How Does Calcium Trigger Neurotransmitter Release? George J Augustine
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320 How does calcium trigger neurotransmitter release? George J Augustine Recent work has established that different geometric A brief introduction to diffusion of Ca2+ arrangements of calcium channels are found at different To appreciate how much Ca2+ is required to trigger trans- presynaptic terminals, leading to a wide spectrum of calcium mitter release, it is important to understand the diffusion signals for triggering neurotransmitter release. These calcium of Ca2+ once it enters the presynaptic terminal. signals are apparently transduced by synaptotagmins — Mathematical models indicate that diffusion yields an calcium-binding proteins found in synaptic vesicles. New immediate accumulation of calcium ions within an area biochemical results indicate that all synaptotagmins undergo covering tens of nanometers around the mouth of open calcium-dependent interactions with membrane lipids and a Ca2+ channels [2,3]. This calcium signal can be damped by number of other presynaptic proteins, but which of these the buffering action of Ca2+-binding proteins [4]. Such uni- interactions is responsible for calcium-triggered transmitter tary Ca2+ influx events will result in qualitatively different release remains unclear. types of intracellular signals, depending on the spatial arrangement of Ca2+ channels relative to each other and to Addresses the relevant Ca2+-binding proteins (Figure 1; and [5,6]). Department of Neurobiology, Box 3209, Duke University Medical Center, Durham, NC 27710, USA; e-mail: [email protected] If the Ca2+-binding proteins are within a few nanometers of 2+ Current Opinion in Neurobiology 2001, 11:320–326 open Ca channels, the signaling arrangement is called a ‘calcium domain’ [2] or, in reference to the spatial dimen- 0959-4388/01/$ — see front matter sions involved, a ‘nanodomain’ [5]. In a single nanodomain, © 2001 Elsevier Science Ltd. All rights reserved. local Ca2+ concentrations vary steeply with distance but can Abbreviations be as high as hundreds of micromolar [3]. When many Ca2+ SNAP-25 25 kDa synaptosomal associated protein channels are clustered together in a membrane area of SNARE soluble NSF-attachment protein receptor roughly 1 µm2, a Ca2+-binding protein positioned within the cluster will sense a ‘microdomain’ of Ca2+ [5,7]. Free Introduction Ca2+ concentration within microdomains can range from Synaptic transmission results from the calcium-regulated tens of micromolar to hundreds of micromolar, depending release of quantal packets of neurotransmitter, with on the amount of summation of individual nanodomains release arising from the exocytotic discharge of neuro- arising from multiple Ca2+ channels [7,8]. transmitter after the fusion of synaptic vesicles with the presynaptic plasma membrane [1]. Given these facts, one A third type of Ca2+ signal arises when Ca2+ channels are might imagine that it would be straightforward to purify 1 µm or further from target Ca2+-binding proteins. In this sit- synaptic vesicles, identify their molecular constituents, uation, ‘radial gradients’ of concentration occur as Ca2+ and thereby understand the molecular basis of neuro- diffuses from the plasma membrane into the interior of the transmitter release. cell [9,10]. Because of the resulting dilution, Ca2+ levels at the target proteins will be much lower than in the previous Unfortunately, the problem has turned out to be more com- two cases and may be in the range of a few micromolar or less plex than appreciated initially. Nearly all of the proteins of synaptic vesicles have been identified and sequenced. Highly localized Ca2+ signaling in presynaptic Many of their binding partners within the presynaptic ter- terminals minal — either on the vesicles, the plasma membrane, or Until recently, there was relatively good agreement that the presynaptic cytoplasm — are now known. Despite this the release of fast-acting neurotransmitters (such as gluta- largess of information, a satisfying picture of the molecular mate and acetylcholine) relies on nanodomain signaling basis of exocytosis has so far eluded us. [11,12]. This consensus was constructed from many lines of evidence. First, injection of Ca2+ buffers with different Part of the problem is that neurotransmitter release rates of Ca2+ binding produces differential effects on involves many different trafficking reactions and each of transmitter release. Specifically, BAPTA, a rapidly binding these requires a substantial number of protein–protein chelator, efficiently blocks release, whereas EGTA, a slow- interactions. In this review, I would like to try to reduce er chelator, does not [13]. Second, high presynaptic Ca2+ the problem of calcium-regulated neurotransmitter release concentrations, of the order of 100–200 µM, are necessary to its simplest possible terms by concentrating on the final to produce rapid rates of secretion [14,15]. Third, titration exocytotic reactions that are triggered by calcium ions of presynaptic Ca2+ signals, by injecting Ca2+ buffers with (Ca2+). My focus will be on recent progress that addresses varying affinities, yields similar estimates of Ca2+ levels two related questions: first, how much Ca2+ is needed to during transmitter release [13]. Fourth, measurements of trigger transmitter release? Second, to which protein(s) local Ca2+ concentration with a low-affinity indicator does Ca2+ bind to trigger release? protein suggest very high Ca2+ concentrations during How does calcium trigger neurotransmitter release? Augustine 321 Figure 1 Types of presynaptic Ca2+ signals. Nanodomain Microdomain Radial gradient ‘Nanodomains’ arise from local diffusion from single open Ca2+ channels, ‘microdomains’ Synaptic Synaptic from multiple open Ca2+ channels, and ‘radial vesicle Synaptic vesicles gradients’ from long-distance movements of vesicles Ca2+ away from the channels. CBP indicates a Ca2+-binding protein that translates Ca2+ CBP entry into vesicle fusion. Modified with Plasma permission from [6]. Plasma membrane membrane Calcium channel Multiple calcium channels Current Opinion in Neurobiology transmitter release [7]. Fifth, the use of Ca2+-activated release at Ca2+ levels as low as a few micromolar, and saw potassium channels as monitors suggests that single action signs of saturation at Ca2+ concentrations greater than potentials raise presynaptic Ca2+ concentration to about 20–30 µM (Figure 2a). This relationship is markedly differ- 150 µM [16••]. Sixth, modifying the number of open Ca2+ ent from the Ca2+ requirements for transmitter release from channels causes changes in transmitter release that are other synapses; for example, approximately 20-fold higher consistent with nanodomain Ca2+ signaling [11,17]. Last, Ca2+ concentrations are required at the goldfish bipolar cell measurements of the opening of individual Ca2+ channels terminal (Figure 2a). suggest that only one channel needs to open to release transmitter [12]. Although these results indicate that a low concentration of Ca2+ is needed to trigger transmitter release, they do not From these findings, the evidence seemed strong that necessarily mean that small Ca2+ signals are produced dur- nanodomains trigger the rapid release of fast-acting ing transmitter release. It is possible that large rises in Ca2+ transmitters. Equally compelling data indicate that concentration still occur, but saturate the Ca2+-dependent microdomain-type Ca2+ signals mediate the secretion of release apparatus. Other experiments in these papers indi- transmitters at terminals that may release transmitter toni- cate that this is not the case [22••,23••]. This point was cally, such as sensory synapses [8,18]. Furthermore, radial established by comparing the rates of neurotransmitter shell-type Ca2+ signals seem to operate at peptide-secreting release to those produced by elevating presynaptic Ca2+ to terminals [19,20]. Thus, the picture seemed clear: very local, various concentrations. This null-point method provided nanodomain Ca2+ signals are used when optimizing for estimates of 5–25 µM for the levels of Ca2+ reached during speed, microdomains are used at slightly slower synapses, a presynaptic action potential. and radial shells mediate the delayed secretion of peptides. Again, this contrasts with comparable experiments done in Microdomain Ca2+ signaling at the Calyx of Held other systems; for example, uncaging 80 µM Ca2+ at the Recent studies have turned this simplification on its head, squid giant synapse causes a lower rate of transmitter however, by providing strong evidence that microdomain release than is produced by an action potential [15].Thus, Ca2+ signaling triggers the very rapid secretion of transmitter presynaptic Ca2+ signals differ even among fast-releasing at the Calyx of Held synapse in mammals. This specialized synapses. At the Calyx presynaptic terminal, presynaptic ‘giant’ synapse serves as a key relay in the auditory system. Ca2+ concentration reaches only relatively low levels dur- One early indication that things may be different from the ing an action potential. But the relatively high Ca2+ affinity picture described above came from the observation that of exocytosis allows this small signal to release large EGTA is almost as potent as BAPTA in blocking transmitter amounts of transmitter. release at this synapse [21]. This suggests that there is a sub- stantial distance between the open Ca2+ channels and the These results are compatible with a microdomain style of relevant Ca2+-binding protein, as would be expected if influx Ca2+ signaling, with several channels