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Home , Exocytosis

Volume 268, number 2, 394-399 1”EBS 08586 August 1990

Neurotransmitter release

Received 9 May 1990

Axon terminals release more than one physiologically active substance. Synaptic messengers may be stored in two different types of vesicles. Small electron-lucent vesicles mainly store classical low molecular weight transmitter substances and the larger electron-dense granules store and release and peptides. Release of the two types of substances underlies different physiological control. Release of messenger molecules from terminals is triggered by influx of Ca’+ through voltage sensitive CT+ channels and a rise in cytosolic Cal- concentrations. Neither the i~lrnediate Ca” target(s) nor the molecular species involved in docking, fusion and retrieval are known. It is. however, hkely that steps involved in the molecular cascade of transmitter release include liberation of vesicles from their association with the cytonet and phosphorylatlotl hy kinasc C of proteins which have the ability to alter between bound and cytoplasmic forms and thus facilitate or initiate the molecular interaction between synaptic vesicles and the plasma membrane.

Exocytosis; ; Protein C; Release: ; Vesicle

1. SITE AND CELLULAR SOURCE OF to be the result of the exocytotic of the con- NEUROTRANS~ITTER RELEASE tents of individual synaptic vesicles. In addition to this phasic release mechanism there is a small but con- I. 1. Types 0s refease and types of released messengers tinuous release of neurotra~~sI~itter from nerve ter- Upon arrival of an the phasic release minals which has also been referred to as transmitter of a quantity of neurotransmitt~r is evoked from the leakage [3-61. terminal axon segment. This is the general physico- chemical mechanism underlying the fast processing of I .2. Consensw for exocyrosis information between nerve cells and also the transfer of Exocytosis of neurotransmitter involves the release of signals from nerve to muscles and glands. soluble synaptic vesicle contents after controlled fusion It is likely that all neurons release at their axon ter- with the presynaptic plasma membrane. Support for minals more than one physiologically active substance. this view comes from ultrastructurai analyses of nerve In addition to the classical low molecular weight terminals on induced transmitter release, uptake of ex- messenger molecules like acetylcholine, noradrenaline tracellular markers into synaptic vesicles, im- and various ammo acids also proteins and peptides are munocytochemicai analyses, the comparison of synap- secreted [1,2]. This observation has abolished the tic vesicle contents and products released, biochemical former one-neuron-one-transmitter doctrine. Whether studies of synaptic vesicle life cycle and synaptic vesicle the substances coreleased with the classical transmitter heterogeneity, and the quanta] nature of the postsynap- substances serve as cotransmitters sustaining signal tic signal [6-131. transfer or rather as modulators of the synaptic signal Using rapid freezing techniques it could be or even act at non-synaptic sites has to be discussed demonstrated at the frog that together with the classical transmitter regarding both the exocytotic fusion of chofinergic synaptic vesicles oc- the cellular preconditions and the molecular cascade of curs about 0.5 ms before the onset of the postsynaptic neurotransmitt~r release. signal. When the synapse is intensely activated and As has been shown for a variety of neurotransmitter synaptic vesicle recycling is impaired, the loss in synap- substances, classical are released in tic vesicle numbers can be related directly to the counts the form of small quanta1 packages and this is thought of postsynaptically recorded transmitter quanta. If synaptic vesicle recycling is completely blocked, the in- corporation of the synaptic vesicle membrane compart- Correspondence: H. Zimmermann, AK Neurachemie, Zoologisches lnstitut der J.W. Goethe-Universitat, Siesmayerstr. 70, D-6000 ment into the presynaptic plasma membrane can be Ftrankfurt am Main, FRG visualized by immunocytochen~ical methods using an-

Published bv Elsevier Science Publishers B. V. (Btomedical Division) 394 00145793/90/$3.50 b 1990 Federation of European Biochemical Societies Volume 268, number 2 FEBS LETTERS August 1990

tibodies against synaptic vesicle membrane proteins dense or dense-cored vesicles (also referred to as fl2f. Whereas it had been assumed for a long time that granuIes) which store a variety of proteins and peptides a singie synaptic vesicle gives rise to a postsynaptic and possibiy also smaller molecular weight quantum, the observation at the neuromuscular junc- neurotransmitters. The function of these granules is tion that the classical quanta1 event may in fact consist thought to be mainly in the release of neuropeptides. of subquanta has come as a challenge to the original The smaller electron-lucent vesicles do not store pro- vesicle hypothesis (14,15]. Does one quantum corres- teins and their function is in the rapid release of low pond to the release of the contents of a single or of molecular weight messengers. Both types reiease their several synaptic vesicles? Are synaptic vesicles func- contents by controlled exocytosis. This means, that ax- tionally heterogeneous with different release pro- on terminals possess a differential organelle outfit for babilities for different vesicle pools f3,73? the release of high and low molecular weight For the neurotransmitter acetylcholine it has also substances. The two types of cell organelles differ not been suggested [ 161 that it is not released by exocytosis only with regard to their contents. At present there is of synaptic vesicles but through pores in the presynapti~ some debate as to what extent they carry common mem- plasma membrane which are formed by the association brane proteins. Whereas there are common features like of membrane proteins referred to as mediatophore. The the presence of a proton pumping ATPase [26], the Ca2 + -activated opening of a mediatophore complex presence of other common components is interpreted wouId give rise to the release of a quantum of differently whether biochemical or ultrastructural acetylcholine. Similarly, the evoked release of amino evidence is used as the main argument [10,27-291. A acid transmitters from cytoplasmic rather than from possible reason for some of the djs~repan~y may come synaptic vesicle stores has long been inferred. The pro- from difficulties in comparing the adrenergic vesicle tein subunit of the mediatophore complex has now been types ‘large-dense cored’, ‘small dense-cored’ and in identified as a component of the synaptic vesicle proton addition ‘electron-~u~ent~ to the two principal vesicle pumping ATPase [ 171. From studies on the vesicular types of other neurons. uptake and storage of amino acids [lS] and on the Ca2 * It is, however, clear that the release characteristics of dependency of amino acid release from brain synap- low and high nloIe~ular weight messengers differ with tosomes [5] strong evidence could be derived for ex- regard to both the activation pattern of the axon and ocytotic release of amino acid transmitters. the Ca2 * dependency ]6,30]: at low action potential fre- quencies release of low molecular weight messengers 1.3. Non-neuronal model systems (classical transmitters) predominates whereas release of Thus, the basic mechanism of evoked neurotransmit- peptides depends on higher frequencies. Furthermore, ter release is analogous or perhaps identical to controfl- differential Ca2+ channels appear to be involved in trig- ed secretory release from other secretory systems in- gering exocytosis from the two organellar sources [3 11. cluding endocrine and exocrine gland cells and possibly This is paralleled by the observation that the small also mast cells, neutrophils or even oocytes or electron-lucent vesicles release their contents at mor- Paramaecium. There has been an increasing interest in phologically specified release sites of the presynaptic non-neuronal model systems for studying the molecular membrane whereas the larger peptide-loaded granules mechanism underlying exocytosis in general but also to release their contents distal to the synaptic cleft [32]. understand more fully neuronal secretion mechanisms Furthermore, the association with surface proteins for [19]. Immediate access to the exocytotic release the linkage to the cytonet is different between granules mechanism of these cells could be obtained by elec- and smaller vesicles. 1 which is thought to con- tropermeabilization [20], by permeabilization with nect electron-lucent synaptic vesicles to actin and digitonin and more recently with bacterial exotoxins tubulin filaments [34] and which is involved in the con- 121-231 or with the patch clamp technique f24,25]. trol of neurotransnlitter release ]35] is not associated with the membrane of protein-storing secretory granules [36]. All these observations suggest that there 2. MORE THAN ONE TYPE OF VESICLE AND are differences in the cellular and molecular precondi- MORE THAN ONE TYPE OF EXOCYTOTfC tions for the release of low and high molecular weight CONTROL? messengers and thus for granules and small electron- Before discussing some of the key mechanisms in- fucent vesicles. Whether there are also differences in the volved in exocytosis, a number of basic features of the final pathway of the exocytotic fusion event needs to be neuronal secretory machinery have to be evaluated. elucidated. This is important if it comes to a comparison with other ft has also been suggested that neurons contain only secretory systems and the deduction of consensus one principal type of vesicle, the dense-cored granule. mechanisms. After release of its peptide and protein contents and Neurons possess in their two principal struc- local membrane recycling this would turn into a smaller tural types of secretory vesicles: the larger electron- eiectron-lucent vesicle to be used in further cycles of

395 Volume 268, number 2 FEBS LETTERS August 1990 release and reloading of small messengers 1371. This stores in rnodulatir~g or - under certain experin~ental would, however, need to involve also alterations in the conditions - in sustaining axonal transmitter release. membrane properties of the organelfe. It is of interest The concentration of Ca” + in the nerve terrn~i~al is con- that the small electron-lu~ent vesicle type has now been trolled by a complex system of soluble buffers, identified as a separate organelle also in neurosecretory organellar stores and systems with axon terminals using vesicIe-specific antibodies [36] and varying Ca2+ transport affinities and capacities and in non-neuronal cells in tissue culture transfected with a every distortion will result in a modulation of transmit- gene coding for a synaptic vesicle-specific protein ter release 166,671, Ca’ ’ is sequestered inside the nerve [38,39]. Thus, large dense-cored granules and small terminal in the smooth endoplasmic reticulum, synaptic electron-lucent vesicles may represent different vehicles vesicles, mito~hondr~a, and possibly also calcisomes. for exocytotic release in a variety of cellular systems. Furtllermor~, there exist intracellular buffering systems like Ca’ * -binding proteins [68,69]. Inositol polyphosphates have been assigned a key role in Ca’+ 3. THE MOLECULAR CASCADE UNDERLYING i~ltra~eIluIar homeostasis. lnositol 1,4,5-trispIlosphate EXOCYTOSIS is formed on receptor-mediated and G-protein- controlled turnover of j~hosphatidylirlositol Due to the inaccessibility of the terminal axon seg- 4,5-bisphosphate together with diacylglycerol, an ac- ment and the small size of isolated synaptosomes, infor- tivator of protein kinase C. The inositol trisphosphate mation concerning the molecular mechanism of functions via specific receptors in the release of Ca”+ transmitter release has also been gained from studies of from intracellutar stores, presumably the en- model systems like the adrenal medulla or neutrophils doplasmatic reticulum and/or the calcisome [70]. In and mast cells. It has to be seen to what extent both the axon terminals receptor-mediated turnover of synaptic mechanisms regulating exocytosis and the molecular phosphoinositides for mobilizing Ca’+ from internal cascade of and fission are identical in all stores may serve mainly as a secondary and modulatory of these secretory systems and even between the two mechanism. It is a major disadvantage that we still do principal synaptic vesicle types. not know the immediate Ca’” target(s). The number of molecular events which are implicated in the mechanism of synaptic vesicle exocytosis is ex- 3.3. Molecular cascade downstream of the CU’~ in_flux panding rapidly. They include an increase in the An early step in the initiation of vesicle exocytosis is cytosolic Ca2 + concentration into the micromolar presumably the mobilization of synaptic vesicles from range 140,411 or even higher 1311, Mg-ATP and the ac- their association with the cytonet [8,34] and possibIy tivation of protein , in particular of protein the removal of a cytoskeletal undercoating of the kinase C [40,42-441, synaptic vesicle-associated pro- presynaptic release site 1231. This may explain the in- teins like I and II or a chromaffin granule- volvement of synapsin I and various cytoskeletal pro- binding protein [45-471, the phosphoproteins B-50 teins in the release process. Ca” and protein (GAP-43) [48] and P92 (possibly the MARCKS protein) phosphorylation play a major role in the control of [81>cytoskeletal proteins like actin and fodrin these processes [23,45,71]. Furthermore, protein kinase [23,50-543, GTP-binding proteins [55-591, arachidonic C-dependent protein phosph~rylation is likely to piay a acid, and even others [24,60,61]. It is, however, not key role in the initiation of the esocytotic event. There clear how these various molecular components interact is strong evidence that activation of protein kinase C and how their interaction is regulated. potentiates transmitter release 172-751. This enzyme is unique in that its soluble form is capable of being 3.2. The senfral role of Ca2 + translocated to a membraI~e-bound form [76,77]. The In action potential evoked synaptic transmission the endogenous activators of the enzyme are thought to be elevation of the intracellular Ca2 ’ Ievel following Ca* +’ diacylglycerol and Ca’+ 1781. Two of its presynapti~ influx through voltage-gated Ca2 + channels is presum- targets which are by now we11 defined are the B-SO pro- ably the very first step of the cascade [4l]. In this tein [79,X0] and the MARCUS protein ]81]. We still respect the nerve terminal differs from non-neuronal know, however, very little of how the substrate proteins secretory systems where exocytosis is initiated by the ac- for protein kinase C interpose betvveen synaptic vesicles tion of receptors at the 162). However, it and the presynaptic plasma metnbrane. A protein of Mr could be demonstrated that in a number of experimen- 92 000 (P92, presumably identical with the MARCKS tal conditions neurotransmitter can also be released in protein) associated with synaptic plasma the absence of extracellular Ca2 ’ [63,64]. There is even has been found to bind to synaptic vesicites after protein evidence that neurotransmitter released in the absence kinase C-dependent phosphorylation 18,821. It may of Ca2+ may be derived from a different intracellular thus act as a mediator for docking the synaptic vesicle pool than that in the presence of Ca”’ j6.5). Together to its release site. Other evidence demonstrates that an- this documents the potential of intracellular Ca’” tibodies against the B-50 protein introduced into brain

396 Volume 268, number 2 FEBS ~,ETTERS August 1990

synaptosomes inhibit transmitter release implicating the control the availability of extraceIluIar Ca’; for i~voIvement of this protein in exocytosis [48]. Similarly transmitter release. External messengers may, however, exocytosis is inhibi&!d by in~ra~~llul~ application of also act via receptor-~~ctiva~ed G-proteins controllirrg antibodies against the ~hromaffin-granule binding pro- phosphoiipase C activity and thus the molecular tein f46]. cascade involved in vesicle exocytosis. This wouid result. Phosphor~lation of the ~euro~~spec~fic B-50 protein in changes in the intraterminal Ca”; level, the concen- has been correlated to transmitter release, long-term tration of dia~~lgI~~eroI for a~t~v~t~~~of protein kinase potentiation and also neurite outgrowth [44,83,84f. The C, and possibly other messenger systems f51 ,lOZ]. in B-SO protein has been reported to bind to the inside of order to fully understand the molecular cascade the plasma membrane [85] to the actin rich neuronal underlying exocytosis at rhe it may thus membrane skeleton [86] and also to ~aImod~lin [87f, be necessary to clearly differentiate between two events: Similarly the MARCKS protein, which is enriched in the cascade following the arrival of the action potential brain but can also be detected in a number of other and Ca’ + influx leading to exocytotic release on the one tissues, becomes phosphorylated on induced transmit- hand, and additional mechanisms which are involved in ter release from synaptosomes and on activation of n~od~lating this event (e.g. by presynapt~~ receptor ~hrornaff~n cells [74,88-901. Both the B-40 and the mediated ~~ecba~~srns~ and causing plasticity MARCUS protein are fatty-acylated [80,91], and in the pllei~omena like potentiati~n or possibly also decrement fatty-acylated form they could be membrane anchored. on the other. Since secretion from model systems like Fatty-ac~rlatio~ and thus membrane anchoring may be mast cells or neutrophifs is activated under regufated by protein kinase C-dependent phos~horyIa- ~~ysiologi~aI conditions by receptor-mediated tion [92,93]. However, additionat properties of the pro- mechanisms only (and not by action potentiaIs~, the tein may be essential for its targeting ta specific do- sensitivity and preponderance of the molecular mains of the nerve cell membrane f94J. Regarding the mechanisms involved in release may differ [l@&fOrl]. strong evidence for protein phosp~oryIat~o~ in n~ur~trai~smitter exocytosis, the importance of 5. RESTING RE.LEASE de~hosphorylation events in this process cannot be overestimated f95,96]. At least for low molecular weight transmitter When docking of synaptic vesicles is achieved, this substances there is in addition to quanta1 also a non- step is followed by an as yet undefined mechanism of quanta1 ~ra~smi~~~rrelease j4,5]. bin-quanta release is membrane fusion and fission. It is thought that the in- preponderant under resting conditions, independent of teraction of the two membrane compartments is in- externaI Ca” + , and results in a steady molecular leakage itiated by a ~~an~~l-like structure possibly i~voIv~~~ of transmitter [I@]. Whereas quanta1 retease is poten- corresponding protein channels in the synaptic vesicle t&ted on depolarization of the axon terminxt this is not ~l~rnbran~ and the presynaptic plasma membrane the case for non-quanta1 release. Neither the [8,97,98]. Eventually soluble messengers may be releas- mechanism of this Ca’+ -independent leakage nor its ed through a short-lived ‘fusion pore’ f24,991 and the p~~s~ologi~aI impIi~ations are clear. At the synaptic vesicle membrane may be recycled for re-use ~e~romus~uIar junction it has been suggested to result [ lOtI]. From electrophysiological experiments it has from the transient incorporation of the vesicular been concIuded that only about 100-200 ps elapse be- acetylcholine transporter into the presynaptic plasma tween the influx of Ca’ * and transmitter reiease riots. f~~e~lbrane on vesiofe exocytosis [I&$ This would aflow Thus, the process leading to the fusion of small the transmitter to leave the nerve terminal by a carrier- eIectr~n~Iu~e~t synaptic vesicles must be very rapid. mediated mechanism and, accordingly, the released This may be another aspect where exoocytosis of larger neur~tr~nsn~itter would be derived from a non-vesicIe granules and small synaptic vesicles differ. bound, cytopfasmic poet.

4. POPULATION OF TRA~S~~ITTER RELEASE 6. SYNOPSIS Synaptic transmission modulates and is subject to r~oduIation. This is of great importance e.g. for synap- Whilst by now many of the prin~~p~I bnjld~~g-stones tic information processing and modulation processes making up the molecular con&d mechanisms involved involved in behav~oural plasticity phenomena. Reieas- in neurotransmitter release are known, they still have to ing nerve terminals possess receptors for other transmit- be placed into the right order _The moIecuIar cascade of ter substances, and pra~ti~aIly alf neur~tra~sm~tt~r the exocytotic event at the nerve terminal remains an substances also act back to the terminals from which enigma. In particulars the Ca* + target(s) and the they were reIeased via presynapt.ic receptors ~autore~ep” molecular (protein and/or ) components directly tars). The modulation may involve receptor-mediated involved in synaptic vesicle dockjng, fusion and modifications of e.g, K + or Cazc channefs and thus retrievat need co be identified.

397 Volume 268, number 2 FEBS LETTERS August 1990

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