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The Calcium Signal for Transmitter Secretion from Presynaptic Nerve Terminals" GEORGE J. AUGUSTINE,bd E. M. ADLER,C.d,eAND MILTON P. CHARLTONC,d bDepartment of Neurobiology Duke UniversityMedical Center Bryan Neuroscience Building Durham, North Carolina 27710 CDepartrnentof Physiology University of Toronto Toronto, Ontario MSS l A 8 Canada dMarine Biological Laboratory Woods Hole, Massachusetts 02543 Although it has been known for more than 30years that Ca ions trigger the exocytotic release of neurotransmitters,' it still is not clear how a rise in Ca concentration ([Ca],) within a presynaptic terminal is transduced into an increased probability of exocytois.^.' One of the reasons that we do not know how Ca causes exocytosis is that we do not know how much Ca is needed for exocytosis. Clarification of this question will define the Ca binding properties of the receptor molecule that triggers exocytosis and provide an important clue to the identity of the receptor molecuIe.4 This article addresses the calcium requirements for transmitter release by summarizing some recent insights into the magnitude and spatio-temporal dimensions of the presynaptic Ca signal that triggers transmitter release. In experimental terms, there are two ways that this question can be approached. First, one could manipulate [Ca], and ask how high it must be raised in order to mimic physiological rates of secretion.596Alternatively, one can ask how high [Cali rises during the secretion event. The latter approach, as applied to the unique 'giant' presynaptic terminal of squid,' will provide the theme for our article.

DIRECT MEASUREMENTS OF PRESYNAPTIC CALCIUM CHANGES DURING TRANSMJITER RELEASE The most obvious way to ask how high [Ca], gets during transmitter release is to use an intracellular Ca indicator to directly measure presynaptic [Ca], during stimuli that trigger release. The fluorescent dye, fura-2,' is presently the indicator of choice for measuring the magnitude of the presynaptic [Ca], changes associated with aThis work was supported by NIH Grant NS-21624 to G. J. Augustine and an MRC Grant to M. P. Charlton. ePresent address: Laboratory of Molecular Neurobiology, Massachusetts General Hospital East, 149 13th Street, Charlestown, MA 02129. 365

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transmitter release. Applications of earlier, less precise Ca indicators have been summarized in reference 2. At the squid giant synapse, photomultiplier measurements of fura-2 fluorescence allow quantification of the presynaptic [Ca], changes caused by action potentials. Such measurements reveal that a single presynaptic action potential raises [Ca],by a surprisingly small amount, approximately 5 nM from a resting level of approximately 50-100 nM.9 One important consideration in evaluating such measurements is that they reflect a spatial average, so that spatial gradients of [Ca], within the presynaptic terminal will not be detected. In particular, it is possible that [Ca], changes in the vicinity of sites of secretion may be higher than those in the bulk cytoplasm, because the voltage-gated Ca channels that are the source of the [Ca], signal are clustered in the vicinity of the active zones, the sites of secretion in these In order to address this possibility, video-imaging methods have been used to make spatially resolved measurements of [Ca], within small presynaptic compartments in the vicinity of the sites of exocytosis. Such measurements, however, still indicate nM changes in [Ca], in response to a single presynaptic action potential.” Two features of the time-course of the [Ca], changes measured with videoimaging techniques suggest limitations even in these measurements of presynaptic [Ca],. First, the decay time of the measured [Ca], changes do not coincide with those expected at the release sites: presynaptic [Ca], changes evoked by action potentials decay over tens of seconds, even though transmitter release ends within a couple of milliseconds following an action potential.‘ Second, the pattern of Ca signals evoked by trains of action potentials is unexpected: [Ca], gradually rises in a ramp-like fashion, so there are no changes with kinetic properties similar to an action potential. Part of the solution to these two problems could be the limited time resolution (ca. 100 ms) of the video cameras used to make spatial maps of [Ca], in these experiments. However, similar slow changes in [Ca], are detected even when using faster detectors such as a photomultiplier or photodiode. A more likely explanation can be found in the limited spatial resolution of the imaging methods, which is on the order of a few micrometers in this tissue.” It is likely that the spatial compartmentalization of the presynaptic [Ca], signal exceeds this limit, and thus the [Ca], signal at the secretory sites still is underestimated by our imaging measurements. Theoretical studies of the diffusion of Ca ions into presynaptic cytoplasm suggest that presynaptic Ca signals drop off steeply in the vicinity of open Ca FIGURE1 is a cartoon that summarizes the expected changes in [Ca], in two presynaptic compartments. The idea is that near an open Ca channel, [Ca], should rapidly rise to high levels and then decline very rapidly when the channel closes. At some distance away from the channel, however, the change in [Ca], will be much smaller and much slower because this signal is low-pass filtered by diffusion throughout the terminal. The video camera is likely to be measuring the [Ca], away from the open channels, because most of the volume of the presynaptic terminal is far from any Ca channels. Thus, this measured signal will be relatively small and slow in comparison to the signal near the open Ca channels, which is presumably the signal that triggers release. Thus, the imaging probably is only revealing the proverbial tip of the iceberg. In fact, depending upon the distances and volumes involved, the actual value of [Ca], changes at active zones could be orders of magnitudes higher than a few nM per action potential. So the problem we faced is how to selectively measure [Ca],within the minute microenvironment of the active zones.

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CALCIUM BUFFERS AS PROBES OF THE PRESYNAPTIC CALCIUM SIGNAL

The only sensor that selectively detects [Ca], at the release sites is the Ca receptor that triggers secretion. We therefore have used the secretory event as an assay to tell us something about the relative [Ca], levels at the release site. Armed with this assay, we then microinjected Ca buffers to try to compete with this receptor and used the resultant changes in secretion, along with the known Ca binding properties of these buffers, to deduce the magnitude and time-course of the Ca signal for secretion." 2. This Examples of results obtained with this approach are shown in FIGURE FIGURE illustrates the differential effects of EGTA and BAPTA, two buffers with

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Time (ms) FIGURE 1. Schematic of [Ca], changes occurring within a presynaptic terminal during an action potential. In the vicinity of the Ca channels opened by the action potential, [Ca],will rise rapidly to high levels and decline rapidly after the channels close. Farther away from the Ca channels, the [Ca], changes will be smaller and slower.

similar affinities for calcium. When injected into presynaptic terminals at concentrations estimated to be in excess of 10 mM, BAF'TA produced a prompt and reversible reduction in action potential-evoked transmitter release, whereas EGTA had negligible effects (see also ref. 19). This shows that despite their similar affinities for Ca, BAF'TA can successfully compete with the Ca receptor for Ca ions, whereas EGTA cannot. This unexpected difference in buffer efficacy presumably is due to differences in the kinetics of Ca binding by the two buffers.*' Previous voltage-clamp experiments at the squid giant synapse show that the minimal delay between Ca entry into the presynaptic terminal and initiation of postsynaptic electrical responses is on the order of a few hundred micro~econds.'~~~' The Ca binding properties of these two Ca

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buffers during this brief time period is illustrated in FIGURE3. Under conditions where the concentration of buffer is high relative to the magnitude of the presynaptic [Ca], change, BAPTA should bind Ca within a fraction of a ps, whereas the time constant for Ca binding to EGTA is on the order of 400 times slower.18It therefore appears that EGTA is too slow to bind Ca before the Ca receptor does, so that release can proceed undiminished. The more rapid kinetics of BAPTA allow it to successfully compete with the receptor and attenuate transmitter release. This suggests that the Ca receptor must bind Ca very rapidly, in the order of tens of

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FIGURE 2. Differential effects of EGTA and BAPTA on transmitter release. Either EGTA (top) or BAPTA (bottom) were microinjected into squid presynaptic terminals to estimated concentrations in excess of 10 mM. Although EGTA had no effect on postsynaptic responses (Post) elicited by presynaptic action potentials (Pre), BAPTA produced a progressive reduction in synaptic transmission. Traces 1 and 9 in the BAPTA experiment indicate postsynaptic responses evoked after 1 and 9 had brief injections of BAPTA. (Adler et al.'* With permission from the Journal of Neuroscience.)

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Time ( p s e c ) FIGURE 3. BAPTA and EGTA bind Ca at different rates. At concentrations of 50 mM, BAPTA will bind Ca with a time constant of less than 1 ps (dashed line), whereas EGTA will bind 400 times more slowly (solid line).

microseconds, because EGTA will start binding Ca at later times. Thus, it appears that the Ca binding step that triggers release is a relatively small fraction of the total, few hundred microsecond-long synaptic delay. Further, because Ca has a finite rate of diffusion, this also means that the Ca receptor responsible for triggering exocytosis must be within a few tens of nanometers of the open Ca channel. This analysis also suggests that BAPTA binding to Ca may be so fast as to be at equilibrium during a BAPTA-buffered release event. Under such conditions, one can then chemically alter the Ca affinity of the BAPTA molecule and determine how changes in affinity alter the ability of BAPTA to attenuate transmitter release. We found that BAPTA derivitives with different affinities (Kds)for Ca have different abilities to compete with the release receptor (FIG. 4). The most effective was dibromoBAPTA (Kd = 4.9 pM); dimethylBAPTA (Kd = 0.2 kM) and BAPTA (Kd = 0.5 pM) were next in potency, whereas dinitroBAPTA, with a Kdof approximately 30 mM, was much less effective. A SIMPLE NUMERICAL MODEL FOR DIFFERENTIAL BUFFER EFFICACY

To translate this pattern of buffer efficacy into information regarding [Ca], levels at the release site, we used a simple numericaI model of Ca-buffer interactions. In this model the primary free parameter was the size of the Ca concentration change to which the buffers were responding. If the presynaptic concentration and Kd of a buffer are known, and the reaction between calcium and buffer reaches equilibrium during the transient rise in [Ca], produced by an action potential, then the relative attenuation of the [Ca], change produced by the buffer can be estimated from the following steady-state equations:

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[call =

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[Catotal] = [Ca],+ [CaBuffer] [Buffer,,,,,] = [CaBuffer]+ [Buffer],

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in which [Catotal] is the level of [Ca], produced by an action potential before injection of buffer; [CaBuffer] and [Ca], are the fraction bound to buffer and remaining free,

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FIGURE 4. Relative efficacy of BAPTA derivatives in attenuating transmitter release. Release, measured as amplitude of postsynaptic response evoked by a presynaptic action potential, decreased with higher concentration of buffer within the presynaptic terminal. DMB = dimethylBAF'TA and DBB = dibromoBAPTA. (Adler er at.'* With permission from theJournal of Neuroscience. )

respectively; [Buffer,,,,,] is the total concentration of buffer injected into the terminal; and [Buffer] is the fraction remaining uncomplexed with Ca. The equations can be combined to determine [Ca], (see refs. 22 and 23): [([Cat"ta'] - Kd - [Buffer,,,,,]) rf: [(Kd + - [ ~ a ~ ~+ , ~~~d[C~tota~11"21 ~l)Z [ca],= (4) 2

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This treatment assumes that the concentration of exogenous buffer is much higher than that of any endogenous buffer, so that the contribution of such a putative endogenous buffer under these conditions is negligible. Equation 4 was used to predict the relative reduction in presynaptic [Ca], produced by different concentrations of the various buffers. The results of this calculation are that the buffers should reduce transmitter release roughly in proportion to their KD values, with buffers having lower KDs more effective in reducing transmitter release. These differences between the theory and experimental observations indicate that equation 4 is not sufficient to predict the ability of a buffer to reduce evoked transmitter release. One limitation of the above calculation is that it does not account for buffer binding to calcium present in the resting nerve terminal (resting [Ca],) prior to nerve stimulation. With a resting [Ca], of 50-100 nM,9 the reduction in free buffer concentration for a buffer load of 300 pM to several millimolar is negligible if the chelation of resting [Ca], is uncompensated and [Ca], is allowed to drop to levels on the order of M. Studies in other systems (see references in ref. 24), however, suggest that homeostatic mechanisms can maintain resting [Ca], at normal levels in the presence of several mM exogenous calcium buffer. If it is assumed that resting [Ca], gravitates to a set point, with Ca from the extracellular fluid and intracellular stores replacing that which is chelated by the buffer, a certain fraction of the buffer will be bound to resting Ca and will therefore be unavailable to chelate the additional Ca added by an action potential, that is, [Catota,].The exact fraction of a given buffer load that is still available to chelate [Ca],,,,] will depend on the buffer Kd and the resting [Ca], set point. Under equilibrium conditions, free buffer concentrations will be predicted by the following equation:

in which [Cartst]is the set point for resting [Ca],. If a correction is made for the reduction of free buffer by resting Ca, and a term added to equation 4 so that [Ca] during the transient cannot be buffered to levels below 5 x lo-* M, predicted [Ca] for the range of concentrations of the BAPTA-family buffers resembles the pattern of buffer efficacy in reducing transmitter release observed experimentally (FIG.5A). The resemblance is apparent regardless of whether it is assumed that the relationship between [Ca], and transmitter release is a linear f ~ n c t i o n or ’ ~ a power function.2125.26 The exact form of the predicted relationship between buffer concentration and [Ca], is strongly dependent on the specific values chosen for parameters, such as the exact size of the [Ca], change produced by an action potential and the resting [Ca], level. Therefore the predictions of the model can be used to estimate these values. In particular, if the peak [Ca], change during an action potential is less than 100 pM, dimethylBAPTA is predicted to be more effective that dibromoBAPTA, which was not observed. Thus, our results are consistent with [Ca], levels on the order of one-hundred micromolar in the vicinity of the release sites during an action potential. One consideration ignored in the above calculations is the influence of endogenous buffers on presynaptic [Ca], and the effect of injected buffers on [Ca],. Although relatively little is known about the properties of such a buffer, if its concentration and Kd are known, then equations 1-3 can also be used to evaluate the impact of this endogenous buffer on [Ca],. For the purposes of calculation, we assumed that the

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presynaptic terminal might have 1mM of an endogenous buffer having a Kdof 1 pM. After including this endogenous buffer, the minimum [Catota,] needed to replicate the observed order of buffer efficacy was 2 mM, with [Caliin this situation being 1.1 mM or greater after complexation by the endogenous buffer (FIG. 5B). Thus, any endogenous buffer present in the presynaptic terminal will raise the total amount of Ca needed to account for our experimental results. The results of these simple calculations force us to conclude that the presynaptic Ca change caused by an action potential is on the order of one-hundred p M or higher, depending on the amount of endogenous buffer in the presynaptic terminal. Similar conclusions have been reached in other secretory cells. By using Ca-activated

FIGURE 5. Numerical model of attenuation of the presynaptic Ca signal by different BAPTA derivitives. A Reduction in [Ca], predicted with [Catota,]= 700 pM, [Ca,,,] = 100 nM. B Reduction in [Ca], predicted with [CatotsJ= 2 mM, [Carest]= 100 nM, and the addition of 1 mM endogenous buffer with a Kdof 1 p M .

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K channels as indicators of [Ca], in hair cells, Roberts et al.” suggest that [Ca], in the secretory region of these cells may reach 100 p M or even higher during depolarization. A similar use of the secretory response as an indicator of [Ca], in adrenal chromaffin cells also suggests levels on the order of 10-100 pM during depolarization.6Thus, three different technical approaches applied to three different experimental systems all suggest that [Ca], at secretory sites is fairly high during exocytosis, perhaps as high as several hundred pM. This indicates that the measurements of a few nM change in [Ca], during a presynaptic action potential were indeed severe underestimates of the magnitude of the changes in the vicinity of the secretory sites

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and that steep spatial gradients of [Ca], occur in the presynaptic terminal during an action potential.

HIGH PRESYNAPTIC CALCIUM LEVELS ARE PRODUCED BY DISCRETE DOMAINS OF CALCIUM ELEVATION We will end our discussion of the presynaptic [Ca], signal by considering how the presynaptic terminal is able to generate such a large change in Ca concentration. We believe that the answer lies in localized “domains” of elevated [Ca], in the immediate vicinity of open Ca channels. The relevant calculations for such a situation (first performed as reported in refs. 28, 16, and 17) present two possible scenarios for generating tens or hundreds of FM Ca2+at a release site. One possibility, schematized in FIGURE 6A, is that [Ca], can reach these concentrations within a single domain if the release site is only a few nanometers away from the open channel (FIG. 6B). This small separation is consistent with the spacing estimates derived from the lack of effect of EGTA, as described above. An alternative possibility is that even if the release site is not so close to a single open Ca channel, high [Ca], levels could result if many open Ca channels are clustered close together. This possibility is diagrammed in FIGURE6C. In this situation, the resulting domains will overlap and produce a generalized rise in Ca, throughout the active zone (FIG. 6D). The clustering of Ca channels illustrated in FIGURE 6C is consistent with video-imaging observations indicating the clustering of Ca channels at secretory ~ i t e sand ’ ~ also is consistent with freeze-fracture measurements of the spacing between large intramembranous particles in the presynaptic membrane.10.z9 The key distinction between these two possibilities is whether or not the [Ca], domains associated with open Ca channels overlap with each other. We have performed experiments designed to look for signs of domain overlap by again using transmitter release as an indicator of relative [Ca], levels at the active zones. In these experiments the number of domains was increased by prolonging the duration of the presynaptic action potential, using pharmacological agents that block the voltagegated K channels responsible for the repolarization phase of the action potential.” Such broadening increases the macroscopic Ca current by increasing the number of open Ca channels, which results in an increased number of domains being generated. Interpretation of these experiments relies on the cooperative behavior of transmitter r e l e a ~ e * ’to , ~yield ~ ~ ~ information ~ on the relative level of [Ca], at the release sites. The assumption is that there is a nonlinear, cooperative relationship between the amount of Ca entering into a domain and the release that results (FIG.7A). The best experimental support for this assumption comes from voltage-clamp experiments performed on the squid giant synapse.26In these experiments, presynaptic Ca influx was varied by changing the external Ca concentration, while repeatedly depolarizing the presynaptic terminal to a constant membrane potential to open a constant number of Ca channels (FIG.7B). Under these conditions the number of domains produced is constant, while the single channel Ca current, and thus the size of the [Ca], signal within each domain, is reduced as [Ca], is lowered. The resultant power-function relationship between the size of the presynaptic Ca current and amount of transmitter release can be interpreted as being due to changes in the size of the [Ca], signal within each domain. These results therefore illustrate that the cooperative nature of transmitter release causes increases in [Ca], within a domain to produce exponential increments in

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transmitter release. Similar observations are made when the presynaptic Ca channels are opened with action potentials instead of voltage-clamp pulse^.*^*^'^^^ Based on such considerations, the cooperative triggering of transmitter release within single domains can be used to ask how [Ca],within domains changes when the number of domains is increased by broadening the presynaptic action potential. If the domains do not overlap, then release will depend on the number of new domains produced and will increase in proportion to the increment in the magnitude of the macroscopic Ca current (FIG. 8A). Conversely, if domains overlap then release will increase more than the number of domains, because the additional overlap produced by recruitment of new domains will elevate [Ca], within these domains and cause the cooperative relationship between [Call and release to be expressed. In the extreme case of complete overlap between domains, then release will increase in proportion to the fourth power of the increment in macroscopic Ca current (FIG. 8B). Thus, the relationship between the increment in macroscopic Ca current and transmitter release provides a means of determining whether or not domains overlap.

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FIGURE 7A. Cooperative triggering of release within a single domain. Left: schematic diagram of distribution of [Ca], changes (bold line) within a domain centered over a single Ca channel. Right: cooperative relationship between [Ca], and probability that release will take place within the domain.

At the squid giant synapse, the K channel blockers, tetraethylammonium and 3,4-diaminopyridine, were used to increase the duration of the presynaptic action potential and thereby increase the number of domains generated by the action potential?” Broadening of the presynaptic action potential caused large increases in transmitter release, as assayed by an increase in the magnitude of the postsynaptic current evoked by a presynaptic action potential (FIG. 9A). At other synapses, K channel blockers also enhance transmitter release (e.g. ref. 33). A plot of the relationship between the duration of the action potential and the amplitude of the postsynaptic current yields a linear relationship, with a slope of approximately 6.3% change in release for a 1%increase in action potential duration (FIG.9B). In order to estimate the magnitude of the macroscopic Ca current flowing into the presynapse during these action potentials, a numerical simulation3’ was performed based on the Hodgkin and Huxle? mathematical model of an action potential and previous voltage-clamp measurements of the voltage-dependent gating of the Ca channels of the squid presynaptic terminal?5r36 This kind of simulation, with no free parameters except for the duration of the presynaptic action potential, indicates that the magnitude of the macroscopic Ca current greatly increases as the

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FIGURE 8. Predicted effects of increased number of domains depend upon whether or not domains overlap. A: If domains do not overlap, then increasing the number of domains will produce a linear rise in release. B: If domains overlap, then release will increase in a supralinear fashion, due to the increment in [Ca], (bracket) at the release sites within the domains.

action potential broadens (FIG. 10A). The quantitative relationship between the duration of the action potential and the integral of the Ca current is a linear function, with an approximate slope of 7% change in Ca influx per 1% change in action potential duration (FIG. 10B). Knowledge of the relationship between action potential duration and magnitude of the macroscopic Ca current (FIG. 10B) allows the relationship shown in FIGURE 9B to be expressed in terms of Ca influx rather than action-potential duration. Such a

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AP duration (%control) FIGURE 9. A Broadening presynaptic action potentials (VPJ by treatment with 3,4diaminopyridine causes large increases in postsynaptic current responses (PSC) evoked by the action potentials. B: Linear relationship between presynaptic action potential (AP)duration and peak amplitude of PSCs. (Augustine?" With permission from the Journal of Physrobg~ (London).)

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FIGURE 10. Numerical simulation of effects of presynaptic action potential broadening on Ca current. A: Increasing the duration of the simulated presynaptic action potential (VPJ causes a large increase in the amplitude of the presynaptic Ca current (Ic,). B Predicted relationship between presynaptic action potential (AP)duration and presynaptic Ica. (Augustine?' With permission from the Journul ofPhysiologv (London).)

transformation reveals that transmitter release increases in direct proportion to the amount of Ca entry (FIG. ll), as has also been reported by Llings et 1 2 1 . ~This ~ relationship is as predicted for the case where domains do not overlap (FIG.8A). Thus, these results suggest that the high [Ca], levels generated at active zones during action potentials are generated within single, nonoverlapping domains. Similar conclusions have been reached on the basis of different experiments performed at frog neuromuscular synapses." Both sets of results suggest that the Ca receptor that triggers release must be very near (within a few nanometers) the voltage-gated Ca channel, because the [Ca], signal cannot reach levels on the order of 100 pM at distances greater than a few nanometers away from the channel (e.g. FIG.6B). At odds with these conclusions are two suggestions of apparent domain overlap in presynaptic terminals. First, high local [Cali levels in active zones of hair cells 300 n

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appear to be caused by domain overlap,” but the Ca channels in this cell appear to be packed in a very high density in comparison to the squid terminal.” Thus, Ca channels of hair cells, but perhaps not those of most presynaptic terminals, may be specially organized to produce overlap. Second, at voltage-clamped squid synapses the relationship between macroscopic Ca current and postsynaptic response has been reported to be a high-order, power function when the number of domains is increased by voltage-clamp depolarizations to different membrane potentials.21,28 This suggests a high degree of spatial overlap of domains (as in FIG. 8B). The 11, however, is difference between these end results and those shown in FIGURE probably due to differences in the number of domains generated by these two kinds of stimuli: during an action potential, only about 10% of the Ca channels are opened,” whereas much larger numbers of channels were probably opened in the voltage-clamp experiments because they used depolarizations much longer-lasting than an action potential. Thus, at the squid synapse it seems that during an action potential the average distance between open Ca channels is sufficiently large to prevent overlap, but during voltage-clamp pulses this distance is decreased and allows overlap to occur. Getting back to the question posed at the beginning of this article, it seems that the receptor to which Ca binds to trigger release experiences high [Ca], levels, perhaps on the order of hundreds of pM, and binds Ca within a few tens of microseconds. Because of the concentrations and times involved, as well as the apparently nonoverlapping nature of the local [Ca], domains within an active zone, this receptor must be located within a few tens of nanometers of the voltage-gated Ca channels of the active zone. The cooperative triggering of release within a single domain suggests the presence of multiple Ca binding sites on one or more molecules important in release, perhaps simply on the Ca receptor itself. These considerations should help molecular biologists to identify more plausible candidates for the Ca receptor that mediates release.

ACKNOWLEDGMENT

We thank Shelli Sedlak for typing this manuscript.

REFERENCES 1. KATZ, B. 1969. The Release of Neural Transmitter Substances. Liverpool University

Press. Liverpool. 2. AUGUSTINE, G. J., M. P. CHARLTON & S. J. SMITH. 1987. Calcium action in synaptic transmitter release. Annu. Rev. Neurosci. 1 0 633-693. 3. KELLY,R. B. 1988. The cell biology of the nerve terminal. Neuron 1: 431-438. S. J. & G. J. AUGUSTINE. 1988. Calcium ions, active zones and synaptic transmitter 4. SMITH, release. Trends Neurosci. 11: 458-464. K. R. & R. S. ZUCKER. 1990. Calcium released by photolysis of DM-nitrophen 5. DELANEY, stimulates transmitter release at squid giant synapse. J. Physiol. (Lond.) 426 473-498. 6. AUGUSTINE, G. J. & E. NEHER.1991. Calcium requirements for secretion in bovine chromaffin cells. J. Physiol. (Lond.) In press. 7. LLINLS, R. R. 1982. Calcium in synaptic transmission. Sci. Am. 247: 56-65. 8. GRYNKIEWICZ, G., M. POENIE & R. Y. TSIEN. 1985. A new generation of Caz+indicators with greatly improved fluorescence properties. J. Biol. Chem. 260 3440-3450. 9. ZIPSER, K., G. J. AUGUSTINE & J. DEITMER. 1991. Na/Ca exchange regulates presynaptic calcium levels. Biophys. J. 5 9 594a.

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The calcium signal for transmitter secretion from presynaptic nerve terminals.

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