]i response to glucose but not to KCl. In summary, the results show that inhibition of calpain blocks spreading and insulin secretion of primary pancreatic beta-cells. It is therefore suggested that calpain could be a mediator of Ca(2+)-induced-insulin secretion and beta-cell spreading.     

Insulin secretory deficiency and glucose intolerance in Rab3A null mice

Insulin secretory dysfunction of the pancreatic beta-cell in type-2 diabetes is thought to be due to defective nutrient sensing and/or deficiencies in the mechanism of insulin exocytosis. Previous studies have indicated that the GTP-binding protein, Rab3A, plays a mechanistic role in insulin exocytosis. Here, we report that Rab3A(-/-) mice develop fasting hyperglycemia and upon a glucose challenge show significant glucose intolerance coupled to ablated first-phase insulin release and consequential insufficient insulin secretion in vivo, without insulin resistance. The in vivo insulin secretory response to arginine was similar in Rab3A(-/-) mice as Rab3A(+/+) control animals, indicating a phenotype reminiscent of insulin secretory dysfunction found in type-2 diabetes. However, when a second arginine dose was given 10 min after, there was a negligible insulin secretory response in Rab3A(-/-) mice, compared with that in Rab3A(+/+) animals, that was markedly increased above that to the first arginine stimulus. There was no difference in beta-cell mass or insulin production between Rab3A(-/-) and Rab3A(+/+) mice. However, in isolated islets, secretagogue-induced insulin release (by glucose, GLP-1, glyburide, or fatty acid) was approximately 60-70% lower in Rab3A(-/-) islets compared with Rab3A(+/+) controls. Nonetheless, there was a similar rate of glucose oxidation and glucose-induced rise in cytosolic [Ca(2+)](i) flux between Rab3A(-/-) and Rab3A(+/+) islet beta-cells, indicating the mechanistic role of Rab3A lies downstream of generating secondary signals that trigger insulin release, at the level of secretory granule transport and/or exocytosis. Thus, Rab3A plays an important in vivo role facilitating the efficiency of insulin exocytosis, most likely at the level of replenishing the ready releasable pool of beta-granules. Also, this study indicates, for the first time, that the in vivo insulin secretory dysfunction found in type-2 diabetes can lie solely at the level of defective insulin exocytosis.     

Calcium-dependent activator protein for secretion 2 interacts with the class II ARF small GTPases and regulates dense-core vesicle trafficking

The Ca(2+) -dependent activator protein for secretion (CAPS) family consists of two members (CAPS1 and CAPS2) and regulates the exocytosis of catecholamine-containing or neuropeptide-containing dense-core vesicles (DCVs) at secretion sites such as nerve terminals. A large fraction of CAPS1, however, is localized in the cell soma, and we have recently shown the possible involvement of somal CAPS1 in DCV trafficking in the trans-Golgi network. CAPS1 and CAPS2 are differentially expressed in various regions of the mouse brain but exhibit similar expression patterns in other tissues, such as the spleen. Thus, in the present study we analyzed whether CAPS2 displays similar subcellular localization and functional roles in the cell soma as CAPS1. We found that somal CAPS2 is associated with the Golgi membrane, and mediates binding and recruitment of the GDP-bound form of ARF4 and ARF5 (members of the membrane-trafficking small GTPase family) to the Golgi membrane. CAPS2 knockdown and overexpression of CAPS2-binding-deficient ARF4/ARF5 both induced accumulation of the DCV resident protein chromogranin?A around the Golgi apparatus. CAPS2 knockout mice have dilated trans-Golgi structures when viewed by electron microscopy. These results for CAPS2 strongly support our idea that the CAPS family proteins exert dual roles in DCV trafficking, mediating trafficking at both the secretion site for exocytosis and at the Golgi complex for biogenesis.     

Resident CAPS on dense-core vesicles docks and primes vesicles for fusion

The Ca²⁺-dependent exocytosis of dense-core vesicles in neuroendocrine cells requires a priming step during which SNARE protein complexes assemble. CAPS (aka CADPS) is one of several factors required for vesicle priming; however, the localization and dynamics of CAPS at sites of exocytosis in live neuroendocrine cells has not been determined. We imaged CAPS before, during, and after single-vesicle fusion events in PC12 cells by TIRF micro­scopy. In addition to being a resident on cytoplasmic dense-core vesicles, CAPS was present in clusters of approximately nine molecules near the plasma membrane that corresponded to docked/tethered vesicles. CAPS accompanied vesicles to the plasma membrane and was present at all vesicle exocytic events. The knockdown of CAPS by shRNA eliminated the VAMP-2–dependent docking and evoked exocytosis of fusion-competent vesicles. A CAPS(ΔC135) protein that does not localize to vesicles failed to rescue vesicle docking and evoked exocytosis in CAPS-depleted cells, showing that CAPS residence on vesicles is essential. Our results indicate that dense-core vesicles carry CAPS to sites of exocytosis, where CAPS promotes vesicle docking and fusion competence, probably by initiating SNARE complex assembly.     

Two distinct secretory vesicle�Cpriming steps in adrenal chromaffin cells

Priming of large dense-core vesicles (LDCVs) is a Ca²⁺-dependent step by which LDCVs enter a release-ready pool, involving the formation of the soluble N-ethyl-maleimide sensitive fusion protein attachment protein (SNAP) receptor complex consisting of syntaxin, SNAP-25, and synaptobrevin. Using mice lacking both isoforms of the calcium-dependent activator protein for secretion (CAPS), we show that LDCV priming in adrenal chromaffin cells entails two distinct steps. CAPS is required for priming of the readily releasable LDCV pool and sustained secretion in the continued presence of high Ca²⁺ concentrations. Either CAPS1 or CAPS2 can rescue secretion in cells lacking both CAPS isoforms. Furthermore, the deficit in the readily releasable LDCV pool resulting from CAPS deletion is reversed by a constitutively open form of syntaxin but not by Munc13-1, a priming protein that facilitates the conversion of syntaxin to the open conformation. Our data indicate that CAPS functions downstream of Munc13s but also interacts functionally with Munc13s in the LDCV-priming process.     

New insights concerning the glucose-dependent insulin secretagogue action of glucagon-like peptide-1 in pancreatic beta-cells.

The GLP-1 receptor is a Class B heptahelical G-protein-coupled receptor that stimulates cAMP production in pancreatic beta-cells. GLP-1 utilizes this receptor to activate two distinct classes of cAMP-binding proteins: protein kinase A (PKA) and the Epac family of cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs). Actions of GLP-1 mediated by PKA and Epac include the recruitment and priming of secretory granules, thereby increasing the number of granules available for Ca(2+)-dependent exocytosis. Simultaneously, GLP-1 promotes Ca(2+) influx and mobilizes an intracellular source of Ca(2+). GLP-1 sensitizes intracellular Ca(2+) release channels (ryanodine and IP (3) receptors) to stimulatory effects of Ca(2+), thereby promoting Ca(2+)-induced Ca(2+) release (CICR). In the model presented here, CICR activates mitochondrial dehydrogenases, thereby upregulating glucose-dependent production of ATP. The resultant increase in cytosolic [ATP]/[ADP] concentration ratio leads to closure of ATP-sensitive K(+) channels (K-ATP), membrane depolarization, and influx of Ca(2+) through voltage-dependent Ca(2+) channels (VDCCs). Ca(2+) influx stimulates exocytosis of secretory granules by promoting their fusion with the plasma membrane. Under conditions where Ca(2+) release channels are sensitized by GLP-1, Ca(2+) influx also stimulates CICR, generating an additional round of ATP production and K-ATP channel closure. In the absence of glucose, no "fuel" is available to support ATP production, and GLP-1 fails to stimulate insulin secretion. This new "feed-forward" hypothesis of beta-cell stimulus-secretion coupling may provide a mechanistic explanation as to how GLP-1 exerts a beneficial blood glucose-lowering effect in type 2 diabetic subjects.     

CAPS acts at a prefusion step in dense-core vesicle exocytosis as a PIP2 binding protein

CAPS-1 is required for Ca2+-triggered fusion of dense-core vesicles with the plasma membrane, but its site of action and mechanism are unknown. We analyzed the kinetics of Ca2+-triggered exocytosis reconstituted in permeable PC12 cells. CAPS-1 increased the initial rate of Ca2+-triggered vesicle exocytosis by acting at a rate-limiting, Ca2+-dependent prefusion step. CAPS-1 activity depended upon prior ATP-dependent priming during which PIP2 synthesis occurs. CAPS-1 activity and binding to the plasma membrane depended upon PIP2. Ca2+ was ineffective in triggering vesicle fusion in the absence of CAPS-1 but instead promoted desensitization to CAPS-1 resulting from decreased plasma membrane PIP2. We conclude that CAPS-1 functions following ATP-dependent priming as a PIP2 binding protein to enhance Ca2+-dependent DCV exocytosis. Essential prefusion steps in dense-core vesicle exocytosis involve sequential ATP-dependent synthesis of PIP2 and the subsequent PIP2-dependent action of CAPS-1. Regulation of PIP2 levels and CAPS-1 activity would control the secretion of neuropeptides and monoaminergic transmitters.