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Synaptotagmin Modulation of Fusion Pore Kinetics in Regulated Exocytosis of Dense-Core Vesicles

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Science  02 Nov 2001:
Vol. 294, Issue 5544, pp. 1111-1115
DOI: 10.1126/science.1064002

Abstract

In the exocytosis of neurotransmitter, fusion pore opening represents the first instant of fluid contact between the vesicle lumen and extracellular space. The existence of the fusion pore has been established by electrical measurements, but its molecular composition is unknown. The possibility that synaptotagmin regulates fusion pores was investigated with amperometry to monitor exocytosis of single dense-core vesicles. Overexpression of synaptotagmin I prolonged the time from fusion pore opening to dilation, whereas synaptotagmin IV shortened this time. Both synaptotagmin isoforms reduced norepinephrine flux through open fusion pores. Thus, synaptotagmin interacts with fusion pores, possibly by associating with a core complex of membrane proteins and/or lipid.

The fusion pore represents a pivotal intermediate in Ca2+-triggered neurotransmitter exocytosis. Contact sites between the vesicle and the plasma membrane presumably contain special components capable of forming a fusion pore after the binding of Ca2+. The fusion pore has been likened to the channels that control ion flux across cell membranes (1), and like ion channels, single fusion pore activity can be observed in high-resolution electrical measurements (2–5). Whereas plasma membrane ion channels are formed by well-characterized proteins, the structural components of the fusion pore have yet to be identified. A molecule likely to be intimately associated with fusion pores is synaptotagmin, a synaptic vesicle protein that binds Ca2+ and phospholipid (6). Genetic and biochemical studies have suggested a number of possible functions for synaptotagmin in vesicle trafficking and fusion (7–10). One appealing scenario is that solubleN-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) form fusion pores. These proteins catalyze the fusion of lipid vesicles (11), and putative fusion pores formed by these proteins could be regulated by synaptotagmin through rapid Ca2+-stimulated binding to the SNARE complex (12). Here, we examined the role of synaptotagmin in specific steps of secretion in norepinephrine (NE)–secreting PC12 cells using amperometry to monitor release from individual dense-core vesicles.

When an amperometry electrode was positioned at the surface of a PC12 cell, exocytosis of a single vesicle registered as a spike of current as NE was oxidized at the electrode surface (3,13) (Fig. 1). These spikes were elicited by depolarization, in this case induced by rapid local application of KCl (Fig. 1) (14). Responses to KCl were characterized by a latent period with no spikes, followed by a period of spikes at irregular intervals. Recordings such as these provide information about the kinetics of exocytosis, allowing us to compare control PC12 cells (Fig. 1A) with cells transfected with either synaptotagmin I (Fig. 1B), synaptotagmin IV (Fig. 1C) (15), or the α1A (P/Q-type) Ca2+ channel (Fig. 1D). Because of the irregular nature of the release process in a single trial, we counted spikes after a given time interval and constructed normalized waiting time distributions (Fig. 1E). These plots provide an overall time course for secretion that is in qualitative agreement with previous studies of dense-core vesicle secretion in PC12 cells (16–18). Synaptotagmin I did not alter the time course significantly. By contrast, secretion was reduced by synaptotagmin IV and enhanced by Ca2+ channels. Thus, the level of secretion in PC12 cells falls in a range amenable to regulation in either direction.

Figure 1

Amperometric recordings of NE release from PC12 cells (depolarization with KCl indicated by bars). (A) Control cells. (B) Cells transfected with synaptotagmin I. (C) Cells transfected with synaptotagmin IV. (D) Cells transfected with α1A Ca2+ channels. (E) Distributions of spike times for control, synaptotagmin I, synaptotagmin IV, and α1A. The distributions were normalized to the number of trials (42 for control, 30 for synaptotagmin I, 38 for synaptotagmin IV, and 11 for α1A). Mean (F) frequency and (G) spike latency. Frequency was calculated as the maximum slope from the plots in (E); first-spike latency was averaged from all recordings. Means are of >500 events from 11 to 42 cells, in experiments from two to eight transfections. **, P < 0.01; ***, P < 0.001.

The spike frequency (maximum slopes in Fig. 1E) was doubled by Ca2+ channels, unchanged by synaptotagmin I, and reduced 2.6-fold by synaptotagmin IV (Fig. 1F). Each change in frequency was accompanied by a change in latency (time from onset of stimulation to first spike) in the opposite direction (Fig. 1G). Other spike properties such as amplitude and quantal size, which are not directly related to the kinetics of exocytosis, were not altered by synaptotagmin I or IV (19). The results with Ca2+ channels indicate sensitivity to Ca2+current, but the reduction in exocytosis caused by synaptotagmin IV transfection cannot be explained by changes in Ca2+current. Ca2+ current was unaffected by transfection with synaptotagmin I or IV (20). The mean peak Ca2+current elicited by voltage steps from −80 to +10 mV was 53 ± 17 pA in control cells (N = 6), 47 ± 10 pA in cells transfected with synaptotagmin I (N = 7,P = 0.38), and 53 ± 11 pA in cells transfected with synaptotagmin IV (N = 7, P = 0.35). As expected, transfection with Ca2+ channels increased Ca2+ current to 104 ± 9 pA (N = 5, P < 0.05). The time constants for Ca2+ current inactivation ranged from 350 to 450 ms for control and synaptotagmin-transfected cells and were statistically indistinguishable. Thus, although synaptotagmin is known to interact with Ca2+ channels (21–23), no effect was observed here.

The vesicle size distribution is altered in Drosophilalacking synaptotagmin I (24). We examined the distribution of quantal size, as this should reflect dense-core vesicle size (provided that vesicular NE concentration remains the same). Spike area (cube root transformed to be proportional to vesicle diameter) appeared to be normally distributed and was indistinguishable between controls and synaptotagmin I or IV (19). Ca2+ channel transfection produced a small but significant increase in spike amplitude and quantal size as well as a small shift in the distribution. PC12 cells contain a heterogeneous population of vesicles (25), and this analysis indicated that synaptotagmin transfection did not redistribute vesicles between size groups.

The different vesicles of PC12 cells undergo exocytosis with different kinetics (16, 17). To determine whether transfected proteins were targeted to dense-core vesicles, we prepared fusion proteins of cyan fluorescent protein (CFP) linked to the NH2-terminus of synaptotagmin I and IV (26). Cells transfected with these constructs exhibited CFP fluorescence concentrated in highly mobile puncta, consistent with a location on vesicles. Ca2+-triggered exocytosis was readily seen in these cells. Cells were fixed and stained with antibodies against CFP and the dense-core vesicle protein chromogranin (Fig. 2). Chromogranin and synaptotagmin exhibited the same pattern of fluorescence, and overlays showed that both synaptotagmin I and IV colocalized with chromogranin (Fig. 2). Thus, both isoforms were targeted to dense-core vesicles.

Figure 2

Synaptotagmin on dense-core vesicles. CFP-tagged synaptotagmin and the dense-core vesicle protein chromogranin were visualized by double immunofluorescence (26) in cells transfected with CFP-synaptotagmin I (A) and CFP-synaptotagmin IV (B). CFP-synaptotagmin is shown as green (left) and chromogranin as red (center). Merged images (right) show colocalization as yellow. Cells that did not take up cDNA for the fusion protein stain only for chromogranin and appear red in the merged images.

If synaptotagmin participates in membrane fusion, then transfecting different isoforms should alter fusion pore kinetics. We therefore examined the “foot” current, which immediately precedes the steeply rising spike (Fig. 3A). Foot onset signals fusion pore opening, and feet are seen in association with ∼80% of spikes ≥ 20 pA. The amplitude of ∼3 pA was similar to that in chromaffin cells, but the duration was considerably shorter (3, 27). We examined the lifetimes of feet to determine whether synaptotagmin influences the stability of the fusion pore. As with ion channels (28,29), the transitions of fusion pores are stochastic in nature, so we used lifetime distributions to characterize fusion pore kinetics (Fig. 3B). These were well fitted by a single exponential (P < 0.0001 for each fit), as previously reported (3). Thus, the open fusion pore can be represented by a single kinetic state. The time constant obtained from these exponential fits is equal to the mean open time, which was 1.38 ± 0.04 ms in control cells, 1.76 ± 0.06 ms with synaptotagmin I, and 0.97 ± 0.02 ms with synaptotagmin IV. These values are significantly different (Fig. 3C, left), indicating that exogenous synaptotagmin alters the stability of the fusion pore. Vesicles containing excess synaptotagmin I formed more stable fusion pores, and vesicles containing excess synaptotagmin IV formed less stable fusion pores. The lifetime of 1.70 ± 0.05 ms with Ca2+ channels was similar to that with synaptotagmin I, suggesting that enhancing Ca2+ entry and enhancing Ca2+ sensing have similar effects on fusion pore dynamics.

Figure 3

(A) Foot current indicates fusion pore opening. Different scales illustrate the whole spike (left) and the foot (right). The foot regions (shaded) are delimited by the baseline and spike onset (scale bars: 10 pA, 2 ms, left; 5 pA, 1 ms, right). (B) Fusion pore open-time distributions. Single exponential fits yielded the mean open time, τ (τfp in text). (C) τ (left) and mean NE flux (right). Flux was computed as foot area divided by duration. **, P < 0.01; ***,P < 0.001. All means were of ∼100 events.

The foot current amplitude, by analogy with the ion channels, can be influenced by the shape of the fusion pore and by the dynamics of very rapid transitions between permeation states. Ca2+ channels, synaptotagmin I, and synaptotagmin IV all reduced the foot amplitude (Fig. 3C, right). This suggests that these proteins either influence the structure of the fusion pore, possibly by narrowing or lengthening it, or modulate a dynamic pore-gating process that is too fast to see with our instrumentation.

The foot current is thought to terminate by fusion pore dilation, during which transmitter rapidly escapes from a vesicle in a spike of current. This phase of release is controlled by NE escape from an intravesicular matrix (27), as well as diffusion when exocytosis occurs at sites away from the electrode (30). Transfection with proteins altered spike shape (Fig. 4). Synaptotagmin IV accelerated the rise time (Fig. 4B) but left the decay times unchanged (Fig. 4, C and D). Slightly more than 50% of the spike decays were double exponential, with the fast component dominating. Ca2+ channels and synaptotagmin I had no effect on the rise time (Fig. 4B) but slowed the fast component of decay (Fig. 4C). The slow component of decay was markedly slowed by synaptotagmin I (Fig. 4D). MUNC18 was also recently reported to alter spikes, reducing the half-width and quantal size of spikes in chromaffin cells (31). Because spike shape and size are governed by events after fusion pore dilation, these results are not what one would expect for proteins involved in membrane trafficking. Thus, the factors controlling transmitter efflux after fusion pore dilation require further study.

Figure 4

(A) Spike shapes and decays. Spikes are shown alongside double exponential fits to the decay phase (scale bars: 10 pA, 1 ms). (B) Spike rise times (35 to 90%). (C) The time constant for the fast component of spike decay, τ1 (decays of single exponential fits were similar to the decays of the fast component of double exponential fits, so the values were combined). (D) Time constants for the slow component of decay, τ2, from decays that were double exponential. *,P < 0.05; **, P < 0.01 [N = ∼100 in (B), ∼100 in (C), and ∼25 in (D)].

Synaptotagmin I and IV both altered fusion pore lifetime (Fig. 3), indicating that this protein regulates the fusion step. Because our measurements are at the single-channel level, we used ideas developed from channel kinetics to interpret the results (28,29). We consider assembly of a closed fusion pore, reversible opening and closing, and irreversible open pore dilation in a kinetic scheme.Embedded ImageThe open state (O) produces foot current, and the dilating state (D) produces the spike. k a is the assembly rate constant, k o and k c are the opening and closing rate constants, and k dis the rate of entering the dilating state. The open-time distribution will then be a single exponential with a time constant:Embedded ImageBoth k d and k ccontribute to τfp, because both processes terminate the open fusion pore state. According to this model, synaptotagmin I reduced the sum k d +k c, whereas synaptotagmin IV increased it.

Changes in k a, k o,k d, k c, or the number of membrane-associated vesicles can all account for the changes in time course of exocytosis in Fig. 1. Increasing k cwould make exocytosis slower by terminating more fusion pore openings without producing spikes, leaving stand-alone feet, which often elude detection (32). It is important that increasingk c reduces the frequency and increases the latency, because it can also account for the reduction in τfp. This provides a simple explanation for the direction of the observed changes produced by synaptotagmin IV in three different measurements. Synaptotagmin I increased the fusion pore lifetime (Fig. 3) but left the latency and frequency unchanged (Fig. 1). According to the above model, this would result from 20% reductions in bothk d and k c. This will not alter spike frequency or latency (Fig. 1, F and G) because the fraction of open fusion pores that dilate,k d/(k c +k d), remains unchanged. Thus, the results of both synaptotagmin I and IV are most parsimoniously explained by changes in rate constants directly associated with the open fusion pore.

PC12 cells express synaptotagmin I (33,34) and IX (35) and contain synaptotagmin III mRNA (36). Transfection with synaptotagmin I should increase the ratio of synaptotagmin I to other isoforms and may increase the number of copies of synaptotagmin per vesicle as well. The parallel changes in k d andk c just discussed for synaptotagmin I would then reflect these changes. Enhancing Ca2+ entry with extra Ca2+ channels altered the stability of the fusion pore in the same way as extra synaptotagmin I, as expected if synaptotagmin I acts as a Ca2+ sensor. Synaptotagmin IV levels are normally very low in PC12 cells (34). In light of the weak Ca2+ binding of this protein (37), the reduced exocytosis seen with synaptotagmin IV further supports synaptotagmin's role as a Ca2+ sensor. Overexpression of synaptotagmin IV in Drosophila reduces synaptic transmission (38), and this may reflect the reduction in Ca2+-triggered fusion observed here.

Synaptotagmin influences the stability of open fusion pores and is therefore likely to be intimately associated with a molecular complex that performs the function of fusing vesicle and plasma membranes. Changes in fusion pore dynamics have recently been proposed to play a role in synaptic plasticity (39), and the control of synaptic vesicle fusion pores by synaptotagmin could provide a molecular mechanism. Synaptotagmin oligomerizes (40) and binds lipids (6), syntaxin (41), SNAP-25 (42), and the SNARE complex (12), all in a Ca2+-dependent manner. Because SNARE proteins fuse membranes (11), the synaptotagmin-mediated alteration in fusion pore open time may represent a functional consequence of these interactions. Mutations in synaptotagmin that impair Ca2+-triggered lipid binding (9) and oligomerization (10) reduce synaptic transmission, and the present approach offers a means of assessing whether these mutations act through a change in fusion pore kinetics. Future studies on how the control of fusion pores by synaptotagmin is regulated by Ca2+ and SNARE proteins may provide a precise picture of how this protein transduces Ca2+ signals during excitation-secretion coupling.

  • * To whom correspondence should be addressed. E-mail: mjackson{at}physiology.wisc.edu

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