Cellular and molecular biology of the presynaptic nerve terminal.

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BIOLOGY OF THE PRESYNAPTIC NERVE TERMINAL William S. Trimble, Michal Linial, * and Richard H. Scheller

Department of Biological Sciences, Stanford University, Stanford, California 94305 KEY WORDS:

exocytosis, membrane fusion, neurotransmitter release, recycling, synaptic vesicles.

INTRODUCTION

Chemical neurotransmission represents the primary form of intercellular communication in the nervous system, yet relatively little is known about the molecular processes involved. The vesicle hypothesis states that spe cialized organelles located in the synaptic region are responsible for the accumulation, storage, and release of neurotransmitters in discrete packets called "quanta." These synaptic vesicles, or their components, are thought to be assembled in the cell body and transported to the terminals via fast axonal transport. At the nerve terminal, vesicles interact with the cytoskeleton and with soluble vesicle-binding proteins prior to docking at specialized membrane sites known as active zones. Following voltage gated Ca 2+ influx, when the local intracellular Ca 2+ concentrations ([Ca 2+]i) may reach several hundred micromolar, the vesicle and the plasma membrane fuse, releasing the vesicle contents into the synaptic cleft. A typical synaptic vesicle from a motoneuron releases approximately 5000 molecules of acetylcholine, and each nerve impulse releases 100-200 quanta at a representative neuromuscular synapse. Following exocytosis,

.. Present Address: Department of Biological Chemistry, Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel.

93 0147--fJ06X/91/030 I--fJ093$02.00

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TRIMBLE, LINIAL & SCHELLER

vesicle membrane is then recycled at the nerve terminal and new vesicles are formed. An alternative model for transmitter release proposes that nonvesicular, cytoplasmic pools of acetylcholine serve as the direct source of quantal release while vesicles function as acetylcholine storage sites. Although not totally discounted, this model has not been supported by recent mor phological or electrophysiological data (for discussion see Rash et al 1988) and therefore is not discussed further. Many of the specific processes involved in membrane trafficking, includ ing exocytosis and endocytosis, are likely to be similar in all cell types. From yeast to mammalian cells, the seemingly diverse systems of consti tutive secretion and interorganelle transport share fundamental molecular properties (for review see Balch 1989). Although the unique features of neurons undoubtedly require specialized modifications of these processes, events such as membrane fusion at the synapse may be similar to mem brane fusion in yeast. Unfortunately, the tremendous diversity of synaptic structures, including the large number of neurotransmitter types, combined with the lack of useful cell lines and the as yet limited success of genetic analysis, makes neuronal secretion a difficult subject for molecular analy sis. Hence, our understanding of the cellular and molecular biology of the synapse will require the consolidation of information from many sources as diverse as the regulatcd sccrction in chromaffin cells to constitutive secretion in yeast. Tn this review we attempt to coalesce the studies from a variety of systems to formulate a picture of the events likely to be important in the regulated release of neurotransmitters. The review follows the life cycle of a synaptic vesicle through biogenesis, exocytosis, and recycling. Where possible we discuss specific proteins and the stages at which they may act.

FORMATION AND TRANSPORT OF SYNAPTIC VESICLES

Neurons are characterized by their irregular shapes, elongated axon(s), and branched dendrites. In some instances the dendritic tree and axon occupy more than 99% of the cell volume. Furthermore, although the axoplasm is similar to the cytoplasm in the cell body, it lacks the machinery necessary for the synthesis of proteins. Hence, neurons have a specialized transport system to move the proteins synthesized in the cell body rapidly to the nerve terminals. This process, termed axonal transport, is discussed in more detail elsewhere (Vallee 1991, in this volume). The components of synaptic vesicles must also be transported to the


SYNAPTIC VESICLE EXOCYTOSIS

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terminals, either in the form of functional vesicles, or as individual com ponents that will be assembled at the terminal. Clearly, dense-cored vesicles containing peptide transmitters are synthesized in the cell body (for review see Sossin et al 1989). Some proteolytic processing of neuropeptide pre cursors occurs within the mature vesicles, and in some cases the final maturation steps are thought to take place during transport. Whether dense-cored vesicles require modification upon arrival at the synapse to become competent for secretion is not known. The synthesis and site of origin of the small, clear vesicles that contain the so-called "classical" transmitters is less clear. These vesicles are generally thought to be loaded with transmitter at the nerve terminal, but the question of whether they are transported empty or are formed by the recycling of the membranes of the dense-cored vesicles remains open. As discussed previously (Kelly 1988), the membranes of dense-cored vesicles contain many of the components of small, clear vesicles (Lowe et al 1988), although at a much lower concentration than those of small vesicles. Despite their differential localization at release sites (Zhu et al 1986) and differential sensitivity to a-latrotoxin-induced release (Matteoli et aI1988), it has been suggested that the small, clear vesicles result from the enrich ment of vesicle proteins recycled following the exocytosis of dense-cored vesicles (Lowe et al 1988). This question has been addressed by studying the location and nature of vesicle proteins after ligature of an axon. In this way Kiene & Stadler (1987) demonstrated that proteins specific to small, clear vesicles moved along the axon by fast anterograde transport in organelles that had biochemical properties similar to those demonstrated for recently recycled "empty" vesicles. These vesicles have a density similar to synaptic vesicles and can take up acetylcholine and ATP in vitro (Stadler & Kiene 1 987). Hence, these studies support the model that, at least in Torpedo electromotoneurons, small, clear vesicles are transported as relatively mature, preformed organelles. Although this study cannot rule out the mixing of membrane comonents from dense-cored vesicles with those of small, clear vesicules, dense-cored vesicle membranes probably cannot account for the formation of all small, clear vesicles. The movement of preformed vesicles along the axons implies the exist ence of specific transport receptors on the vesicular membrane surface, either as temporary or permanent constituents of the vesicle. Purified synaptic vesicles from the electric organ of marine rays are competent for movement along axoplasmic fibers in a crude squid giant axon exudate (Schroer et al 1985) but incompetent for kinesin-mediated movement along purified micro tubules (Schroer et al 1988). Thus, some component found in the exudate, in addition to kinesin, is necessary for vesicle movement (see Vallee 1991, this volume).


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Unlike dense-cored vesicles, small, clear vesicles are probably not filled with neurotransmitter until they reach the nerve terminal. In the case of Torpedo electromotor neurons, both newly synthesized and recycled vesicles have been shown to take up acetylcholine in vitro (Stadler & Kiene 1987). The acetylcholine storage system of vesicles appears to involve an ATPase that pumps protons into the vesicles, and an acetylcholine transporter that utilizes this proton motive force to translocate acetyl choline (for review see Marshall & Parsons 1987). The characterization of proton pump components from chromaffin granules (Wang et al 1988, Mandel et al 1988) and the availability of a drug (AH5183) capable of blocking acetylcholine translocation (Marshall & Parsons 1987) should permit the complete elucidation of this process.

CYTOARCHITECTURE OF THE NERVE TERMINAL

The directed exocytosis that occurs in neurons during neurotransmission undoubtedly requires specialized structures unique to the nerve terminal. Transmission electron microscopy has revealed extensive patches of elec tron-dense material co-localized on the presynaptic membrane, the post synaptic membrane, and the junctional cleft in virtually all types of synapses. Around these so-called active zones in the presynaptic axoplasm are clustered large numbers of uniform 80-100 nm membrane-bound vesicles (for review see Landis 1988). Statistical analyses of electron micro graphs suggest that vesicles are likely to be associated with filamentous molecules (Smith 1988). Also, the active zone density appears to be made up of fibrillar molecules extending from the plasma membrane to the vesicles. Freeze-fracture analyses of synaptic plasma membranes have revealed a number of large 90 A intramembranous particles within the active zones. Their concentration at 1500 per /tm 2 synaptic plasma mcmbrane in the squid giant synapse is consistent with the number of Ca 2+ channels predicted (Pumplin et al 1981), thus suggesting that each particle may represent a single calcium channel. In addition, these molecules may contribute to the observed presynaptic density. Biochemical analyses have revealed that the predominant macro molecules present in the nerve terminals are F-actin, microtubules, neuro filaments, fodrin (brain spectrin), and the synapsins. Immunofluorescence microscopy on Torpedo electric organs has shown that microtubules and neurofilaments do not extend far into the nerve terminals (Walker et al 1985). Mitochondria and endoplasmic reticulum-like structures often



97

found in the terminals are excluded from the direct vicinity of the active zones (Landis 1988). One molecule that appears to cross-link the vesicles to each other and to the cytoskeleton is synapsin. Synapsin is the name given to a family of neuronal phosphoproteins associated with small, clear vesicles (for review see De Camilli & Greengard 1986). This family consists of four closely related forms called synapsin Ia and Ib and synapsin IIa and IIb (Siidhof et aI1989). Synapsins Ia and Ib derive from the alternate processing of a single gene (Haas & DeGennaro 1988, Siidhof et al 1989) and the same is thought to be true of synapsins IIa and lIb (Siidhof et al 1989b). Most of what is known about the biochemical and functional properties of synapsins derives from the study of synapsin I. Synapsin I has been shown to bind to microtubules and neurofilaments in vitro (Baines & Bennett 1986, Goldenring et al 1986) but, as mentioned above, these components do not penetrate far into the nerve terminals. More import antly perhaps, synapsin I binds to fodrin, which appears to be present at approximately equimolar ratios with synapsin I (Baines & Bennett 1985). Also, synapsin I binds and bundles F-actin, and binds to small, clear vesicles in vitro (Schieb1er et aI1986). Synapsin I is phosphorylated at three sites: Site 1 in the collagenase resistant globular head is phosphorylated by cAMP-dependant protein kinase and Ca 2+ /calmodulin-dependant protein kinase I (Ca 2+ /CM kinase I); sites 2 and 3 are phosphorylated by Ca 2+ /CM kinase II and are located in the collagenase-sensitive tail region (for details see De Camilli & Green gard 1986, Czernik et aI1987). Phosphorylation of sites 2 and 3 within the tail region reduces the affinity of binding to small, clear vesicles (Schiebler et a1 1986). F-actin bundling is inhibited by phosphorylation at site 1 and is abolished by phosphorylation at sites 2 and 3 (Bahler & Greengard 1987). The hypothesis that the phosphorylation state of synapsin T is involved in regulating neurotransmission was tested by direct injection of synapsin or Ca 2+ /CM kinase II into the preterminal digit of squid giant axons (Llinas et al 1985). Under these circumstances, injection of the dephos phorylated form of synapsin inhibited neurotransmission, whereas phos phorylated synapsin had no effect and Ca 2+ /CM kinase II facilitated transmission. When introduced into extruded squid axoplasm, dephos phorylated synapsin inhibited the movement of organelles within the interior of the axoplasm, whereas Ca 2+ /CM kinase II removed this inhi bition (McGuinness et al 1989). Another approach to the study of nerve terminal structure has come from the use of quick-freeze etching techniques prior to rotary replication and electron microscopy. Taking advantage of the fact that certain mol-


98


ecules have characteristic morphologies when viewed with this technique, two groups have developed generalized models for the architecture of active zones (Landis et al 1988, Hirokawa et al 1989). Both have found that actin filaments appear to form a network closely associated with the active zone. Short, 30 nm strands appear to link the vesicles to the actin network and to each other through their globular head groups. These strands closely resemble, in size and appearance, purified, rotary shadowed synapsin I. Taken together, these results suggest that the tail of synapsin binds vesicles, whereas the globular head group binds actin or the head of another synapsin molecule, cross-linking the vesicles. A third type of structure found in these experiments was a long (200 nm), thin strand that resembled fodrin and was seen to radiate from the active zone membrane both to synapsin-like and actin-like filaments. Statistical analyses dem onstrated that the vesicles were usually at least 30 nm away from the membrane (Hirokawa et al 1989). Taking the ultrastructural and phos phorylation results together, a model similar to that proposed by Llimis et al (1985) can be proposed in which synaptic vesicles are attached to each other and to the F-actin network by the nonphosphorylated form of synapsin I. This cross-linking is thought to decrease vesicle mobility in axoplasm and their delivery to the active zone. The vesicle-cytoskeleton complex is then anchored to the synaptic membrane by fodrin filaments that radiate from the active zones. Phosphorylation of synapsin I by Ca2+ /CM kinase II reduces its vesicle and F-actin binding affinity, increas ing vesicle movement within the cytoplasm. Although the rate of phos phorylation is probably too slow for changes in the phosphorylation state of synapsin to drive exocytosis, these changes might play a modulatory role in the subsequent activity of the synapse by regulating vesicle docking at the active zone. A great deal of information has also been obtained on the role of the cytoskeleton in exocytosis in chromaffin cells, and some parallels may be drawn between the two systems. In chromaffin cells, a highly organized cytoskeletal network is localized beneath the plasma membrane (for review see Aunis & Bader 1988). This network contains actin, fodrin, caldesmon, and vinculin. Again in this case, fodrin is thought to link the actin network to the plasma membrane, while the other molecules function as cross linkers. The network is presumed to serve as a physical barrier to exocytosis, as actin filament reorganization or disassembly appears to be necessary to allow the granules access to the sites of exocytosis. Proteases such as calpain are activated by micromolar calcium levels and are efficient at hydrolyzing structural proteins (see Melloni & Pontremoli 1989). Hence, degradation of such a barrier could be involved in changing the pool size of available, fusion-competent vesicles for future rounds of release.



99

Although calpains have been detected in neurons, whether their local ization will be consistent with a role in neurotransmission remains to be determined. Interestingly, treatment of permeabilized chromaffin cells with anti bodies specific for fodrin partially blocks exocytosis, but antibodies specific for other cytoskeletal proteins have no effect (Perrin et aI1987). This could be intcrpreted to mean that the antibody blocks actin disassembly, prevents Ca2+-mediated proteolysis of fodrin (Burgoyne & Cheek 1987a), or steri cally blocks vesicle movement to the fusion sites. Presumably the blockage is only partially effective because some of the granules are already docked near the membrane beyond the F-actin barrier. A tempting proposal is that the cytoskeletal barrier in chromaffin cells might modulate the number of vesicles available for release. Unlike the model proposcd above for synapsin, however, the rate of release from chromaffin cells is much lower than from neurons, and changes in the cytoskeletal sequestration of the vesicles could occur rapidly enough to influence ongoing rather than future release rates. EXOCYTOSIS

Exocytosis is the process by which molecules are secreted from eukaryotic cells. The process is thought to involve the fusion of vesicles with the plasma membrane and the release of vesicle contents into the extraccllular space. In most cells these processes are constitutive, but neuronal and other cell types have an additional pathway of regulated release whereby the exocytotic event is coupled to a stimulus. Studies of the process of exocytosis have been greatly aided by the use of two powerful tools. Permeabilized cell systems mediated by the addition of detergents (e.g. digitonin) or pore-forming toxins (e.g. staphylococcal IX-toxin) allow the introduction of ions, nucleotides, and in some cases even macromolecules without significant leakage of the essential cellular components. Patch-clamping techniques provide direct measurements of single fusion events and provide a means for the introduction of defined solutions into the cell interior. These results must be viewed with caution, however, as some loss of cellular macromolecules must occur and the extent of these effects is undoubtedly affected by the method of per meabilization. The rate of exocytosis between excitable and non-excitable cells differs by several orders of magnitude. There is, therefore, no reason to assume that the mechanisms underlying secretion are identical. However, the study of these related systems may allow the formulation of testable hypotheses that will be constructive once technical advances permit the direct inves-


100


tigation of secretory mechanisms in neurons. Penner & Neher (1989) have suggested that in nonexcitable cells undergoing slow release, control of exocytosis might be maintained by enzymes and modulated by Ca 2+ levels, whereas in fast-acting neurons, [Ca 2+J elevation is the rate-limiting step, while enzymatic proccsses regulate the slower, modulatory events. Somewhat surprisingly, the pathways leading to exocytosis appear to differ widely, depending on the cell system studied. For example in PC12 cells, as in most neurons, exocytosis can be stimulated by increases in [Ca 2+]j alone (Ahnert-Hilger et al 1987). Chromaffin cells, on the other hand, require Ca 2+ and Mg 2+/ATP (Bader et al 1986). In both cell types, the non-hydrolyzable GTP analog GTPyS inhibits Ca 2+ -triggered exocytosis (Knight & Baker 1985, Ahnert-Hilger et al 1987). In contrast, Ca 2+-triggered release in human platelets is stimulated by GTPyS (Haslam & Davidson 1984). Similarly, botulinum toxin D inhibits secretion in chromaffin cells (Adam-Vizi et a1 1988) but potentiates secretion in plate lets (Banga et al 1988). Provision of GTP and Ca 2+ is both sufficient and necessary for exocytosis from permeabilized neutrophils and mast cells (Barrowman et al 1986, Cockroft et al 1987). Despite the discrepancies across experimental systems, these results suggest a role for Ca 2+- and GTP-regulaled processes in exocytosis. Therefore, GTP- and Ca 2+-bind ing proteins implicated in exocytosis are discussed below. Other proteins thought to be involved in exocytosis include protein kinases, phospho lipases, and ion channels associated with osmotic swelling. These are also briefly discussed. GTP-Binding Proteins

In mast cells, which secrete histamine in response to IgE, an IgE receptor coupled G-protein activates the phospholipase-C second-messenger path way, thus leading to the conversion of phosphatidylinositol-4,5-bis phosphate to diacylglycerol and inositol- l ,4,5-trisphosphate. This lattcr product is responsible for the release of calcium from the intracellular stores. In mast cells, phospholipase C can also be activated by the nonhydrolyzable GTP analog GTPyS. However, addition of neomycin (an inhibitor of phospholipase C) was unable to block the effectiveness of GTPyS in stimulating release of histamine from permeabilized mast cells (Barrowman et al 1986). This result suggested that GTP was involved at two steps: in the activation of phospholipase C and at a point subsequent to phosphoJipase-C-induced [Ca 2+]j increases. The GTP-binding protein presumed to be acting at this latter step was termed Ge for exocytosis (Barrowman et al 1986). Several groups have recently reported finding GTP-binding proteins on secretory granules and synaptic vesicles. Toutant et al (1987) have



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described three pertussis toxin substrates (Go and two others) that have molecular weight of 39, 40, and 41 kD and co-purify with chromaffin granule membranes. On human neutrophil granules a pertussis toxin sub strate immunologically related to Gi• has been identified (Retrosen et al 1988). A GTP-binding, pertussis toxin substrate of about 40 kD co-purifies with synaptic vesicles from Torpedo electric organ (Ngsee et al 1 990). The role of these G. subunits in exocytosis is unk n own. Using GTP-overlay and ADP-ribosylation assays, several groups have also detected a number of small ras-like GTP-binding proteins (20-25 kD) tightly associated with the membranes of secretory organelles (Burgoyne & Morgan 1989, Doucet et al 1989, Ngsee et aI1990). These proteins are members of a large and growing family of molecules in which there are now more than 20 members. As with the large G proteins, the role of ras like molecules in exocytosis is currently unknown. However, several lines of evidence point to a possible involvement in vesicular transport. One of these ras-like proteins, mammalian rhoC, is the ADP ribosylation substrate of botulinum toxin C3 and appears to play a role in actin microfilament assembly (Chardin et al 1 989). As a botulinum substrate has been found to purify with synaptic vesicles from marine rays (Ngsee et al 1990), it is tempting to speculate that this molecule could be rhoC and that it participates in some aspect of the association between vesicles and the cytoskeleton. Furthermore, evidence that the ras oncogene itself might play a Tole in vesicle transport has been obtained. Microinjection of purified H-ras pro tein into fibroblasts led to a marked increase in fluid phase pinocytosis, possibly due to the activation of phospholipase Az (Bar-Sagi.& Feramisco 1986). In contrast, microinjection of H-ras into mast cells resulted in massive degranulation (Bar-Sagi & Gomperts 1988). Analogy with the elongation factor Tu led Bourne (1988) to speculate that the small ras-like GTP-binding proteins might function to regulate the vectorial transport of membrane vesicles through the cell. Bourne's model states that the GTP-bound form of the molecule is bound to vesicles and remains bound until the vesicles reach their target site. At this point the GTP is hydrolyzed to GDP and the GDP-bound protein is released from the vesicle. This GDP-bound form of the molecule probably returns to the donor membrane from which the vesicles originate, exchanges GDP for GTP i" n an enzyme-catalyzed reaction, and repeats the process. The high cytoplasmic GTP: GDP ratio would ensure a unidirectional process by shifting the equilibrium coe"fficient in favor of the forward reaction. Interestingly, independent studies have found that proteins called GTPase activating proteins (GAPs) are responsible for promoting 'GTP hydrolysis (for review see McCormick 1989). At present, the role of these


102


proteins is not known, although two possibilities have been suggested. GAPs could serve as the downstream targets for the function of ras-like proteins (McCormick 1989). Alternatively, these proteins might serve as feedback inhibitors of ras activity by decreasing the duration of the GTP bound "on" state (Ballester et aI1989). Several different GAPs apparently exist (Trahey et al 1988) and it is possible that each distinct type of small GTP-binding protein could associate with only one or a small subset of these GAPs (Garrett et al 1989). This specificity could be responsible for insuring the directed nature of vectorial transport or the specificity of down regulation. Of interest is the observation that the ability of GAPs to stimulate GTPase activity is inhibited by certain lipids in vitro (Tsai et al 1989a). Furthermore, comparison of R-ras and its GAP with rho and rho GAP revealed that inhibitory lipids differed for each (Tsai et al 1989b). These inhibitory lipids, such as phosphatidic acid, arachadonic acid, and phosphatidylinositol monophosphate are often the products of mitogenic and other stimuli, a fact that suggests a biological control of the activity of these small GTP-binding proteins through lipid metabolism. GAPs are also substrates for tyrosine phosphorylation and, in quiescent fibroblasts, PDGF receptor phosphorylation of GAP results in its translocation from the cytosol to the plasma membrane (Molloy et al 1 989). Translocation of these enzyme activators could regulate either the site or extent of their activity. Secretory defects in yeast strains YPTl and SEC4 both contain mutations within small ras-like GTP-binding proteins. The yptl mutant is defective in the transport of vesicles from the endoplasmic reticulum to the Golgi apparatus, while in sec4 mutants, vesicles from the Golgi appar atus fail to fuse with the plasma membrane to secrete their contents. Mutational analysis of the Sec4p product has allowed Walworth et al (1989) to develop a protein that fails to bind GTP. This protein results in a dominant loss of function in wild type yeast and it is thought that the mutant molecules are trapped in an active conformation in the absence of bound GTP. These experiments suggest that the mutant molecules can attach to vesicles and target them to the membrane but cannot be inac tivatcd. It is further proposed that the mutation is dominant because the membrane target sites get saturated with nonfunctional Sec4p and therefore these sites are unavailable for normal Sec4p function. From the perspective of a neuron, the Sec4p function might be analogous to a prefusion docking step, in which the vesicle would be targeted to a site in the active zone from which it could fuse readily upon Ca 2+ influx. Genetic techniques permit yeast researchers to search directly for genes that interact with the Sec4p product. Through such a search it was found that the product of another secretory mutation in yeast, SEC15, waf



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probably acting directly downstream from SEC4 (Salminen & Novick 1989). Although this gene product showed no direct homology to any knowD proteins, it could represent a new member of the diverse GAP family of molecules, or could be a unique target molecule. The search for SEC15 homologues in neurons may thereby lead to the discovery of specialized docking components or novel GAP-like molecules at the mem brane. Although SEC4 and rhoC may represent two of the vesicle-associated GTP-binding proteins, the plurality of these proteins on vesicles suggests that this class of molecule might regulate a number of other functions as well. In different cell types and different intracellular locations, GTP binding proteins almost certainly regulate a variety of cellular processes. Calcium Influx

In neurons, Ca 2+ enters the cytoplasm from the extracellular milieu through voltage-gated Ca2+ channels. Three classes of physiologically distinct Ca2+ channels have been identified to date, namely the T, L, and N type channels (for review see Tsien et a1 1988, Smith & Augustine 1988). Although different channel types appear to act in different neurons, the most consistent generalization is that N-type channels are involved in mediating the release of SCVs, whereas dense-cored vesicles secretion follows Ca2+ entry through the high-conductance, slow-inactivation L type channels. Though both classical and peptide neurotransmitters may be released from the same nerve terminal, higher frequencies of stimulation may be required to elicit peptide release. This may reflect intrinsic differ ences in their release mechanisms or, as peptide release occurs away from the active zone, this may reflect the extensive stimulation required to achieve [Ca 2+]i increases at significant distances from the channels them selves. Despite the development of Ca 2+ -sensitive fluorescent probes, measure ment of changes in the presynaptic [Ca 2+ ]i within the necessary temporal and spatial levels remains technically difficult. Currently available flu orescent markers can measure changes in [Ca 2+]i with millisecond time limits and 1-2 ,urn resolution. However, presynaptic release requires only 0.1-1 msec, and Ca2+ diffusion is likely to occur over much less than 1 ,urn. Hence, theoretical models have been the only means of predicting the nature of the Ca 2+ influx. A discussion of these models has recently been presented (Smith & Augustine 1988). Although the spatial distribution of Ca 2+ binding proteins and Ca 2+ pumps will significantly influence local [Ca2+]i, simplistic uppcr estimates of [Ca2+]i during depolarization can be generated. The resting cytoplasmic [Ca 2+]i has been estimated to be in the range of 1O-7M (Di Polo & Beauge 1983). Based on models in which Ca2+


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channels are packed in compact arrays with 40 nm spacing at the active zone, Ca 2+ concentrations close to the membrane and between the chan nels would reach steady state levels of 40-400 pM within 1 msec, depending upon the fraction of channels opened during the action potential. Local [Ca2+]j in chromaffin cells, which lack the ordered packing of Ca2+ chan nels, is likely to be much lower. The action potentials that open Ca 2+ channels, and allow a local increase in [Ca 2+]j, may also play a role in mediating neurosecretion (for review see Augustine et al 1987). For example, models have been proposed in which the action potential results in a voltage-mediated conformational change in a membrane protein, which increases its sensitivity to calcium. The use of the photolabile calcium chelator nitr-S in conjunction with voltage clamp techniques allows the two properties to be examined inde pendently. Zucker & Haydon (1988) have reported that, in slow releasing Helisoma snail neurons in culture, increasing presynaptic Ca2+ levels to the micromolar level was sufficient to elicit neurotransmission, and that presynaptic voltage had no direct effect. In contrast, Hochner et al (1989) suggest that this result was caused by an increase in spontaneous release. Furthermore, they show that in a conventional, fast neuromuscular prep aration from the first walking leg of Procambarus clarkii crayfish, action potentials resulted in synchronous release under conditions of raised [Ca 2+]j when entry of extracellular Ca2+ was blocked. Further studies will be necessary to resolve this issue. Calcium-Binding Proteins

Once the [Ca2+]j is elevated, free diffusion is controlled by calcium-binding proteins in the vicinity of the active zone. It is reasonable to assume that Ca2+-dependent exocytosis might be mediated by molecular changes occurring within calcium-binding molecules following Ca2+ influx. At present, the nature of these calcium-responsive exocytosis "trigger" mol ecules is unknown. Although the role of specific membrane lipids cannot be ruled out, the observation that the necessary factors eventually diffuse out of permeabilized cells argues that the molecules are probably soluble proteins. Several approaches have been taken to study the proteins that have a direct role in responding to [Ca 2+], changes. These include the isolation of proteins that bind to chromaffin granule membranes in the presence of micromolar [Ca2+]j (Creutz et aI1983), and the characterization of proteins and factors that are lost from cells under detergent-permeabilized con ditions and that can be added back in reconstitution experiments (Sarafian et al 1987). Together, these approaches have resulted in the discovery of a large and growing family of calcium-binding proteins.



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For Ca 2+ -binding proteins to play a role in Ca 2+ -regulated exocytosis, they must meet several criteria. Firstly, they must reside in the cytoplasm, or possess Ca2+-binding sites accessible to the cytoplasm. Secondly, their Ca 2+-binding affinity must be consistent with the [Ca2+]j predicted for the active zone following depolarization. Thirdly, the specificity of the mol ecule for Ca2+ over other divalent cations is critical. Finally, the proteins must be spatially distributed within the cells in a pattern consistent with a role in promoting release. The function of these calcium-binding proteins could be to promote the association of membranes prior to fusion, to act directly as calcium-induced fusogens, or to function as Ca2+-regulated fusion pores between the vesicle and plasma membranes. Below we discuss several molecules that have been proposed to serve as calcium-regulated exocytosis mediators. As the precise stage at which Ca2+ acts is not known, any or all of these molecules could participate in the release process. ANNEXINS

This generic name has been proposed (Geisow et al 1987) to encompass the large group of calcium-binding proteins that reversibly associate with biomembranes. Members of this family include the lipo cortins, chromobindins, calpactins, calelectrins, endonexins, and synexins. Because they were discovered by a variety of means, the resulting ambi guities in the naming and classification of these proteins has produced a great deal of confusion in the literature. As molecular cloning and bio chemical analyses proceed, the relationships between many of these mol ecules are becoming clarified. The properties of some of them have been listed in Table lA. A 17-animo-acid consensus sequence is moderately conserved between members of the annexin family and is present in mul tiple copies within them (Geisow et aI1986). The evolutionary importance of these molecules is evidenced by the fact that one annexin, chromobindin A, has bccn found to be conserved from mammals to yeast (Martin et al 1989). All annexins appear to bind lipids in a specific manner; phos phatidylserine and phosphatidylinositol are preferred targets and there is no measurable binding to phosphatidylcholine. Chromaffin granule membranes possess Mg2+ and ATP-dependent aminophospholipid trans locase activity that maintains cytoplasmic orientations of pho sphatidylserine (Zachowski et al 1 989). Whether this activity accounts for the ATP and Mg2+ dependence of chromaffin granule exocytosis is not known. All annexins also appear capable of inhibiting phospholipase A2 activity, presumably by binding to the substrate rather than the enzyme. Most bind Ca2+ in the low micromolar ranges but require much higher [Ca 2+1 to promote membrane aggregation. Synexin I was the first of the annexins shown to bind chromaffin granule membranes (Creutz et al 1978). This 47 kD molecule promotes

SYNEXIN

Table IA

Calcium- and membrane-binding proteins


Name calbindin caldesmon calelectrina (mammalian) calelectrina

Other names

M/

Kd'

28

?

Vitamin D dependent

Hunzinker et al 1 98 3

p70

70

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