Exocytosis: a Molecular Review and Physiological Perspective

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Neuron, Vol. 17, 1049–1055, December, 1996, Copyright 1996 by Cell Press Exocytosis: A Molecular Review and Physiological Perspective

Robert S. Zucker random number (about ten on average) of such com- Division of Neurobiology plexes. This hypothesis can account quantitatively for University of California the highly nonlinear Ca2ϩ-dependence of rates and ex- Berkeley, California 94720–3200 tents of secretion of cortical granules in sea urchin eggs, where live exocytosis of single granules can be visual- ized in isolated membrane patches from these eggs. From 1952 to about 1985, exocytosis in neurons was At synapses between nerve cells, high concentrations 2ϩ studied mainly at neuromuscular junctions and selected of [Ca ]i occur only during action potentials and are giant synapses by electrophysiology and electron mi- localized at active zones where vesicles are docked in croscopy. Early advances were followed by a period the neighborhood of clusters of Ca2ϩ channels.Evidence of relative stagnation as these techniques appeared to for the existence of such “Ca2ϩ microdomains” has reach their full potential. Then, in the last few years, come from three types of experiments: 1) Simulations everything changed. New methods for measuring and of Ca2ϩ diffusion from Ca2ϩ channel clusters in the pres- 2ϩ ence of a slowly diffusible buffer predict a complex pat- controlling presynaptic calcium concentration ([Ca ]i) 2ϩ using fluorescent dyes and photosensitive Ca2ϩ-binding tern of peaks and troughs of [Ca ]i transients in active compounds emerged, as did techniques for simulating zones during action potentials (Figure 1). In these simu- 2ϩ the diffusion of Ca2ϩ in the presynaptic neuron. New lations, [Ca ]i reaches about 150 ␮M in the spaces 2ϩ methods of monitoring secretion were developed, in- between rows of Ca channels where vesicles are cluding measurement of presynaptic membrane area by docked. These predictions have been confirmed in ac- means of membrane capacitance, and of uptake and tual experiments on frog saccular hair cells by measur- release by means of fluorescent dyes trapped in endocy- ing the activity of Ca2ϩ-activated potassium channels tosed vesicles. Modern methods of membrane biology that are co-localized with presynaptic Ca2ϩ channels and genetic engineering were applied to proteins (Roberts, 1994). 2) Local Ca2ϩ domains also have been thought to be involved in neurosecretion. Meanwhile the observed in turtle cochlear hair cells by confocal im- patch clamp permitted electrophysiological analysis of aging of fast-binding low affinity fluorescent Ca2ϩ buff- previously inaccessible tissues. As a result, exocytosis ers (Tucker and Fettiplace, 1995) and at squid giant emerged from a backwater in relative obscurity to be- synapses by imaging phosphorescence from low affinity come a new frontier in biological research. This review photoproteins (Llina´ s et al., 1995). 3) Secretion is often 2 summarizes some of the recent advances and indicates triggered by Ca ϩ entering through mixtures of N- and 2ϩ current directions of research in this active field. P/Q-type high-voltage-activated Ca channels, as- sessed by actions of specific channel blockers on transmission (Wheeler et al., 1994; Mintz et al., 1995). The ,Ca2؉ and Secretion: Cooperativity nonlinear dependence of spike-evoked secretion on the Domains, and Shells number of channels opened indicates that secretion is At fast synapses, exocytosis requires high [Ca2ϩ]. Pho- i triggered by Ca2ϩ microdomains arising from multiple tolysis of the light-sensitive caged Ca2ϩ compound DM- clustered Ca2ϩ channels. nitrophen has been used to elevate [Ca2ϩ] rapidly while i These results from neurons contrast with those ob- monitoring secretion postsynaptically or by electroni- tained from endocrine cells. In endocrine cells, secretion cally measuring changes in presynaptic capacitance un- can be measured on a fast time scale by either mi- der whole-cell voltage clamp. In goldfish retinal bipolar neurons, Heidelberger et al. found that [Ca2ϩ] had to croamperometric detection of biogenic amines with car- i bon fiber electrodes or by changes in membrane capaci- rise to over 50 ␮M before the rates of secretion matched tance. In these cells, a higher Ca2ϩ-sensitivity than in those achieved by depolarization (Heidelberger et al., neurons (Heinemann et al., 1994), a lower maximal rate 1994). At the crayfish neuromuscular junction, a tran- of secretion during depolarizations (Chow et al., 1994), sient rise in [Ca2ϩ] to about 75 ␮M triggered neurosecre- i and a persistent secretion, which outlasts Ca2ϩ influx tion with magnitude and kinetics similar to the phasic (Chow et al., 1996), have been observed. All of these release accompanying an action potential (Lando` and properties indicate a loose coupling of Ca2ϩ channels Zucker, 1994). These experiments demonstrated di- to the secretory machinery of endocrine cells, with exo- rectly a sharply nonlinear dependence of secretory rate cytosis being triggered by a submembrane shell with a on the level of [Ca2ϩ], and suggest that at least four Ca2ϩ i concentration of [Ca2ϩ] of only about 5 ␮M. ions must bind with positive cooperativity and affinities i from 10–150 ␮M to a target complex to trigger rapid secretion. This is a significant elaboration of the original Short-Term Synaptic Enhancement: Facilitation, proposal of Ca2ϩ cooperativity by Dodge and Rahami- Augmentation, and PTP moff (1967), based on the nonlinear dependence of Following one or more presynaptic action potentials, transmitter release on extracellular [Ca2ϩ]. However, transmitter release per spike is often increased by a other schemes are also possible. For example, Vogel et number of processes differing in their characteristic dual. (1996) propose that secretion is accelerated by the rations—facilitation lasting about 1 s, augmentation last- Ca2ϩ-dependent activation of vesicular fusion com- ing about 30 s, and potentiation or posttetanic potentia- plexes, where each vesicle contains a rather large but tion (PTP) lasting several minutes. It has long been Neuron 1050

2ϩ Figure 1. Simulation of a [Ca ]i Micro- Domain The calculated spatial distribution of time- 2ϩ averaged submembrane [Ca ]i is shown for a depolarized active zone in a frog saccular hair cell. The locations of Ca2ϩ channels (solid circles) and Ca2ϩ-dependent Kϩ channels (open circles) are shown on a projection 2ϩ above the [Ca ]i profile. The predicted aver- 2ϩ age level of [Ca ]i was confirmed by measurement of Ca2ϩ-dependent Kϩ current. Spheres show thelocations of docked synap- 2ϩ tic vesicles in the troughs of [Ca ]i between rows of Ca2ϩ channels. Adapted from Rob- erts, 1994.

thought that these effects of repeated presynaptic ac- Ravin et al., unpublished data) or by steady weak photol- tion potentials were the result of presynaptic accumula- ysis of DM-nitrophen (Kamiya and Zucker, 1994). Substi- 2ϩ 2ϩ 2ϩ tion of residual [Ca ]i during conditioning stimulation tution of Sr for Ca depresses phasic release while (Katz and Miledi, 1968; Magleby and Zengel, 1982). Re- increasing asynchronous release and facilitation (Goda cent evidence confirms this hypothesis: there is a close and Stevens, 1994), suggesting that facilitation and 2ϩ 2ϩ correlation between the decay of residual free [Ca ]i asynchronous release reflect a different target of Ca and of each process (augmentation and PTP in Aplysia action from phasic exocytosis. This Ca2ϩ target, like that central synapses and crayfish NMJ: Kretz et al., 1982; involved in phasic secretion, seems to require coopera- Connor et al., 1986; Delaney et al., 1989; Delaney and tive action of Ca2ϩ for its activation. It is interesting that 2ϩ Tank, 1994; and facilitation in cerebellar synapses: Atluri at goldfish bipolar synapses, micromolar levels of [Ca ]i 2ϩ and Regehr, 1996). Furthermore, residual [Ca ]i can be fail to trigger any detectable secretion, as monitored by eliminated rapidly by photolysis of diazo-2, which re- presynaptic capacitance change (Heidelberger et al., leases a BAPTA-like Ca2ϩ chelator. At the crayfish neuro- 1994). Either these tonically transmitting synapses lack muscular junction, this maneuver reduces all three forms the sorts of short-term synaptic plasticities described of synaptic enhancement (Kamiya and Zucker, 1994). It at phasic synapses, or perhaps a low rate of secretion was originally thought that residual Ca2ϩ acted at the is obscured by simultaneous endocytosis (see Vesicle site triggering exocytosis to enhance release by simply Pools below). 2ϩ adding to the transient rise in [Ca ]i occurring during Flash photolysis of diazo-2 rapidly reduces residual 2ϩ conditioned action potentials (Miledi and Thies, 1971). [Ca ]i and in so doing eliminates most facilitation But the many-fold increase in transmission often ob- equally rapidly. Therefore, facilitation must be due to served during facilitation, augmentation, and PTP can- Ca2ϩ binding and activating its target with rapid kinetics. not arise from such a mechanism (Yamada and Zucker, When residual Ca2ϩ is chelated with EGTA-AM in cere- 2ϩ 1992) because residual [Ca ]i is usually below 1 ␮M bellar synapses (Atluri and Regehr, 1996), the decay 2ϩ while the transient rise in [Ca ]i required to trigger exo- time constant of facilitation is shortened from 200 ms cytosis during action potentials reaches 100 ␮M or more to 40 ms. In these synapses, 40 ms may be the decay in active zones. This indicates that short-term synaptic rate of the facilitation process itself when it is activated 2ϩ 2ϩ plasticity is due to Ca acting on targets distinct from only by the brief local rise in [Ca ]i during an action the molecular Ca2ϩ-receptor triggering phasic secretion. potential, while the duration of facilitation under normal Unlike the low affinity target(s) involved in triggering circumstances is determined by the time it takes to 2ϩ 2ϩ fast exocytosis, the large efficacy of low [Ca ]i levels remove residual [Ca ]i from these nerve terminals. At observed during the decay of facilitation, augmentation, the crayfish neuromuscular junction, diazo-2 photolysis and PTP suggests that there must be one or more tar- also eliminates augmentation and PTP, some of it rapidly gets with high Ca2ϩ affinity. but most with slow kinetics (time constant about 350 Short-term enhancement of phasic release underlying ms). The similar kinetics and sensitivities of augmen- 2ϩ PSPs is accompanied by a concurrent increase in spon- tation and PTP to residual [Ca ]i (about ten-fold en- 2ϩ taneous release rate or frequency of miniature postsyn- hancement per ␮M residual [Ca ]i; Delaney and Tank, aptic potentials (mPSPs), often called asynchronous re- 1994), and the fact that facilitation and augmentation/ lease. This may reflect Ca2ϩ action at one of the sites PTP interact multiplicatively (Magleby and Zengel, 1982) involved in short-term synaptic plasticity. Indeed, a high suggest that augmentation/PTP are due, at least in part, 2ϩ 2ϩ affinity site relating [Ca ]i to mPSP frequency is indi- to Ca acting at a site with higher affinity and slower 2ϩ 2ϩ cated when [Ca ]i is elevated to about 1 ␮MbyCa - kinetics than the site triggering facilitation. The pro- transporting ionophores (Delaney and Tank, 1994; R. longed persistence of PTP after long tetani is partly Review 1051

What are the functions of short-term synaptic enhancement in the neural processing of information? Syn- aptic transmission which requires facilitation, augmentation, or PTP to reach postsynaptic threshold for generating action potentials, produces an effective filter that responds only to sustained high frequencies; this property can be used to extract signals from noise. Two more concrete applications of synaptic plasticity have recently come to light. In the first, the polysynaptic component of the Aplysia siphon withdrawal reflex habitu- ates to repeated tactile stimulation, and this behavior is due to PTP in an inhibitory interneuronal feedback circuit, which progressively reduces the gain of excit- atory interneuronal input to the motor neurons (Fischer and Carew, 1993). In the second, computer simulations of the activity within hypothetical neuronal circuits show that inclusion of facilitation in selected synapses permits the circuit to transform different temporal patterns of afferent activity into distinct spatial patterns of neuronal activation (Buonomano and Merzenich, 1995). Recently, experimental evidence has been obtained for just such transformations in the CA3 region of the hippocampus (D. V. Buonomano, P. W. Hickmott, and M. M. Merzenich, Figure 2. Possible Sites of Ca2ϩ Action in Exocytosis and Short- in preparation). Term Synaptic Plasticity Undocked, docked, and exocytosing synaptic vesicles are shown in a synaptic terminal, along with open Ca2ϩ channels admitting Is Fusion Always Complete or Can Ca2ϩ into the terminal. Exocytosis is triggered by Ca2ϩ binding at It Be Transient? site I, perhaps to synaptotagmin I or affecting the interaction be- Neurosecretion might involve complete fusion of vesi- tween Ca2ϩ channels and syntaxin or SNAP-25. Facilitation is activated by Ca2ϩ ions at site II, which may be another vesicular protein cles with the plasma membrane and subsequent endo- such as synaptotagmin III or VII. Augmentation/PTP is caused by cytosis, or a transient “kiss-and-run” fusion could allow Ca2ϩ acting at site III, which may be a vesicular or cytoplasmic vesicle contents to escape through a briefly opened protein involved in docking vesicles at active zones. fusion pore (Figure 3). Early ultrastructural and tracer uptake studies provide support for both modes of opera- tion under different stimulus conditions. More recent due to presynaptic Naϩ accumulation and consequent evidence for complete fusion comes from staining and retardation of Ca2ϩ removal by Naϩ/Ca2ϩ exchange (Mul- subsequent destaining of internalized membranes in key and Zucker, 1992), and partly to posttetanic leakage nerve terminals with FM1–43 during electrical stimula- of Ca2ϩ from mitochondria that are loaded only during tion. The finding that staurosporine can abolish FM1–43 long tetani (Y.-G. Tang and R.S. Zucker, in preparation). destaining and slow down staining with little effect on transmitter release (Henkel and Betz, 1995) suggests that secretion at frog motor nerve terminals can switch .Where Are These Ca2؉ Targets Located That between complete and transient fusion modes Are Involved in Exocytosis, Facilitation, Analysis of rise times of mPSPs at the frog neuromus- and Augmentation/PTP? cular junction indicates that acetylcholine escapes The shortness of the synaptic delay, 0.2 ms from the through a fusion pore similar to that measured electri- opening of Ca2ϩ channels to the beginning of transmitter cally during fusion of mast cell granules (van der Kloot, release (Llina´ s et al., 1981), requires that exocytosis be 1995), rather than by instantaneous complete fusion of triggered by Ca2ϩ acting in the immediate neighborhood vesicles with the surface membrane. A lack of effect of (within tens of nanometers) of Ca2ϩ channel clusters. monovalent and divalent cations on rise time of mPSPs This locates the exocytotic targets of Ca2ϩ to membrane indicates that, unlike the release of serotonin from mast proteins near Ca2ϩ channels or to proteins in nearby cells, acetylcholine may not be released from an in- docked vesicles (see Molecular Components below). travesicular ion exchange matrix. Finally, amperometric Since facilitation is maximal shortly after an action po- measurements of release of serotonin from cultured tential, Ca2ϩ must be acting reasonably near Ca2ϩ chan- leech neurons allowed measurement of the amount and nels, perhaps on a different protein in the docking/fusion kinetics of quantal release (Bruns and Jahn, 1995). Small complex, a protein involved in attaching vesicles to the responses presumably arising from fusion of small vesi- plasma membrane. The slow kinetics of augmentation/ cles were due to release of about 5,000 molecules within PTP allow for the involvement of second messengers, 100 ␮s, consistent with a rapid opening of a preassem- and even for such processes as the movement (or mobi- bled fusion pore. Large responses presumably arising lization, as it is called) of predocked vesicles to docking from fusion of large dense core vesicles were due to sites at the plasma membrane (Figure 2). release of about 80,000 molecules through a pore that Neuron 1052

another 190 vesicles more slowly (␶ϭ430 ms, Hsu and Jackson, 1996). Goldfish retinal bipolar cells contain 55 ribbon synapses, to each of which are attached 100 vesicles, 20 of them docked at the membrane (von Gers- dorff et al., 1996). The 1100 docked vesicles (20 ϫ 55) can be released rapidly (␶ϭ0.5 ms) by depolarization, and this pool is refilled after depolarization with a 5 s time constant (Mennerick and Matthews, 1996, [this issue of Neuron]). The remaining 5000 ribbon vesicles are released more slowly (␶ϭ300 ms) during prolonged depolarization, perhaps reflecting the rate at which these can replace the docked vesicles. Since this is faster than the 5 s recovery rate for the fast release of docked vesicles at rest, and since presynaptic perfusion of EGTA blocks the slow component during persistent depolarization, Ca2ϩ may mobilize vesicles to docking sites at the membrane. In bovine chromaffin cells, Ca2ϩ also appears to mobilize vesicles (Neher and Zucker, 1993; von Ru¨ den and Neher, 1993) from a reserve pool of about 1000 (␶ϭ500 ms) to a fully releasable pool of 100–200 (emptiable with ␶ϭ2 ms, Heinemann et al., 1994). In chromaffin cells and melanotrophs, the immediately releasable and fully primed pool corresponds to about 10–20% of morpho- logically docked vesicles (Parsons et al., 1995). In contrast to these examples, frog saccular hair cells appear Figure 3. Two Modes of Exocytosis capable of sustained high levels of quantal release— In “kiss-and-run,” transmitter is released through a transient open- 500/s/active zone for up to 2 s without apparent deple- ing of a fusion pore from the vesicle through the plasma membrane. In “complete fusion,” the vesicle membrane fuses with and becomes tion—despite the fact that in active zones (defined as embedded in the plasma membrane following release of transmitter. having a dense body) only about 30 of 80 vesicles are docked at the membrane (Parsons et al., 1994). These studies raise again the important, but still unre- opened initially like that of small vesicles but then pro- solved question of whether mobilization of vesicles from gressively dilated. It is not yet clear whether in most reserve pools to docking sites, or the priming of docked circumstances this pore subsequently closes or dilates vesicles, correspond to particular phases of short-term synaptic enhancement such as facilitation or augmen- fully to produce complete fusion. tation/PTP, as proposed in Figure 2. In most of these studies, secretion was monitored as Vesicle Pools an increase in membrane capacitance, and a rise in Much attention has been focused on pools of transmitter capacitance to a new steady level was taken as indicat- that are to varying degrees available or competent for ing secretion occurring during the rise. A final flat trace release. Classically, this question has been approached is assumed to represent cessation of exocytosis, but by attributing different kinetic components of depres- it could equally well represent the delayed onset of a sion of repeated PSPs to depletion of transmitter stores balanced rate of endocytosis. Simultaneous use of ca- successively more distant from release sites. Recently, pacitance and either amperometry or FM1–43 staining release has been activated by continuous depolariza- (Chow et al., 1994, 1996; Smith and Betz, 1996) allow 2ϩ tion, hypertonic stimulation, or steps of [Ca ]i imposed distinction between these alternatives. At salamander 2ϩ by photolysis of caged Ca compounds, while secretion rod photoreceptor synapses, a sudden elevation of has been monitored by mPSP frequency, presynaptic 2ϩ 2ϩ [Ca ]i by photolysis of caged Ca compounds led to capacitance increase, or FM1–43 destaining. Often an a step rise in capacitance, while sudden reduction in early phase of rapid secretion occurs, followed by one 2ϩ 2ϩ [Ca ]i by photolysis of the caged Ca -absorbing chela- or more slower phases, and in “fire and reload” models tor diazo-2 led to a step drop, showing that the resting of secretion, these are interpreted as reflecting release steady capacitance represents continuous balanced of fully docked and primed vesicles followed by (a re- rates of exo- and endocytosis (Rieke and Schwartz, loading and) release of vesicles progressively less com- 1996). petent for fusion. In cultured rat hippocampal neurons, single boutons For example, chick retinal amacrine cells subjected containing one active zone can be activated by focal to depolarizing pulses release 0 or 1 quantum, equal in perfusion of hyperosmotic solutions or solutions con- size to a spontaneous mPSP, from each of 10 release taining high-Kϩ/high-Ca2ϩ. Single 5 s stimuli (Stevens sites at a rate of 150/s/site (with a decay constant of and Tsujimoto, 1995), as well as trains of action poten- depleting this store, ␶ϭ7 ms). Subsequent release oc- tials (Rosenmund and Stevens, 1996), evoke release of curs at 22/s/site from a much larger store (Borges et al., a readily releasable pool of 15 quanta at 20/s; this pool 1995). Rat pituitary synaptosomes initially release 40 can be replenished with a 10 s time constant. One sec- vesicles to a depolarization (␶ϭ14 ms), and then release ond hyperkalemic stimuli repeated at 0.2–0.5 Hz, which Review 1053

permit replenishment of this pool, release 90 quanta, injection of fragments of the C2 domains (which might corresponding to the total number of vesicles in a bou- act as competitive blockers of the action of synaptotag- ton; refilling this pool takes about 40 s (Liu and Tsien, min) blocks transmitter release without altering Ca2ϩ 1995). This corresponds to the time required for endocy- influx or vesicle docking (Bommert et al., 1993). Release tosis of released vesicles and their reentry into a releas- is also blocked by polyclonal antibodies made to a frag- able pool, as measured by FM1–43 destaining (Ryan ment that includes the C2A domain, which shows Ca2ϩ- and Smith, 1995). In this preparation, action potentials dependent binding, but not antibodies to the C2B do- only release one quantum per release site, release oc- main, which does not bind Ca2ϩ (Mikoshiba et al., 1995). curs only about 25% of the time, and release sites remain Cultured hippocampal neurons from mice with synapto- refractory for 5–20 ms afterwards (Allen and Stevens, tagmin I deletion mutants show a specific defect in pha- 1994; Stevens and Wang, 1995). If a quantal PSP is due sic transmitter release (Geppert et al., 1994). The syn- to release of 1 vesicle, this would correspond to a vesicle chronized release of quanta immediately following an depletion rate of 1 per 4 spikes or 0.25% of the total action potential that constitutes the fast PSP was ab- vesicle pool. This is close to estimates of the rate of sent, but asynchronous release, the delayed increase in vesicle depletion per action potential from FM1–43 de- mPSP frequency following action potentials, persisted. staining experiments (Ryan and Smith, 1995), and this This is consistent with synaptotagmin acting either as a 2ϩ 2ϩ correspondence is one of the strongest arguments for Ca -sensitive activator of release or as a Ca -inhibited the common assumption that single vesicles are respon- brake on spontaneous or constitutive release. It will be sible for the quantal components of transmitter release. interesting to see if these defects can be rescued by At the squid giant synapse, photolysis of caged Ca2ϩ replacement of synaptotagmin or by use of conditional 2ϩ knockouts. generates a step rise in [Ca ]i but only a transient postsynaptic depolarization reflecting secretion decaying Synaptotagmin is not, however, the only molecule as- 2ϩ with a time constant of 30 ms (Hsu et al., 1996). A series sociated with vesicle fusion showing a Ca -sensitivity. 2ϩ of step rises in [Ca2ϩ] led to a series of transient releases N-type Ca channels bind separately to syntaxin and i to SNAP-25 independently of synaptotagmin; this bind- of transmitter. Many of the results can be quantitatively ing is strongly activated by increasing [Ca2ϩ]to20␮M, explained by depletion of an immediately releasable and inhibited at higher [Ca2ϩ] (Sheng et al., 1996). This transmitter pool, with a compensatory Ca2ϩ-dependent biphasic Ca2ϩ-dependence could underlie a dual-phase mobilization into that pool (R. Zucker,unpublished data). “cock-and-fire” triggering of secretion by Ca2ϩ. Interac- Hsu et al. prefer an alternative interpretation, that the tions in the reverse direction also exist (Bezprozvanny secretory process displays a sort of adaptation in re- et al., 1995): syntaxin coexpressed in oocytes with sponse to step rises in [Ca2ϩ]. If the latter interpretation i N- and Q-type Ca2ϩ channels stabilizes channel inactiva- is correct, the Ca2ϩ-dependence of secretion is likely to tion. A fragment of the protein binding region of L-type prove even more complex than is now believed. Ca2ϩ channels dissociates the channels from syntaxin and inhibits phasic transmitter release following action Molecular Components potentials without altering Ca2ϩ influx when injected into Molecular biology has allowed the identification and cultured sympathetic neurons (Mochida et al., 1996); cloning of a number of proteins involved in vesicle dock- asynchronous release and synaptic facilitation, how- ing and fusion. Some synaptic vesicle proteins (synapto- ever, were enhanced. These effects, similar to those tagmin and synaptobrevin) form tight associations with seen in synaptotagmin deletion mice, could reflect the plasma membrane proteins (syntaxin, SNAP-25, and disruption of the tight association of Ca2ϩ channels with 2ϩ N-type Ca channels) to form a protein complex called their molecular targets, removing the targets from the the fusion complex. Other vesicle or soluble proteins shoulders of Ca2ϩ microdomains and leaving them in (NSF, ␣-SNAP, and munc18 or n-sec1) appear to be the valleys where release is triggered only weakly and involved in assembling the complex. Several of these asynchronously by Ca2ϩ influx (Figure 1). In this view, proteins are homologous to yeast proteins involved in synaptotagmin might be the target of these Ca2ϩ micro- membrane trafficking and fusion. Important evidence domains, but so might other Ca2ϩ-binding proteins such for their involvement in neurosecretion came from the as rabphillin, doc2, or scinderin (Calakos and Scheller, inhibition of secretion by proteolytic fractions of botuli- 1996; Zhang et al., 1996). Alternatively, the Ca2ϩ sensor num and tetanus neurotoxins that specifically cleave in secretion could be the channel-syntaxin or channel- synaptobrevin, syntaxin, and SNAP-25, and from the SNAP-25 interaction, and synaptotagmin mutants or discovery that transmission is inhibited by presynaptic inhibitors might disrupt this interaction. Thus, the evi- injection of peptides that compete with the action of dence to date does not point uniquely to a single candi- synaptotagmin, synaptobrevin, and ␣-SNAP (Calakos date as the Ca2ϩ target in neurosecretion (site I in Fig- and Scheller, 1996). ure 2). Of all these proteins, synaptotagmin has received the It is possible that the increased asynchronous release most attention recently. Containing two C2 Ca2ϩ-binding and synaptic facilitation observed on dissociation of domains and being part of the fusion complex, it is an Ca2ϩ channels from syntaxin and in synaptotagmin mu- 2ϩ 2ϩ obvious candidate for transducing an elevation in [Ca ]i tants reflect a separate target of Ca action in these into a secretory trigger. The C2A domain of synaptotag- processes, as proposed above (site II in Figure 2). Candi- min I shows Ca2ϩ-dependent binding to syntaxin and dates for this action include the neural synaptotagmins membrane phospholipids (apparent KDs of about 300 III and VII, since they show a much higher (micromolar) and 5 ␮M, respectively) that may be a first step in trig- Ca2ϩ-sensitivity in syntaxin binding than does synapto- gering membrane fusion (Li et al., 1995). Presynaptic tagmin I (Li et al., 1995). Neuron 1054

It has been suggested that kinetically slow augmen- E. (1996). Mechanisms determining the time course of secretion in tation and PTP are due to Ca2ϩ acting via Ca2ϩ/calmodu- neuroendocrine cells. Neuron 16, 369–376. lin-dependent kinase II (CaMKII) to phosphorylate sy- Connor, J.A., Kretz, R., and Shapiro, E. (1986). Calcium levels mea- napsin I and “mobilize” vesicles tethered to actin sured in a presynaptic neurone of Aplysia under conditions that modulate transmitter release. J. Physiol. 375, 625–642. microfilaments at release sites (site III in Figure 2; Greengard et al., 1993). Presynaptic injection of antibod- Delaney, K.R., and Tank, D.W. (1994). A quantitative measurement of the dependence of short-term synaptic enhancement on presyn- ies to synapsin I leads to a depression of high-frequency aptic residual calcium. J. Neurosci. 14, 5885–5902. transmission and a depletion of vesicles at release sites Delaney, K.R., Zucker, R.S., and Tank, D.W. (1989). Calcium in motor (Pieribone et al., 1995). But mice with synapsin I and nerve terminals associated with posttetanic potentiation. J. Neu- CaMKII gene knockouts show normal or even enhanced rosci. 9, 3558–3567. PTP (Rosahl et al., 1995; Chapman et al., 1995), and Dodge, F.A. Jr., and Rahamimoff, R. (1967). Co-operative action of calmodulin and CaMKII inhibitors fail to block facilita- calcium ions in transmitter release at the neuromuscular junction. tion, augmentation, or PTP (Kamiya and Zucker, 1994). J. Physiol. 193, 419–432. Mice without synapsin II show reduced PTP, but synap- Fischer, T.M., and Carew, T.J. (1993). Activity-dependent potentia- sin II is not phosphorylated by CaMKII. Interestingly, tion of recurrent inhibition: a mechanism for dynamic gain control in facilitation is increased in synapsin I and reduced in the siphon withdrawal reflex of Aplysia. J. Neurosci. 13, 1302–1314. CaMKII knockouts. The results indicate that facilitation Geppert, M., Goda, Y., Hammer, R.E., Li, C., Rosahl, T.W., Stevens, 2ϩ is regulatable independently from PTP and secretion C.F., and Su¨ dhof, T.C. (1994). Synaptotagmin I: a major Ca sensor for transmitter release at a central synapse. Cell 79, 717–727. and suggest that synapsins and CaMKII can influence the rate of supply of primedvesicles available for release Goda, Y., and Stevens, C.F. (1994). Two components of transmitter release at a central synapse. Proc. Natl. Acad. Sci. USA 91, 12942– during repetitive stimulation but do not mediate genera- 12946. tion of synaptic enhancement. Greengard, P., Valtorta, F., Czernik, A.J., and Benfenati, F. (1993). Synaptic vesicle phosphoproteins and regulation of synaptic func- What’s Next? tion. Science 259, 780–785. The immediate future seems to lie with the imagina- Heidelberger, R., Heinemann, C., Neher, E., and Matthews, G. (1994). tive combination of techniques. 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