Multiple stimulation-dependent processes regulate the size of the releasable pool of vesicles

In neuroendocrine cells and neurones, changes in the size of a limited pool of readily releasable vesicles contribute to the plasticity of secretion. We have studied the dynamic alterations in the size of a near-membrane pool of vesicles in living neuroendocrine cells. Using evanescent wave microscopy we monitored the behaviour of individual secretory vesicles at the plasma membrane. Vesicles undergo sequential transitions between several states of differing fluorescence intensity and mobility. The transitions are reversible, except for the fusion step, and even in nonstimulated conditions the vesicles change states in a dynamic equilibrium. Stimulation selectively speeds up the three forward transitions leading towards exocytosis. Vesicles lose mobility in all three dimensions upon approach of the plasma membrane. Their movement is directed and targeted to the docking fusion sites. Sites of vesicle docking and exocytosis are distributed non-uniformly over the studied “footprint” region of the cell. While some areas are the sites of repeated vesicle docking and fusion, others are completely devoid of spots. Vesicular mobility at the membrane is confined, as if the vesicle were imprisoned in a cage or tethered to a binding site. Received: 10 August 1998 / Revised version: 24 September 1998 / Accepted: 24 September 1998     

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.     

Synaptotagmin VII as a plasma membrane Ca(2+) sensor in exocytosis

Synaptotagmins I and II are Ca(2+) binding proteins of synaptic vesicles essential for fast Ca(2+)-triggered neurotransmitter release. However, central synapses and neuroendocrine cells lacking these synaptotagmins still exhibit Ca(2+)-evoked exocytosis. We now propose that synaptotagmin VII functions as a plasma membrane Ca(2+) sensor in synaptic exocytosis complementary to vesicular synaptotagmins. We show that alternatively spliced forms of synaptotagmin VII are expressed in a developmentally regulated pattern in brain and are concentrated in presynaptic active zones of central synapses. In neuroendocrine PC12 cells, the C(2)A and C(2)B domains of synaptotagmin VII are potent inhibitors of Ca(2+)-dependent exocytosis, but only when they bind Ca(2+). Our data suggest that in synaptic vesicle exocytosis, distinct synaptotagmins function as independent Ca(2+) sensors on the two fusion partners, the plasma membrane (synaptotagmin VII) versus synaptic vesicles (synaptotagmins I and II).     

Synaptotagmin IX regulates Ca2+-dependent secretion in PC12 cells.

Synaptotagmin (Syt) I-deficient phaeochromocytoma (PC12) cell lines show normal Ca(2+)-dependent norepinephrine (NE) release (Shoji-Kasai, Y., Yoshida, A., Sato, K., Hoshino, T., Ogura, A., Kondo, S., Fujimoto, Y., Kuwahara, R., Kato, R., and Takahashi, M. (1992) Science 256, 1821-1823). To identify an alternative Ca(2+) sensor, we searched for other Syt isoforms in Syt I-deficient PC12 cells and identified Syt IX, an isoform closely related to Syt I, as an abundantly expressed dense-core vesicle protein. Here we show that Syt IX is required for the Ca(2+)-dependent release of NE from PC12 cells. Antibodies directed against the C2A domain of either Syt IX or Syt I inhibited Ca(2+)-dependent NE release in permeable PC12 cells indicating that both Syt proteins function in dense-core vesicle exocytosis. Our results support the idea that Syt family proteins that co-reside on secretory vesicles may function cooperatively and redundantly as potential Ca(2+) sensors for exocytosis.     

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.     

Activity-dependent IGF-1 exocytosis is controlled by the Ca(2+)-sensor synaptotagmin-10

Synaptotagmins Syt1, Syt2, Syt7, and Syt9 act as Ca(2+)-sensors for synaptic and neuroendocrine exocytosis, but the function of other synaptotagmins remains unknown. Here, we show that olfactory bulb neurons secrete IGF-1 by an activity-dependent pathway of exocytosis, and that Syt10 functions as the Ca(2+)-sensor that triggers IGF-1 exocytosis in these neurons. Deletion of Syt10 impaired activity-dependent IGF-1 secretion in olfactory bulb neurons, resulting in smaller neurons and an overall decrease in synapse numbers. Exogenous IGF-1 completely reversed the Syt10 knockout phenotype. Syt10 colocalized with IGF-1 in somatodendritic vesicles of olfactory bulb neurons, and Ca(2+)-binding to Syt10 caused these vesicles to undergo exocytosis, thereby secreting IGF-1. Thus, Syt10 controls a previously unrecognized pathway of Ca(2+)-dependent exocytosis that is spatially and temporally distinct from Ca(2+)-dependent synaptic vesicle exocytosis controlled by Syt1. Our findings thereby reveal that two different synaptotagmins can regulate functionally distinct Ca(2+)-dependent membrane fusion reactions in the same neuron.     

Neurotransmitter release at rapid synapses

The classical concept of the vesicular hypothesis for acetylcholine (ACh) release, one quantum resulting from exocytosis of one vesicle, is becoming more complicated than initially thought. 1) synaptic vesicles do contain ACh, but the cytoplasmic pool of ACh is the first to be used and renewed on stimulation. 2) The vesicles store not only ACh, but also ATP and Ca(2+) and they are critically involved in determining the local Ca(2+) microdomains which trigger and control release. 3) The number of exocytosis pits does increase in the membrane upon nerve stimulation, but in most cases exocytosis happens after the precise time of release, while it is a change affecting intramembrane particles which reflects more faithfully the release kinetics. 4) The SNARE proteins, which dock vesicles close to Ca(2+) channels, are essential for the excitation-release coupling, but quantal release persists when the SNAREs are inactivated or absent. 5) The quantum size is identical at the neuromuscular and nerve-electroplaque junctions, but the volume of a synaptic vesicle is eight times larger in electric organ; at this synapse there is enough ACh in a single vesicle to generate 15-25 large quanta, or 150-200 subquanta. These contradictions may be only apparent and can be resolved if one takes into account that an integral plasmalemmal protein can support the formation of ACh quanta. Such a protein has been isolated, characterised and called mediatophore. Mediatophore has been localised at the active zones of presynaptic nerve terminals. It is able to release ACh with the expected Ca(2+)-dependency and quantal character, as demonstrated using mediatophore-transfected cells and other reconstituted systems. Mediatophore is believed to work like a pore protein, the regulation of which is in turn likely to depend on the SNARE-vesicle docking apparatus.     

Vesicular Ca(2+) mediates granule motion and exocytosis

Secretory vesicles of chromaffin cells are acidic organelles that maintain an increasing pH gradient towards the cytosol (5.5 vs. 7.3) that is mediated by V-ATPase activity. This gradient is primarily responsible for the accumulation of large concentrations of amines and Ca(2+), although the mechanisms mediating Ca(2+) uptake and release from granules, and the physiological relevance of these processes, remain unclear. The presence of a vesicular matrix appears to create a bi-compartmentalised medium in which the major fractions of solutes, including catecholamines, nucleotides and Ca(2+), are strongly associated with vesicle proteins, particularly chromogranins. This association appears to be favoured at acidic pH values. It has been demonstrated that disrupting the pH gradient of secretory vesicles reduces their rate of exocytosis and promotes the leakage of vesicular amines and Ca(2+), dramatically increasing the movement of secretory vesicles and triggering exocytosis. In this short review, we will discuss the data available that highlights the importance of pH in regulating the association between chromogranins, vesicular amines and Ca(2+). We will also address the potential role of vesicular Ca(2+) in two major processes in secretory cells, vesicle movement and exocytosis.     

Imaging Ca2+ concentration changes at the secretory vesicle surface with a recombinant targeted cameleon.

Regulated exocytosis involves the Ca(2+)-triggered fusion of secretory vesicles with the plasma membrane, by activation of vesicle membrane Ca(2+)-binding proteins [1]. The Ca(2+)-binding sites of these proteins are likely to lie within 30 nm of the vesicle surface, a domain in which changes in Ca2+ concentration cannot be resolved by conventional fluorescence microscopy. A fluorescent indicator for Ca2+ called a yellow 'cameleon' (Ycam2) - comprising a fusion between a cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13 and an enhanced yellow-emitting GFP - which is targetable to specific intracellular locations, has been described [2]. Here, we generated a fusion between phogrin, a protein that is localised to secretory granule membranes [3], and Ycam2 (phogrin-Ycam2) to monitor changes in Ca2+ concentration ([Ca2+]) at the secretory vesicle surface ([Ca2+]gd) through alterations in fluorescence resonance energy transfer (FRET) between the linked cyan and yellow fluorescent proteins (CFP and YFP, respectively) in Ycam2. In both neuroendocrine PC12 and MIN6 pancreatic beta cells, apparent resting values of cytosolic [Ca2+] and [Ca2+](gd) were similar throughout the cell. In MIN6 cells following the activation of Ca2+ influx, the minority of vesicles that were within approximately 1 microm of the plasma membrane underwent increases in [Ca2+](gd) that were significantly greater than those experienced by deeper vesicles, and greater than the apparent cytosolic [Ca2+] change. The ability to image both global and compartmentalised [Ca2+] changes with recombinant targeted cameleons should extend the usefulness of these new Ca2+ probes.     

Interaction between the vesicular glutamate transporter type 1 and endophilin A1, a protein essential for endocytosis

In the nerve terminal, neurotransmitter is actively packaged into synaptic vesicles before its release by Ca2+-dependent exocytosis. The three vesicular glutamate transporters (VGLUT1, -2 and -3) are highly conserved proteins that display similar bioenergetic and pharmacological properties but are expressed in different brain areas. We used the divergent C-terminus of VGLUT1 as a bait in a yeast two-hybrid screen to identify and map the interaction between a proline-rich domain of VGLUT1 and the Src homology domain 3 (SH3) domain of endophilin. We further confirmed this interaction by using different glutathione-S-transferase-endophilin fusion proteins to pull down VGLUT1 from rat brain extracts. The expression profiles of the two genes and proteins were compared on rat brain sections, showing that endophilin is most highly expressed in regions and cells expressing VGLUT1. Double immunofluorescence in the rat cerebellum shows that most VGLUT1-positive terminals co-express endophilin, whereas VGLUT2-expressing terminals are often devoid of endophilin. However, neither VGLUT1 transport activity, endophilin enzymatic activity nor VGLUT1 synaptic targeting were altered by this interaction. Overall, the discovery of endophilin as a partner for VGLUT1 in nerve terminals strongly suggests the existence of functional differences between VGLUT1 and -2 terminals in their abilities to replenish vesicle pools.     

Calcium gradients and exocytosis in bovine adrenal chromaffin cells

The relationship between the localized Ca(2+) concentration and depolarization-induced exocytosis was studied in patch-clamped adrenal chromaffin cells using pulsed-laser Ca(2+) imaging and membrane capacitance measurements. Short depolarizing voltage steps induced Ca(2+) gradients and small "synchronous" increases in capacitance during the pulses. Longer pulses increased the capacitance changes, which saturated at 16 fF, suggesting the presence of a small immediately releasable pool of fusion-ready vesicles. A Hill plot of the capacitance changes versus the estimated Ca(2+) concentration in a thin (100 nm) shell beneath the membrane gave n = 2.3 and K(d) = 1.4 microM. Repetitive stimulation elicited a more complex pattern of exocytosis: early pulses induced synchronous capacitance increases, but after five or more pulses there was facilitation of the synchronous responses and gradual increases in capacitance continued between pulses (asynchronous exocytosis) as the steep submembrane Ca(2+) gradients collapsed. Raising the pipette Ca(2+) concentration led to early facilitation of the synchronous response and early appearance of asynchronous exocytosis. We used this data to develop a kinetic model of depolarization-induced exocytosis, where Ca(2+)-dependent fusion of vesicles occurs from a small immediately releasable pool with an affinity of 1-2 microM and vesicles are mobilized to this pool in a Ca(2+)-dependent manner.     

Munc18-1 promotes large dense-core vesicle docking.

Secretory vesicles dock at the plasma membrane before Ca(2+) triggers their exocytosis. Exocytosis requires the assembly of SNARE complexes formed by the vesicle protein Synaptobrevin and the membrane proteins Syntaxin-1 and SNAP-25. We analyzed the role of Munc18-1, a cytosolic binding partner of Syntaxin-1, in large dense-core vesicle (LDCV) secretion. Calcium-dependent LDCV exocytosis was reduced 10-fold in mouse chromaffin cells lacking Munc18-1, but the kinetic properties of the remaining release, including single fusion events, were not different from controls. Concomitantly, mutant cells displayed a 10-fold reduction in morphologically docked LDCVs. Moreover, acute overexpression of Munc18-1 in bovine chromaffin cells increased the amount of releasable vesicles and accelerated vesicle supply. We conclude that Munc18-1 functions upstream of SNARE complex formation and promotes LDCV docking.     

Botulinum neurotoxin A blocks synaptic vesicle exocytosis but not endocytosis at the nerve terminal

The supply of synaptic vesicles in the nerve terminal is maintained by a temporally linked balance of exo- and endocytosis. Tetanus and botulinum neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins identified as critical for synaptic vesicle exocytosis. We show here that botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis does not arrest vesicle membrane endocytosis. In the murine spinal cord, cell cultures exposed to botulinum neurotoxin A, neither K(+)-evoked neurotransmitter release nor synaptic currents can be detected, twice the ordinary number of synaptic vesicles are docked at the synaptic active zone, and its protein substrate is cleaved, which is similar to observations with tetanus and other botulinal neurotoxins. In marked contrast, K(+) depolarization, in the presence of Ca(2+), triggers the endocytosis of the vesicle membrane in botulinum neurotoxin A-blocked cultures as evidenced by FM1-43 staining of synaptic terminals and uptake of HRP into synaptic vesicles. These experiments are the first demonstration that botulinum neurotoxin A uncouples vesicle exo- from endocytosis, and provide evidence that Ca(2+) is required for synaptic vesicle membrane retrieval.     

Exocytotic release of ATP from cultured astrocytes

Astrocytes appear to communicate with each other as well as with neurons via ATP. However, the mechanisms of ATP release are controversial. To explore whether stimuli that increase [Ca(2+)](i) also trigger vesicular ATP release from astrocytes, we labeled ATP-containing vesicles with the fluorescent dye quinacrine, which exhibited a significant co-localization with atrial natriuretic peptide. The confocal microscopy study revealed that quinacrine-loaded vesicles displayed mainly non-directional spontaneous mobility with relatively short track lengths and small maximal displacements, whereas 4% of vesicles exhibited directional mobility. After ionomycin stimulation only non-directional vesicle mobility could be observed, indicating that an increase in [Ca(2+)](i) attenuated vesicle mobility. Total internal reflection fluorescence (TIRF) imaging in combination with epifluorescence showed that a high percentage of fluorescently labeled vesicles underwent fusion with the plasma membrane after stimulation with glutamate or ionomycin and that this event was Ca(2+)-dependent. This was confirmed by patch-clamp studies on HEK-293T cells transfected with P2X(3) receptor, used as sniffers for ATP release from astrocytes. Glutamate stimulation of astrocytes was followed by an increase in the incidence of small transient inward currents in sniffers, reminiscent of postsynaptic quantal events observed at synapses. Their incidence was highly dependent on extracellular Ca(2+). Collectively, these findings indicate that glutamate-stimulated ATP release from astrocytes was most likely exocytotic and that after stimulation the fraction of quinacrine-loaded vesicles, spontaneously exhibiting directional mobility, disappeared.     

Synaptotagmin C2A loop 2 mediates Ca2+-dependent SNARE interactions essential for Ca2+-triggered vesicle exocytosis

Synaptotagmins contain tandem C2 domains and function as Ca(2+) sensors for vesicle exocytosis but the mechanism for coupling Ca(2+) rises to membrane fusion remains undefined. Synaptotagmins bind SNAREs, essential components of the membrane fusion machinery, but the role of these interactions in Ca(2+)-triggered vesicle exocytosis has not been directly assessed. We identified sites on synaptotagmin-1 that mediate Ca(2+)-dependent SNAP25 binding by zero-length cross-linking. Mutation of these sites in C2A and C2B eliminated Ca(2+)-dependent synaptotagmin-1 binding to SNAREs without affecting Ca(2+)-dependent membrane binding. The mutants failed to confer Ca(2+) regulation on SNARE-dependent liposome fusion and failed to restore Ca(2+)-triggered vesicle exocytosis in synaptotagmin-deficient PC12 cells. The results provide direct evidence that Ca(2+)-dependent SNARE binding by synaptotagmin is essential for Ca(2+)-triggered vesicle exocytosis and that Ca(2+)-dependent membrane binding by itself is insufficient to trigger fusion. A structure-based model of the SNARE-binding surface of C2A provided a new view of how Ca(2+)-dependent SNARE and membrane binding occur simultaneously.     

Bovine chromaffin granule membranes undergo Ca(2+)-regulated exocytosis in frog oocytes

We have devised a new method that permits the investigation of exogenous secretory vesicle function using frog oocytes and bovine chromaffin granules, the secretory vesicles from adrenal chromaffin cells. Highly purified chromaffin granule membranes were injected into Xenopus laevis oocytes. Exocytosis was detected by the appearance of dopamine-beta-hydroxylase of the chromaffin granule membrane in the oocyte plasma membrane. The appearance of dopamine-beta-hydroxylase on the oocyte surface was strongly Ca(2+)-dependent and was stimulated by coinjection of the chromaffin granule membranes with InsP3 or Ca2+/EGTA buffer (18 microM free Ca2+) or by incubation of the injected oocytes in medium containing the Ca2+ ionophore ionomycin. Similar experiments were performed with a subcellular fraction from cultured chromaffin cells enriched with [3H]norepinephrine-containing chromaffin granules. Because the release of [3H]norepinephrine was strongly correlated with the appearance of dopamine-beta-hydroxylase on the oocyte surface, it is likely that intact chromaffin granules and chromaffin granule membranes undergo exocytosis in the oocyte. Thus, the secretory vesicle membrane without normal vesicle contents is competent to undergo the sequence of events leading to exocytosis. Furthermore, the interchangeability of mammalian and amphibian components suggests substantial biochemical conservation of the regulated exocytotic pathway during the evolutionary progression from amphibians to mammals.     

Complexin arrests a pool of docked vesicles for fast Ca(2+)-dependent release

Regulated exocytosis requires that the assembly of the basic membrane fusion machinery is temporarily arrested. Synchronized membrane fusion is then caused by a specific trigger-a local rise of the Ca(2+) concentration. Using reconstituted giant unilamellar vesicles (GUVs), we have analysed the role of complexin and membrane-anchored synaptotagmin 1 in arresting and synchronizing fusion by lipid-mixing and cryo-electron microscopy. We find that they mediate the formation and consumption of docked small unilamellar vesicles (SUVs) via the following sequence of events: Synaptotagmin 1 mediates v-SNARE-SUV docking to t-SNARE-GUVs in a Ca(2+)-independent manner. Complexin blocks vesicle consumption, causing accumulation of docked vesicles. Together with synaptotagmin 1, complexin synchronizes and stimulates rapid fusion of accumulated docked vesicles in response to physiological Ca(2+) concentrations. Thus, the reconstituted assay resolves both the stimulatory and inhibitory function of complexin and mimics key aspects of synaptic vesicle fusion.     

A single vesicular glutamate transporter is sufficient to fill a synaptic vesicle

Quantal size is the postsynaptic response to the release of a single synaptic vesicle and is determined in part by the amount of transmitter within that vesicle. At glutamatergic synapses, the vesicular glutamate transporter (VGLUT) fills vesicles with glutamate. While elevated VGLUT expression increases quantal size, the minimum number of transporters required to fill a vesicle is unknown. In Drosophila DVGLUT mutants, reduced transporter levels lead to a dose-dependent reduction in the frequency of spontaneous quantal release with no change in quantal size. Quantal frequency is not limited by vesicle number or impaired exocytosis. This suggests that a single functional unit of transporter is both necessary and sufficient to fill a vesicle to completion and that vesicles without DVGLUT are empty. Consistent with the presence of empty vesicles, at dvglut mutant synapses synaptic vesicles are smaller, suggesting that vesicle filling and/or transporter level is an important determinant of vesicle size.     

Vesicular Ca(2+) -induced secretion promoted by intracellular pH-gradient disruption

The actions of the protonophore CCCP on intracellular Ca2+ regulation and exocytosis in chromaffin cells have been examined. Simultaneous fura-2 imaging and amperometry reveal that exposure to CCCP not only perturbs mitochondrial function but that it also alters vesicular storage of Ca2+ and catecholamines. By disrupting the pH gradient of the secretory vesicle membrane, the protonophore allows both Ca(2+) and catecholamine to leak into the cytosol. Unlike the high cytosolic Ca2+ concentrations resulting from mitochondrial membrane disruption, Ca2+ leakage from secretory vesicles may initiate exocytotic release. In conjunction with previous studies, this work reveals that catalytic and self-sustained vesicular Ca(2+) -induced exocytosis occurs with extended exposure to weak acid or base protonophores.     

Developmental acquisition of a rapid calcium-regulated vesicle supply allows sustained high rates of exocytosis in auditory hair cells

Auditory hair cells (HCs) have the remarkable property to indefinitely sustain high rates of synaptic vesicle release during ongoing sound stimulation. The mechanisms of vesicle supply that allow such indefatigable exocytosis at the ribbon active zone remain largely unknown. To address this issue, we characterized the kinetics of vesicle recruitment and release in developing chick auditory HCs. Experiments were done using the intact chick basilar papilla from E10 (embryonic day 10) to P2 (two days post-hatch) by monitoring changes in membrane capacitance and Ca(2+) currents during various voltage stimulations. Compared to immature pre-hearing HCs (E10-E12), mature post-hearing HCs (E18-P2) can steadily mobilize a larger readily releasable pool (RRP) of vesicles with faster kinetics and higher Ca(2+) efficiency. As assessed by varying the inter-pulse interval of a 100 ms paired-pulse depolarization protocol, the kinetics of RRP replenishment were found much faster in mature HCs. Unlike mature HCs, exocytosis in immature HCs showed large depression during repetitive stimulations. Remarkably, when the intracellular concentration of EGTA was raised from 0.5 to 2 mM, the paired-pulse depression level remained unchanged in immature HCs but was drastically increased in mature HCs, indicating that the Ca(2+) sensitivity of the vesicle replenishment process increases during maturation. Concomitantly, the immunoreactivity of the calcium sensor otoferlin and the number of ribbons at the HC plasma membrane largely increased, reaching a maximum level at E18-P2. Our results suggest that the efficient Ca(2+)-dependent vesicle release and supply in mature HCs essentially rely on the concomitant engagement of synaptic ribbons and otoferlin at the plasma membrane.