Fast Exocytosis Research Articles

Kinetic efficiency of endocytosis at mammalian CNS synapses requires synaptotagmin I.

At nerve terminals, synaptic vesicle components are retrieved from the cell surface and recycled for local reuse soon after exocytosis. The kinetics of this coupling is critical for the proper functioning of synapses during repetitive action potential firing, because deficiencies in this process lead to abnormal depletion of the releasable vesicle pool. Although the molecular basis of this coupling is poorly understood, numerous biochemical data point to a role for synaptotagmin I (SytI), an essential synaptic vesicle protein required for fast calcium-dependent exocytosis. Here, using synapto-pHluorin in an approach that allows the dissection of endocytosis and exocytosis into separate components during periods of stimulation, we examined exocytic-endocytic coupling in synapses from SytI knockout mice and their WT littermates. We show that endocytosis is significantly impaired in the absence of SytI with the relative rates of endocytosis compared with exocytosis reduced approximately 3-fold with respect to WT. Thus, in addition to regulating exocytosis, SytI also controls the kinetic efficiency of endocytosis at nerve terminals.

Synaptobrevin is essential for fast synaptic-vesicle endocytosis.

Synaptobrevin-2 (VAMP-2), the major SNARE protein of synaptic vesicles, is required for fast calcium-triggered synaptic-vesicle exocytosis. Here we show that synaptobrevin-2 is also essential for fast synaptic-vesicle endocytosis. We demonstrate that after depletion of the readily releasable vesicle pool, replenishment of the pool is delayed by knockout of synaptobrevin. This delay was not from a loss of vesicles, because the total number of pre-synaptic vesicles, docked vesicles and actively recycling vesicles was unaffected. However, altered shape and size of the vesicles in synaptobrevin-deficient synapses suggests a defect in endocytosis. Consistent with such a defect, the stimulus-dependent endocytosis of horseradish peroxidase and fluorescent FM1-43 were delayed, indicating that fast vesicle endocytosis may normally be nucleated by a SNARE-dependent coat. Thus, synaptobrevin is essential for two fast synapse-specific membrane trafficking reactions: fast exocytosis for neurotransmitter release and fast endocytosis that mediates rapid reuse of synaptic vesicles.

Evidence that fast exocytosis can be predominantly mediated by vesicles not docked at active zones in frog saccular hair cells.

The prevailing model of neurotransmitter release stipulates that Ca2+ influx triggers the rapid fusion of vesicles that are docked at presynaptic active zones. Under this model, slower tonic release is supported by vesicles clustered nearby that have to translocate to the release sites before fusion. We have examined this hypothesis at the afferent synapse of saccular hair cells of the leopard frog, Rana pipiens. Detailed morphological measurements at this ribbon synapse show that on average 32 vesicles are docked at each active zone. We show that at this 'graded' synapse, depolarization produces an exocytotic 'burst' that is largely complete within 20 ms after fusion of 280 vesicles per active zone, almost an order of magnitude more than expected. Recovery from paired pulse depression occurs with a time constant of 29 ms, indicating that replenishment of this fast-fusing pool of vesicles is also fast. Our results suggest that non-docked vesicles are capable of fast fusion and that these vesicles constitute the vast majority of the fast-fusing pool. The view that the population of fast-fusing presynaptic vesicles is limited to docked vesicles therefore requires re-evaluation. We propose that compound fusion, i.e. the fusion of vesicles with each other before and/or after they fuse with the membrane can explain multivesicular release at this synapse.

Examining synaptotagmin 1 function in dense core vesicle exocytosis under direct control of Ca2+.

We tested the long-standing hypothesis that synaptotagmin 1 is the Ca2+ sensor for fast neurosecretion by analyzing the intracellular Ca2+ dependence of large dense-core vesicle exocytosis in a mouse strain carrying a mutated synaptotagmin C2A domain. The mutation (R233Q) causes a twofold increase in the KD of Ca2+-dependent phospholipid binding to the double C2A-C2B domain of synaptotagmin. Using photolysis of caged calcium and capacitance measurements we found that secretion from mutant cells had lower secretory rates, longer secretory delays, and a higher intracellular Ca2+-threshold for secretion due to a twofold increase in the apparent KD of the Ca2+ sensor for fast exocytosis. Single amperometric fusion events were unchanged. We conclude that Ca2+-dependent phospholipid binding to synaptotagmin 1 mirrors the intracellular Ca2+ dependence of exocytosis.

Properties of exocytotic response in vertebrate photoreceptors.

Synaptic transmission at the photoreceptor synapse is characterized by continuous release of glutamate in darkness. Release is regulated by the intracellular calcium concentration ([Ca2+]i). We here examined the physiological properties of exocytosis in tiger salamander (Ambystoma tigrinum) retinal rods and cones. Patch-clamp capacitance measurements were used to monitor exocytosis elicited by a rapid and uniform increase in [Ca2+]i by photolysis of the caged Ca2+ compound NP-EGTA. The amplitude of flash-induced increases in membrane capacitance (Cm) varied monotonically with [Ca2+]i beyond approximately 15 microM. The following two types of kinetic responses in Cm were recorded in both rods and cones: 1) a single exponential rise (39% of cells) or 2) a double-exponential rise (61%). Average rate constants of rapid and slow exocytotic responses were 420 +/- 168 and 7.85 +/- 5.02 s-1, respectively. The rate constant for the single exponential exocytotic response was 17.5 +/- 12.4 s-1, not significantly different from that of the slow exocytotic response. Beyond the threshold [Ca2+]i of approximately 15 microM, the average amplitude of rapid, slow, and single Cm response were 0.84 +/- 0.35, 0.82 +/- 0.20, and 0.70 +/- 0.23 pF, respectively. Antibodies against synaptotagmin I, a vesicle protein associated with fast exocytosis, strongly stained the synaptic terminal of isolated photoreceptors, suggesting the presence of fusion-competent vesicles. Our results confirm that photoreceptors possess a large rapidly releasable pool activated by a low-affinity Ca2+ sensor whose kinetic and calcium-dependent properties are similar to those reported in retinal bipolar cells and cochlear hair cells.

Mechanism of Calcium-independent Synaptotagmin Binding to Target SNAREs

Synaptic vesicle exocytosis requires three SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) proteins: syntaxin and SNAP-25 on the plasma membrane (t-SNAREs) and synaptobrevin/VAMP on the synaptic vesicles (v-SNARE). Vesicular synaptotagmin 1 is essential for fast synchronous SNARE-mediated exocytosis and interacts with the SNAREs in brain material. To uncover the step at which synaptotagmin becomes linked to the three SNAREs, we purified all four proteins from brain membranes and analyzed their interactions. Our study reveals that, in the absence of calcium, native synaptotagmin 1 binds the t-SNARE heterodimer, formed from syntaxin and SNAP-25. This interaction is both stoichiometric and of high affinity. Synaptotagmin contains two divergent but conserved C2 domains that can act independently in calcium-triggered phospholipid binding. We now show that both C2 domains are strictly required for the calcium-independent interaction with the t-SNARE heterodimer, indicating that the double C2 domain structure of synaptotagmin may have evolved to acquire a function beyond calcium/phospholipid binding.

Protein Kinase C-Dependent Phosphorylation of Synaptosome-Associated Protein of 25 kDa at Ser187Potentiates Vesicle Recruitment

Activation of protein kinase C (PKC) constitutes a key event in the upregulation of secretory strength in neurons and neurosecretory cells during extensive stimulation, presumably by speeding up vesicle supply. However, the molecular targets and their mode of action remain elusive. We studied the only PKC-dependent phosphorylation site in the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, Ser(187), in synaptosome-associated protein of 25 kDa (SNAP-25). This phosphorylation site is located within the negatively charged C-terminal end of SNAP-25, which has been shown to be of critical importance in calcium-triggered exocytosis. We combined mutational studies that used overexpression in chromaffin cells with capacitance measurements and flash photolysis of caged calcium, allowing for high time resolution during both the stimulation and measurement of exocytosis. Overexpression of mutants simulating the phosphorylated form of Ser(187) accelerated vesicle recruitment after the emptying of the releasable vesicle pools. Overexpression of mutants simulating the nonphosphorylated form, or block of PKC, impaired the refilling of the vesicle pools to similar extents. Biochemical studies verified the phosphorylation of a subpopulation of SNAP-25 after elevation of intracellular calcium concentrations. Some of the mutations led to a moderately decreased fast exocytotic burst component, which did not seem to be associated with the phosphorylation state of SNAP-25. Thus the C terminus of SNAP-25 plays a role for both fast exocytosis triggering and vesicle recruitment, and the latter process is regulated by PKC-dependent phosphorylation.

Structure/Function Analysis of Ca2+Binding to the C2A Domain of Synaptotagmin 1

Synaptotagmin 1, a Ca2+ sensor for fast synaptic vesicle exocytosis, contains two C2 domains that form Ca2+-dependent complexes with phospholipids. To examine the functional importance of Ca2+ binding to the C2A domain of synaptotagmin 1, we studied two C2A domain mutations, D232N and D238N, using recombinant proteins and knock-in mice. Both mutations severely decreased intrinsic Ca2+ binding and Ca2+-dependent phospholipid binding by the isolated C2A domain. Both mutations, however, did not alter the apparent Ca2+ affinity of the double C2 domain fragment, although both decreased the tightness of the Ca2+/phospholipid/double C2 domain complex. When introduced into the endogenous synaptotagmin 1 gene in mice, the D232N and D238N mutations had no apparent effect on morbidity and mortality and caused no detectable alteration in the Ca2+-dependent properties of synaptotagmin 1. Electrophysiological recordings of cultured hippocampal neurons from knock-in mice revealed that neither mutation induced major changes in synaptic transmission. The D232N mutation, however, caused increased synaptic depression during repetitive stimulation, whereas the D238N mutation did not exhibit this phenotype. Our data indicate that Ca2+ binding to the C2A domain of synaptotagmin 1 may be important but not essential, consistent with the finding that the two C2 domains cooperate and may be partially redundant in Ca2+-dependent phospholipid binding. Moreover, although the apparent Ca2+ affinity of the synaptotagmin 1/phospholipid complex is critical, the tightness of the Ca2+/phospholipid complex is not. Our data also demonstrate that subtle changes in the biochemical properties of synaptotagmin 1 can result in significant alterations in synaptic responses.

Short-term synaptic plasticity.

Synaptic transmission is a dynamic process. Postsynaptic responses wax and wane as presynaptic activity evolves. This prominent characteristic of chemical synaptic transmission is a crucial determinant of the response properties of synapses and, in turn, of the stimulus properties selected by neural networks and of the patterns of activity generated by those networks. This review focuses on synaptic changes that result from prior activity in the synapse under study, and is restricted to short-term effects that last for at most a few minutes. Forms of synaptic enhancement, such as facilitation, augmentation, and post-tetanic potentiation, are usually attributed to effects of a residual elevation in presynaptic [Ca(2+)]i, acting on one or more molecular targets that appear to be distinct from the secretory trigger responsible for fast exocytosis and phasic release of transmitter to single action potentials. We discuss the evidence for this hypothesis, and the origins of the different kinetic phases of synaptic enhancement, as well as the interpretation of statistical changes in transmitter release and roles played by other factors such as alterations in presynaptic Ca(2+) influx or postsynaptic levels of [Ca(2+)]i. Synaptic depression dominates enhancement at many synapses. Depression is usually attributed to depletion of some pool of readily releasable vesicles, and various forms of the depletion model are discussed. Depression can also arise from feedback activation of presynaptic receptors and from postsynaptic processes such as receptor desensitization. In addition, glial-neuronal interactions can contribute to short-term synaptic plasticity. Finally, we summarize the recent literature on putative molecular players in synaptic plasticity and the effects of genetic manipulations and other modulatory influences.

Synaptotagmins form a hierarchy of exocytotic Ca(2+) sensors with distinct Ca(2+) affinities.

Synaptotagmins constitute a large family of membrane proteins implicated in Ca(2+)-triggered exocytosis. Structurally similar synaptotagmins are differentially localized either to secretory vesicles or to plasma membranes, suggesting distinct functions. Using measurements of the Ca(2+) affinities of synaptotagmin C2-domains in a complex with phospholipids, we now show that different synaptotagmins exhibit distinct Ca(2+) affinities, with plasma membrane synaptotagmins binding Ca(2+) with a 5- to 10-fold higher affinity than vesicular synaptotagmins. To test whether these differences in Ca(2+) affinities are functionally important, we examined the effects of synaptotagmin C2-domains on Ca(2+)-triggered exocytosis in permeabilized PC12 cells. A precise correlation was observed between the apparent Ca(2+) affinities of synaptotagmins in the presence of phospholipids and their action in PC12 cell exocytosis. This was extended to PC12 cell exocytosis triggered by Sr(2+), which was also selectively affected by high-affinity C2-domains of synaptotagmins. Together, our results suggest that Ca(2+) triggering of exocytosis involves tandem Ca(2+) sensors provided by distinct plasma membrane and vesicular synaptotagmins. According to this hypothesis, plasma membrane synaptotagmins represent high-affinity Ca(2+) sensors involved in slow Ca(2+)-dependent exocytosis, whereas vesicular synaptotagmins function as low-affinity Ca(2+) sensors specialized for fast Ca(2+)-dependent exocytosis.

Endogenous Calcium Buffers Regulate Fast Exocytosis in the Synaptic Terminal of Retinal Bipolar Cells

Calcium-triggered exocytosis at the synapse is suppressed by addition of calcium chelators, but the effects of endogenous Ca 2+ buffers have not been tested. We find that 80% of Ca 2+ binding sites in the synaptic terminal of retinal bipolar cells were associated with mobile molecules that suppressed activation of Ca 2+-sensitive K + channels with an efficiency equivalent to ∼1.2 mM BAPTA. Removing these buffers caused a 30-fold increase in the number of vesicles released by Ca 2+ tail currents lasting ∼0.5 ms and a 2-fold increase in the rapidly releasable pool of vesicles (RRP). The effects of BAPTA and EGTA indicate that vesicles comprising the RRP were docked at variable distances from Ca 2+ channels. We propose that endogenous Ca 2+ buffers regulate the size of the RRP by suppressing the release of vesicles toward the periphery of the active zone.

Fast Exocytosis with Few Ca 2+ Channels in Insulin-Secreting Mouse Pancreatic B Cells

The association of L-type Ca 2+ channels to the secretory granules and its functional significance to secretion was investigated in mouse pancreatic B cells. Nonstationary fluctuation analysis showed that the B cell is equipped with <500 α1 C L-type Ca 2+ channels, corresponding to a Ca 2+ channel density of 0.9 channels per μm 2. Analysis of the kinetics of exocytosis during voltage-clamp depolarizations revealed an early component that reached a peak rate of 1.1 pFs −1 (≈650 granules/s) 25 ms after onset of the pulse and is completed within ∼100 ms. This component represents a subset of ≈60 granules situated in the immediate vicinity of the L-type Ca 2+ channels, corresponding to ∼10% of the readily releasable pool of granules. Experiments involving photorelease of caged Ca 2+ revealed that the rate of exocytosis was half-maximal at a cytoplasmic Ca 2+ concentration of 17 μM, and concentrations >25 μM are required to attain the rate of exocytosis observed during voltage-clamp depolarizations. The rapid component of exocytosis was not affected by inclusion of millimolar concentrations of the Ca 2+ buffer EGTA but abolished by addition of exogenous L C753–893, the 140 amino acids of the cytoplasmic loop connecting the 2 nd and 3 rd transmembrane region of the α1 C L-type Ca 2+ channel, which has been proposed to tether the Ca 2+ channels to the secretory granules. In keeping with the idea that secretion is determined by Ca 2+ influx through individual Ca 2+ channels, exocytosis triggered by brief (15 ms) depolarizations was enhanced 2.5-fold by the Ca 2+ channel agonist BayK8644 and 3.5-fold by elevating extracellular Ca 2+ from 2.6 to 10 mM. Recordings of single Ca 2+ channel activity revealed that patches predominantly contained no channels or many active channels. We propose that several Ca 2+ channels associate with a single granule thus forming a functional unit. This arrangement is important in a cell with few Ca 2+ channels as it ensures maximum usage of the Ca 2+ entering the cell while minimizing the influence of stochastic variations of the Ca 2+ channel activity.

The actions of barium and strontium on exocytosis and endocytosis in the synaptic terminal of goldfish bipolar cells.

1. We investigated the properties of Ca2+-sensitive steps in the cycling of synaptic vesicles by comparing the actions of Ca2+, Ba2+ and Sr2+ in the synaptic terminal of depolarizing bipolar cells isolated from the retina of goldfish. FM1-43 fluorescence and capacitance measurements demonstrated that exocytosis, endocytosis and vesicle mobilization were maintained when external Ca2+ was replaced by either Ba2+ or Sr2+. 2. The rapidly releasable pool of vesicles (RRP) was equivalent to 1.5 % of the membrane surface area when measured in the presence of 2.5 mM Ca2+, but only 0.4 % in 2.5 mM Sr2+. The relative sizes of the RRP in Ca2+, Sr2+ and Ba2+ were 1.0, 0.28 and 0.1, respectively. We conclude that a smaller proportion of docked vesicles are available for fast exocytosis triggered by the influx of Sr2+ or Ba2+ compared to Ca2+. 3. The slow phase of exocytosis was not altered when Ca2+ was replaced by Ba2+, but it was accelerated 1.6-fold in Sr2+. The peak concentrations of Ca2+, Sr2+ and Ba2+ (measured using Mag-fura-5) were approximately 4, approximately 14 and approximately 60 microM, respectively. The order of efficiency for the stimulation of slow exocytosis was Ca2+ approximately Sr2+ > Ba2+. 4. Exocytosis was prolonged after the influx of Sr2+ and Ba2+. Sr2+ was cleared from the synaptic terminal with the same time constant as Ca2+ (1.3 s), but Ba2+ was cleared 10-100 times more slowly. Although Ba(2+) stimulates the slow release of a large number of vesicles, it did so less efficiently than Ca2+ or Sr2+. 5. The recovery of the membrane capacitance was equally rapid in Sr2+ and Ca2+, demonstrating that the fast mode of endocytosis could be triggered by either cation.

The heterotrimeric Gi(3) protein acts in slow but not in fast exocytosis of rat melanotrophs.

Besides having a role in signal transduction some trimeric G-proteins may be involved in a late stage of exocytosis. Using immunocytochemistry and confocal microscopy we found that Gi(3)-protein resides mainly in the plasma membrane, whereas Gi(1/2-)protein is preferentially associated with secretory granules. To study the function of trimeric Gi(3)- and Gi(1/2)-proteins, secretory responses in single rat melanotrophs were monitored by patch-clamp membrane capacitance measurements. We report here that mastoparan, an activator of trimeric G-proteins, enhances calcium-induced secretory activity in rat melanotrophs. The introduction of synthetic peptides corresponding to the C-terminal domain of the (&agr;)-subunit of Gi(3)- and Gi(1/2)-proteins indicated that Gi(3 )peptide specifically blocked the mastoparan-stimulated secretory activity, which indicates an involvement of a trimeric Gi(3)-protein in mastoparan-stimulated secretory activity. Flash photolysis of caged Ca(2+)-elicited biphasic capacitance increases consisting of a fast and a slower component. Injection of anti-Gi(3) antibodies selectively inhibited the slow but not the fast component of secretory activity in rat melanotrophs. We propose that the plasma membrane-bound Gi(3)-protein may be involved in regulated secretion by specifically controlling the slower kinetic component of exocytosis.

Diffusion Barriers Limit the Effect of Mobile Calcium Buffers on Exocytosis of Large Dense Cored Vesicles

Fast exocytosis in melanotropic cells, activated by calcium entry through voltage-gated calcium channels, is very sensitive to mobile calcium buffers (complete block at 800 μM ethylene glycol bis( β-aminoethyl ether)- N, N, N′N′-tetraacetic acid (EGTA)). This indicates that calcium diffuses a substantial distance from the channel to the vesicle. Surprisingly, 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid (BAPTA), having a similar K D for calcium as EGTA but a ∼100 times faster binding rate, blocked exocytosis only twice as effectively as EGTA. Using computer simulations, we demonstrate that this result cannot be explained by free diffusion and buffer binding rates. We hypothesized that local saturation of calcium buffers is involved. A diffusion barrier for both calcium and buffer molecules, located 50–300 nm from the membrane and reducing diffusion 1000 to 10,000 times, generated similar calcium concentrations for specific concentrations of EGTA and BAPTA. With such barriers, calcium rise phase kinetics upon short step depolarizations (2–20 ms) were faster for EGTA than for BAPTA, implying that short depolarizations should allow exocytosis with 50 μM EGTA but not with 25 μM BAPTA. This prediction was confirmed experimentally with capacitance measurements. Coupling exocytosis to calcium dynamics in the model, we found that a barrier with a ∼3000 times reduced diffusion at ∼130 nm beneath the membrane best explains the experimentally observed effects of EGTA and BAPTA on block and kinetics of release.

Rapid Exocytosis in Single Chromaffin Cells Recorded from Mouse Adrenal Slices

We report here that brief depolarizations such as action potentials trigger exocytosis in thin mouse adrenal slices. The secretory rates obtained in membrane capacitance recordings from chromaffin cells in slices are faster than those observed in isolated cells. Fast exocytosis in slices is attributable to the rapid release of a small pool of vesicles. The pool recovers from depletion with a time constant of 10 sec. Recruitment of the rapidly released vesicles is strongly hindered by the fast Ca2+ chelator BAPTA and much less by the slower chelator EGTA. We suggest that these vesicles are located in close proximity to Ca2+ channels. Spatial coupling of Ca2+ entry and exocytosis may be sensitive to cell isolation and culture.

Enhancement of neurite-sprouting by suppression of HPC-1/syntaxin 1A activity in cultured vertebrate nerve cells

HPC-1/syntaxin 1A is a C-terminal anchored neuronal membrane protein, of which all of the N-terminal regions are located on the intracellular side, and it interacts with presynaptic membrane proteins, synaptic vesicle proteins and soluble N-ethylmaleimide-sensitive fusion protein attachment proteins (SNAPs). HPC-1/syntaxin 1A has been proposed to act as a target SNAP receptor (t-SNARE) in the neuron and contributes to the vesicle docking/fusion process during the fast exocytosis at the presynaptic active zone. However, studies using an electron-microscope revealed that HPC-1/syntaxin 1A distributed not only at the presynaptic region but throughout the whole axonal membrane, and the functions of this axonal HPC-1/syntaxin 1A remain completely unknown. To investigate its physiological role, we attempted to inhibit the function of HPC-1/syntaxin 1A in cultured neural cells by following two methods. First, de novo synthesis of HPC-1/syntaxin 1A was inhibited by an application of antisense oligonucleotide in cultured adult rat dorsal root ganglion (DRG) neurons. Second, antibody against HPC-1/syntaxin 1A was applied intra-axonally in the cultured chick retinal ganglion neuron. Both treatments, which were expected to downregulate the function of HPC-1/syntaxin 1A, consistently elicited an enhancement of the axonal sprouting. These results suggest that the axonal HPC-1/syntaxin 1A would physiologically suppress the excess axon-collateral sprouting. Downregulation of HPC-1/syntaxin 1A expression may underlie the control of collateral sprouting and synapse formation during development and memory processes.

Two components of exocytosis and endocytosis in phaeochromocytoma cells studied using caged Ca2+ compounds.

1. Changes in membrane capacitance evoked by the rapid photolysis of a caged Ca2+ compound, DM-nitrophen or nitrophenyl-EGTA, were investigated in undifferentiated PC12 cells. They were interpreted as representing exocytosis and endocytosis. 2. The Ca2+ jumps evoked two components of exocytosis. Slow exocytosis was selectively evoked with small increases in intracellular Ca2+ concentration between 5 and 10 microM, while fast exocytosis preceded the slow one at [Ca2+]i greater than 10 microM. 3. The release rates of the two components of exocytosis depended steeply on [Ca2+]i. A half-maximal release rate was achieved at 8 and 24 microM for the slow and fast exocytoses, respectively. 4. Prior Ca2+ rises did not augment the fast exocytosis. 5. The fast exocytosis was often followed by a rapid decrease in membrane capacitance, representing endocytosis, after a delay of 0.5-2 s. The speed and delay in the fast endocytosis were Ca2+ dependent. Amounts of the fast endocytosis tended to balance with those of the fast exocytosis evoked by the same Ca2+ jumps. 6. The slow exocytosis was followed by a sluggish endocytosis that was associated with large capacitance steps indicative of secretory processes involving large dense-core vesicles. The onset of the slow endocytosis exhibited a complex Ca2+ dependence. The amounts of the slow endocytosis appeared to parallel those of the slow exocytosis. Prior induction of the slow exocytosis gave rise to selective excess retrieval of membrane during the slow endocytosis. 7. These data indicate the existence of two distinct populations of secretory vesicles in PC12 cells. They seem to couple selectively with specific endocytotic mechanisms. Our data suggest that the two vesicles belong to two distinct secretory pathways.

Ca2+-dependent Exocytotic Pathways in Chinese Hamster Ovary Fibroblasts Revealed by a Caged-Ca2+ Compound

Ca2+-dependent exocytosis and endocytosis of Chinese hamster ovary (CHO) fibroblasts were investigated using capacitance measurement and rapid photolysis of a caged-Ca2+ compound, dimethoxynitrophenamine tetrasodium salt. CHO cells exhibited large and fast increases in membrane capacitance (1.9 +/- 1 picofarads, or 13 +/- 7% of total membrane area, mean +/- S.D., n = 37) upon Ca2+ jumps to [Ca2+]i larger than 20 microM. The fast exocytosis occurred with a delay (20-80 ms), and exhibited a rate constant that was strongly dependent on [Ca2+]i. The maximal rate constant of exocytosis was 2.8/s, and a half-maximal rate was achieved at 30 microM. The fast exocytosis was followed by rapid endocytosis in 28% of the cells. The endocytosis often began after a delay of 0.5-2 s. Ca2+ jumps also induced stepwise increases in membrane capacitance of 10-134 femtofarads in 40% of the cells, indicating fusion of large vesicles with diameters of 0.4-1.5 micron. The exocytosis of the large vesicles could selectively be induced with smaller Ca2+ jumps (6-20 microM), and occurred slowly with a rate constant of 0. 3/s. These data indicate that CHO fibroblasts possess Ca2+-dependent exocytotic mechanisms. Moreover, two parallel exocytotic pathways may exist reminiscent of those of neurons and endocrine cells. A kinetic model was constructed to account for the fast exocytosis of CHO cells.

Fast exocytosis and endocytosis triggered by depolarisation in single adrenal chromaffin cells before rapid Ca2+ current run-down.

The kinetics of exocytosis and membrane retrieval (endocytosis) were examined in bovine chromaffin cells using membrane capacitance measurement during whole-cell recording. At early times after breakthrough to the whole-cell recording mode, depolarisation for 1 s resulted in a fast (600 vesicles per s) exocytotic response and efficient membrane retrieval with a time constant of 25 s. The ability to activate fast exocytosis and retrieval was lost during intracellular dialysis, with a time constant of 40 s. At later times, a slow exocytotic response could be elicited with no membrane retrieval following single depolarisations. The wash-out of the responses appeared to be due to a rapid loss of a portion of the Ca2+ current. Trains of depolarisation at late times after breakthrough could elicit a fast (time constant 4 s) retrieval. These data show that in addition to a previously studied slow Ca(2+)-independent retrieval mechanism, chromaffin cells also possess an efficient and rapid retrieval pathway coupled to exocytosis that can be activated following depolarisation. The fast endocytosis appears to have a higher threshold for activation than exocytosis, probably due to a higher Ca2+ requirement. Rapid membrane retrieval appears to occur via a clathrin-independent pathway in chromaffin cells.