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PubMed1998

A novel tetanus neurotoxin-insensitive vesicle-associated membrane protein in SNARE complexes of the apical plasma membrane of epithelial cells.

The importance of soluble N-ethyl maleimide (NEM)-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs) in synaptic vesicle exocytosis is well established because it has been demonstrated that clostridial neurotoxins (NTs) proteolyze the vesicle SNAREs (v-SNAREs) vesicle-associated membrane protein (VAMP)/brevins and their partners, the target SNAREs (t-SNAREs) syntaxin 1 and SNAP25. Yet, several exocytotic events, including apical exocytosis in epithelial cells, are insensitive to numerous clostridial NTs, suggesting the presence of SNARE-independent mechanisms of exocytosis. In this study we found that syntaxin 3, SNAP23, and a newly identified VAMP/brevin, tetanus neurotoxin (TeNT)-insensitive VAMP (TI-VAMP), are insensitive to clostridial NTs. In epithelial cells, TI-VAMP-containing vesicles were concentrated in the apical domain, and the protein was detected at the apical plasma membrane by immunogold labeling on ultrathin cryosections. Syntaxin 3 and SNAP23 were codistributed at the apical plasma membrane where they formed NEM-dependent SNARE complexes with TI-VAMP and cellubrevin. We suggest that TI-VAMP, SNAP23, and syntaxin 3 can participate in exocytotic processes at the apical plasma membrane of epithelial cells and, more generally, domain-specific exocytosis in clostridial NT-resistant pathways.

Amino Acid Sequence↗

PubMed1996

Differential distribution of syntaxin isoforms 1A and 1B in the rat central nervous system.

Syntaxin 1 binds to several proteins of the synaptic terminal and is a central component in the pathway of protein-protein interactions that underlies docking and fusion of synaptic vesicles. Molecular studies revealed the occurrence of two isoforms, syntaxin 1A and syntaxin 1B, which coexpress in neural tissues. However, they display differential expression patterns in endocrine cell types. We generated isoform-specific antibodies that were used in Western blotting and immunocytochemical studies. First, we confirmed the sole presence of syntaxin 1A in endocrine pituitary cells. Second, we found distinctive immunolabelling patterns of each isoform in the rat olfactory system, hippocampus, striatum, thalamus and spinal cord. In addition, the principal white matter commissures displayed distinct immunoreactivity for each isoform. This report shows, for the first time, major differences between the distributions of syntaxin 1A and syntaxin 1B isoforms in the rat central nervous system.

Animals↗

PubMed1997

Regulated secretion in platelets: identification of elements of the platelet exocytosis machinery.

To further characterize the molecular mechanisms of platelet function, we have sought to identify some of the proteins that mediate the secretory events of the platelet release reaction. We report that platelets contain the general elements of the membrane transport apparatus: N-ethylmaleimide sensitive fusion protein (NSF), p115/transcytosis-associated protein (p115/TAP), and the soluble NSF attachment proteins (alpha- and, gamma-SNAP). The cDNAs encoding two of these proteins, alpha- and gamma-SNAP, have been cloned from a human platelet-derived cDNA library. Platelet membrane extracts possess SNAP receptor (SNARE) activity, suggesting that the class of proteins (SNAREs) proposed to provide the specificity for vesicle docking and membrane fusion are present in platelets. To identify these proteins, we have used specific antibodies against known SNAREs to probe platelet extracts. Syntaxin 2 and 4 can be readily detected in platelet membrane preparations and are shown to participate in 20 S complex formation. Syntaxin 1, 3, and 5 could not be detected. Other known SNARE and SNARE-associated proteins such as vesicle-associated membrane protein (VAMP)/synaptobrevin 2, SNAP-25, synaptophysin, or synaptotagmin I could not be immunochemically detected in platelet membrane preparations. The presence of both the general transport proteins (NSF and SNAPs) and specific transport proteins (syntaxin 2 and 4) indicates that platelet exocytosis uses a molecular mechanism similar to other secretory cells such as neurons. However, the subcellular concentrations of these proteins suggest that, unlike neuronal secretion, granule-to plasma membrane docking may be the limiting step in platelet exocytosis.

Amino Acid Sequence↗

PubMed1995

Cellubrevin and synaptobrevins: similar subcellular localization and biochemical properties in PC12 cells.

There is strong evidence to indicate that proteins of the synaptobrevin family play a key role in exocytosis. Synaptobrevin 1 and 2 are expressed at high concentration in brain where they are localized on synaptic vesicles. Cellubrevin, a very similar protein, has a widespread tissue distribution and in fibroblasts is localized on endosome-derived, transferin receptor-positive vesicles. Since brain cellubrevin is not detectable in synaptic vesicles, we investigated whether cellubrevin and the synaptobrevins are differentially targeted when co-expressed in the same cell. We report that in the nervous system cellubrevin is expressed at significant levels only by glia and vascular cells. However, cellubrevin is coexpressed with the two synaptobrevins in PC12 cells, a neuroendocrine cell line which contains synaptic vesicle-like microvesicles. In PC12 cells, cellubrevin has a distribution very similar to that of synaptobrevin 1 and 2. The three proteins are targeted to neurites which exclude the transferrin receptor and are enriched in synaptic-like microvesicles and dense-core granules. They are recovered in the synaptic-like microvesicle peak of glycerol velocity gradients, have a similar distribution in isopycnic fractionation and are coprecipitated by anti-synaptobrevin 2 immunobeads. Finally, cellubrevin, like the synaptobrevins, interact with the neuronal t-SNAREs syntaxin 1 and SNAP-25. These results suggest that cellubrevin and the synaptobrevins have similar function and do not play a specialized role in constitutive and regulated exocytosis, respectively.

Amino Acid Sequence↗

PubMed1998

Interaction of cysteine string proteins with the alpha1A subunit of the P/Q-type calcium channel.

Cysteine string proteins (Csps) are J-domain chaperone proteins anchored at the surface of synaptic vesicles. Csps are involved in neurotransmitter release and may modulate presynaptic calcium channel activity, although the molecular mechanisms are unknown. Interactions between Csps, proteins of the synaptic core (SNARE) complex, and P/Q-type calcium channels were therefore explored. Co-immunoprecipitation suggested that Csps occur in complexes containing synaptobrevin (VAMP), but not syntaxin 1, SNAP-25, nor P/Q-type calcium channels labeled with 125I-omega-conotoxin MVIIC. However binding experiments with 35S-labeled Csp1 demonstrated an interaction (apparent KD = 700 nM at pH 7.4 and 4 degreesC) with a fusion protein containing a segment of the cytoplasmic loop linking homologous domains II-III of the alpha1A calcium channel subunit (BI isoform, residues 780-969). Binding was specific as it was displaced by unlabeled Csp1, and no interactions were detected with fusion proteins containing other calcium channel domains, VAMP, or syntaxin 1A. A Csp binding site on the P/Q-type calcium channel is thus located within the 200 residue synaptic protein interaction site that can also bind syntaxin I, SNAP-25, and synaptotagmin I. Csp may act as a molecular chaperone to direct assembly or disassembly of exocytotic complexes at the calcium channel.

Animals↗

PubMed2000

SNAP-25 regulation during adrenal gland development: comparison with differentiation markers and other SNAREs.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is one of a limited number of soluble N-ethylmaleimide-sensitive fusion attachment protein receptors (SNAREs) that play a major role in membrane docking of synaptic vesicles and secretory granules during regulated exocytosis. We have previously shown that SNAP-25 levels differ between noradrenergic and adrenergic chromaffin cell populations of the adult adrenal gland. We examine SNAP-25 expression by immunofluoresence in cells of the sympathoadrenal lineage in the rat during late embryonic and postnatal development. In parallel, tyrosine hydroxylase was used to identify sympathoadrenal cells, phenylethanolamine N-methyltransferase to distinguish adrenergic from noradrenergic chromaffin cells, and chromogranin A to define the presence of secretory granules. In addition, SNAP-25 protein and mRNA levels were followed in adrenal gland extracts by immunoblotting and reverse transcription-polymerase chain reaction (RT-PCR). Protein levels were compared with those of other molecules also implicated in organelle trafficking, including syntaxin 1 and vesicle-associated membrane protein (VAMP-2) and the nonneuronal analogues SNAP-23 and cellubrevin. This study provides evidence that SNAP-25 is expressed early during development in sympathoadrenal neurons and migrating cells. It is detected in intra-adrenal chromoblasts as soon as they enter the adrenal primordium. Its differential expression between catecholamine chromaffin cell phenotypes is already evident from the 17th embryonic day, future noradrenergic cells appearing to express higher levels than adrenergic cells. The granule maturation marker chromogranin A is expressed in chromaffin cells later than SNAP-25. Both SNAP-25 protein and mRNA increased rapidly in the adrenal gland in the perinatal period to peak during the first postnatal week, after which levels dropped dramatically to adult values. In contrast, levels of both syntaxin and SNAP-23 appeared to remain fairly constant throughout adrenal gland development. VAMP-2 expression increased gradually around birth to reach maximal levels during the first two postnatal weeks, and then decreased slightly. Cellubrevin levels also appeared to increase gradually until adult values were attained by the end of the second postnatal week. The threefold increase of SNAP-25 mRNA shortly after birth compared to the low adult levels suggests that during this period SNAP-25 is implicated in additional functions than regulated secretion, possibly associated with cellular growth or maturation.

Adrenal Glands↗

PubMed2001

Calcium-dependent translocation of synaptotagmin to the plasma membrane in the dendrites of developing neurones.

In neurones, the morphological complexity of the dendritic tree requires regulated growth and the appropriate targeting of membrane components. Accurate delivery of specific supplies depends on the translocation and fusion of transport vesicles. Vesicle SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors) and target membrane SNAREs play a central role in the correct execution of fusion events, and mediate interactions with molecules that endow the system with appropriate regulation. Synaptotagmins, a family of Ca(2+)-sensor proteins that includes neurone-specific members involved in regulating neurotransmitter exocytosis, are among the molecules that can tune the fusion mechanism. Using immunocytochemistry, confocal and electron microscopy, the localisation of synaptotagmin I in the dendrites of cultured rat hypothalamic neurones was demonstrated. Synaptotagmin labelling is concentrated at dendritic branch points, and in microprocesses. Following depolarisation, the N-terminal domain of synaptotagmin was detected at the extracellular surface of the dendritic plasma membrane. The insertion of synaptotagmin in the plasma membrane was elicited by L-type Ca(2+) channel activation and by mobilisation of the internal ryanodine-sensitive Ca(2+)stores. Furthermore, the localisation of L-type Ca(2+) channels and of ryanodine receptors, relative to the localisation of synaptotagmin in dendrites, suggests that both Ca(2+) entry and intracellular Ca(2+) stores may contribute to the fusion of dendritic transport vesicles with the membrane. Fusion is likely to involve SNAP-25 and syntaxin 1 as both proteins were also identified in dendrites. Taken together these results suggest a putative regulatory role of synaptotagmins in the membrane fusion events that contribute to shaping the dendritic tree during development.

Animals↗

PubMed2001

Localization of cellubrevin-related peptide, endobrevin, in the early endosome in pancreatic beta cells and its physiological function in exo-endocytosis of secretory granules.

Cellubrevins are integral membrane proteins expressed in a wide variety of tissues and usually localized in recycling vesicles. Here, we investigated the cellular localization of a cellubrevin-related peptide, endobrevin, in pancreatic (beta) cells and its implication in the exo-endocytosis of insulin and (gamma)-amino butyric acid (GABA). Immunocytochemistry showed that endobrevin is associated with tubulo-vesicular structures, which are colocalized with early endosomes labeled by early endosome antigen (EEA)-1 in insulinoma MIN6 cells. To determine the cellular localization of endobrevin, we appended the green fluorescent protein (GFP) to endobrevin and the fusion protein was introduced into MIN6 cells. The subcellular localization of GFP-endobrevin was visualized by confocal laser microscopy. Colocalization study based on the expressed GFP-endobrevin and endocytosed Texas-Red(Tx-R) labeled transferrin receptor and immunocytochemistry with anti-EEA1 antibody revealed that endobrevin was preferentially localized in the early endosome. Then, we examined the functional role of endobrevin in the exocytosis of insulin and GABA from pancreatic (beta) cells. Endobrevin overexpression increased the amount of GABA released from MIN6 cells; in contrast, it decreased the glucose-stimulated insulin release from rat islets, MIN6 and INS1-D cells to approximately 50% of the control levels. Both in vitro and in vivo binding studies showed that endobrevin binds to syntaxin 1. Finally, using the fluorescent probe FM4-64, it was revealed that endobrevin overexpression accelerates vesicle recycling. We conclude that (1) endobrevin is localized in the early endosome in pancreatic (beta) cells and (2) endobrevin plays a physiological role in the exo-endocytosis of insulin and GABA from pancreatic (beta) cells, probably via an interaction between endocytic vesicles and the endosome.

Animals↗

PubMed2000

The SNARE Vti1a-beta is localized to small synaptic vesicles and participates in a novel SNARE complex.

Specific soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins are required for different membrane transport steps. The SNARE Vti1a has been colocalized with Golgi markers and Vti1b with Golgi and the trans-Golgi network or endosomal markers in fibroblast cell lines. Here we study the distribution of Vti1a and Vti1b in brain. Vti1b was found in synaptic vesicles but was not enriched in this organelle. A brain-specific splice variant of Vti1a was identified that had an insertion of seven amino acid residues next to the putative SNARE-interacting helix. This Vti1a-beta was enriched in small synaptic vesicles and clathrin-coated vesicles isolated from nerve terminals. Vti1a-beta also copurified with the synaptic vesicle R-SNARE synaptobrevin during immunoisolation of synaptic vesicles and endosomes. Therefore, both synaptobrevin and Vti1a-beta are integral parts of synaptic vesicles throughout their life cycle. Vti1a-beta was part of a SNARE complex in nerve terminals, which bound N-ethylmaleimide-sensitive factor and alpha-SNAP. This SNARE complex was different from the exocytic SNARE complex because Vti1a-beta was not coimmunoprecipitated with syntaxin 1 or SNAP-25. These data suggest that Vti1a-beta does not function in exocytosis but in a separate SNARE complex in a membrane fusion step during recycling or biogenesis of synaptic vesicles.

Alternative Splicing↗

PubMed2001

Amyloid beta-peptide inhibits neuronal glucose uptake by preventing exocytosis.

Amyloid beta peptide (Abeta) is suspected as a contributing factor for decreased glucose utilization in the brain of Alzheimer's patients; however, little is known about the regulatory mechanism of neuronal glucose uptake and how Abeta affects such a mechanism. We report that membrane depolarization by 40 mM KCl increases both neuronal glucose uptake and immunolabeling of the exofacial epitope of glucose transporter isoform GLUT3, suggesting that fusion of GLUT3 vesicles with the plasma membrane increases glucose uptake. Abeta25-35 decreased neuronal glucose uptake and this decrease was prevented by exocytosis-enhancing compounds (40 mM KCl, 50 microM ruthenium red). Abeta25-35 also inhibited exocytosis of the fluorescent membrane dye FM1-43 at neuronal cell bodies; however, 40 mM KCl was effective in overcoming this Abeta inhibition. Furthermore, GLUT3 colocalized with SNARE (N-ethylmaleimide-sensitive factor attached protein receptor) complex proteins (SNAP-25 and Syntaxin 1), and cleavage of the v-SNARE, VAMP, reduced glucose uptake. Our findings suggest that neuronal glucose uptake is regulated by SNARE complex-dependent docking and fusion of GLUT3 vesicles with the plasma membrane and that Abeta decreases glucose uptake by inhibiting fusion of these vesicles.

Amyloid beta-Peptides↗

PubMed2001

mRNA expression of membrane-fusion-related proteins in rat parotid gland.

The soluble N-ethylmaleimide-sensitive fusion (NSF) attachment protein (SNAP) receptor (SNARE) hypothesis has been applied to exocytosis in salivary glands. The expression of SNARE proteins has not been well investigated in the parotid gland. In this study, the mRNA expression of SNAREs and membrane-fusion-related proteins were investigated in the rat parotid by reverse transcriptase-polymerase chain reaction (RT-PCR). All syntaxins except syntaxin 1, and the vesicle-associated membrane proteins (VAMP) except VAMP-7, NSF, SNAP-23 and alpha-SNAP, were expressed for the SNAREs in rat parotid. Synaptotagmins 3, 4 and 11, Munc18s (1, 2 and 3), syncollin, prenylated Rab acceptor (PRA1), zygin 1, pantophysin and cellugyrin, which are the other membrane-fusion-related proteins, were also detected, but neither Rim nor rabphilin 3A, which have high specificity of binding to Rab 3A, were found. mRNA expressions of many SNAREs and of the membrane-fusion-proteins suggest novel interactions for the regulation of salivary exocytosis.

Animals↗

PubMed1996

SNAP-25 is present on chromaffin granules and acts as a SNAP receptor.

SNAP-25 is located on the plasma membrane and essential for exocytosis of neurotransmitters. It was suggested that SNAP-25 and syntaxin 1 via the interaction with VAMP-2 located on synaptic vesicles mediate the docking of the vesicles with the plasma membrane. In the present study, by means of biochemical and morphological analyses, we showed that SNAP-25 is present on chromaffin granules as well as on the plasma membrane. Reconstitution and immunoprecipitation analyses revealed that SNAP-25 on chromaffin granules has essentially the same properties as does SNAP-25 on the plasma membrane.

Animals↗

PubMed1999

GS32, a novel Golgi SNARE of 32 kDa, interacts preferentially with syntaxin 6.

Syntaxin 1, synaptobrevins or vesicle-associated membrane proteins, and the synaptosome-associated protein of 25 kDa (SNAP-25) are key molecules involved in the docking and fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, cell biological, and biochemical characterization of a 32-kDa protein homologous to both SNAP-25 (20% amino acid sequence identity) and the recently identified SNAP-23 (19% amino acid sequence identity). Northern blot analysis shows that the mRNA for this protein is widely expressed. Polyclonal antibodies against this protein detect a 32-kDa protein present in both cytosol and membrane fractions. The membrane-bound form of this protein is revealed to be primarily localized to the Golgi apparatus by indirect immunofluorescence microscopy, a finding that is further established by electron microscopy immunogold labeling showing that this protein is present in tubular-vesicular structures of the Golgi apparatus. Biochemical characterizations establish that this protein behaves like a SNAP receptor and is thus named Golgi SNARE of 32 kDa (GS32). GS32 in the Golgi extract is preferentially retained by the immobilized GST-syntaxin 6 fusion protein. The coimmunoprecipitation of syntaxin 6 but not syntaxin 5 or GS28 from the Golgi extract by antibodies against GS32 further sustains the preferential interaction of GS32 with Golgi syntaxin 6.

Amino Acid Sequence↗

PubMed1997

Transient expression of botulinum neurotoxin C1 light chain differentially inhibits calcium and glucose induced insulin secretion in clonal beta-cells.

We have investigated the effect of botulinum neurotoxin (BoNT) C1 light chain (LC) on insulin exocytosis from the clonal beta-cell line HIT-T15. In streptolysin-O permeabilized cells, the beta-cell impermeant BoNT C1 cleaved mainly syntaxin 1 and inhibited Ca2+ as well as GTPgammaS induced exocytosis. To study the effect of BoNTs in intact cells, we transiently coexpressed the BoNT LC together with a reporter gene for insulin release. BoNT C1 inhibited K+ induced insulin secretion by 95% but reduced insulin release stimulated by glucose only by 25%. Thus a component of glucose stimulated insulin release is insensitive to BoNT C1.

Animals↗

PubMed1998

The effects of SNAP/SNARE complexes on the ATPase of NSF.

The ATPase of the N-ethylmaleimide sensitive factor (NSF) appears to be central to the events that culminate in vesicle-target membrane fusion. Complexes containing different combinations of NSF, alpha-SNAP, Vamp-2 (synaptobrevin 2), syntaxin 1, and SNAP-25 were reconstituted and then tested for their effect on the ATPase of NSF. While NSF interacts with all alpha-SNAP-containing complexes, only the alpha-SNAP/t-SNARE complex significantly stimulated ATPase activity. This stimulation was dependent on increasing SNAP/t-SNARE complex and was saturable. The apparent stimulation of ATPase activity is due to a 10-fold increase in initial hydrolysis rate. Complex containing both v- and t-SNAREs bound significantly more alpha-SNAP but did not stimulate the ATPase of NSF.

Adenosine Triphosphatases↗

PubMed1996

The V0 sector of the V-ATPase, synaptobrevin, and synaptophysin are associated on synaptic vesicles in a Triton X-100-resistant, freeze-thawing sensitive, complex.

Anti-synaptobrevin 2 immunoprecipitates obtained from freshly prepared Triton X-100 extracts of rat synaptosomes contained, in addition to synaptophysin, a 10-kDa band, which we identified by peptide sequencing and Western blotting as the c subunit of the vacuolar proton pump (V-ATPase) also called ductin or mediatophore. Ac39 and Ac116, two other transmembrane subunits of the V0 sector of the V-ATPase, were also found by Western blotting to be enriched in the immunoprecipitates. None of these V-ATPase subunits, or synaptophysin, was present in anti-synaptobrevin 2 immunoprecipitates obtained from frozen-thawed Triton X-100 extracts, which were greatly enriched, instead, in SNAP-25 and syntaxin 1. Accordingly, V-ATPase subunit c was found in anti-synaptophysin immunoprecipitates. Thus, the two complexes appear to be mutually exclusive. Subcellular fractionation of rat brain demonstrated that V-ATPase subunit c is localized with synaptobrevin 2 and synaptophysin in synaptic vesicles. The coprecipitation of V-ATPase subunit c with the synaptobrevin-synaptophysin complex suggests that this interaction may play a role in recruiting the proton pump into synaptic vesicles. Freeze-thawing, which involves a mild denaturing step, may produce a conformational change which dissociates the complex and mimics a change which occurs in vivo as a prerequisite to SNARE complex formation.

Amino Acid Sequence↗

PubMed1999

SNARE complex at the ribbon synapses of cochlear hair cells: analysis of synaptic vesicle- and synaptic membrane-associated proteins.

Neurotransmitters are released via exocytosis of synaptic vesicles involving a fusion complex consisting of a set of highly conserved proteins, which form a multiprotein complex resulting in the docking of synaptic vesicles at the site of release. There are three major differences between cochlear hair cell synapses and CNS synapses: (i) hair cells have a specialized structure, the synaptic ribbon, to which synaptic vesicles are attached; (ii) hair cells can maintain high and sustained release of neurotransmitter; and (iii) hair cells lack synaptophysin and synapsin. These differences suggest that an unconventional mechanism of neurotransmitter release may be involved at ribbon synapses. In this study we used different and complementary approaches to determine whether or not ribbon-containing hair cells of the cochlea express any component of the core fusion complex found in conventional synapses. Syntaxin 1, the synaptic membrane synaptosome-associated protein (SNAP)-25 and vesicle-associated membrane protein (VAMP or synaptobrevin) were found to be present in the organ of Corti of both rat and guinea-pig, as shown by reverse transcription polymerase chain reaction and Western blotting. In situ hybridization and immunocytochemistry showed mRNA and protein expression, respectively, in both inner and outer hair cells. Synaptotagmins I and II, generally considered to play major roles in neurotransmitter release at central synapses, were not detected in the organ of Corti.

Animals↗

PubMed1999

Fusion of endosomes involved in synaptic vesicle recycling.

Recycling of vesicles of the regulated secretory pathway presumably involves passage through an early endosomal compartment as an intermediate step. To learn more about the involvement of endosomes in the recycling of synaptic and secretory vesicles we studied in vitro fusion of early endosomes derived from pheochromocytoma (PC12) cells. Fusion was not affected by cleavage of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins synaptobrevin and syntaxin 1 that operate at the exocytotic limb of the pathway. Furthermore, fusion was inhibited by the fast Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid but not by the slow Ca(2+) chelator EGTA. Endosome fusion was restored by the addition of Ca(2+) with an optimum at a free Ca(2+) concentration of 0.3 x 10(-6) M. Other divalent cations did not substitute for Ca(2+). A membrane-permeant EGTA derivative caused inhibition of fusion, which was reversed by addition of Ca(2+). We conclude that the fusion of early endosomes participating in the recycling of synaptic and neurosecretory vesicles is mediated by a set of SNAREs distinct from those involved in exocytosis and requires the local release of Ca(2+) from the endosomal interior.

Animals↗