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Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan
Submitted 24 August 2006; accepted in final form 29 October 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Koh and Bellen 2003; Südhof and Rizo 1996). Recent findings support the idea that Ca2+ binding to the C2B domain is essential for such synaptic transmission (Mackler et al. 2002; Nishiki and Augustine 2004b; Robinson et al. 2002). In addition, Mackler et al. (2002) generated two strains of transformants in each of which two negatively charged aspartates (D) that coordinate the binding of Ca2+ were mutated to asparagines (N). The mutated syt I was expressed in the syt I–null background and synaptic transmission was examined at the neuromuscular junction (NMJ). One transformant, P[syt IB-D3,4N], survived beyond third instars. The mean amplitude of nerve-evoked synchronous synaptic potentials in P[syt IB-D3,4N] was reduced to <1% of the control in which wild-type syt I was expressed in the syt I–null background (P[syt IWT]). The other transformant, P[syt IB-D1,2N], did not survive beyond the embryonic stage and synaptic transmission was studied in third instar larvae expressing syt IWT and P[syt IB-D1,2N]. The mean amplitude of nerve-evoked synchronous synaptic potentials was reduced to <10% of control (Mackler et al. 2002).
In cultured mouse hippocampal neurons, Nishiki and Augustine (2004b) examined the mutations of each of aspartate (D) to asparagine (N) in the C2B domain on nerve-evoked synaptic transmission. When they expressed mutated Syt I(D2N) (D at position 309) or Syt I(D3N) (D at position 363) in syt I knockout synapses, synchronous synaptic currents were virtually abolished, whereas asynchronous release was not enhanced as in syt I knockout synapses. On the other hand, the substitution of D to N at positions 303 (D1), 365 (D4), and 371 (D5) had relatively minor effects on synaptic transmission. They concluded that Ca2+-binding sites in C2B are essential for fast synaptic transmission. Furthermore, because in syt I(D2N) and syt I(D3N), delayed asynchronous release was not enhanced as in syt I knockout synapses, they concluded that Syt I has dual functions: synchronization of fast release and suppression of delayed release.
We sought to address the dual roles of Syt I in synaptic transmission using Drosophila syt I mutants. The previous study (Mackler et al. 2002) was limited in three aspects; 1) Synaptic transmission in P[syt IB-D1,2N] lines could not be studied in the syt I–null background because the mutant did not survive beyond the embryonic stage. 2) In P[syt IB-D3,4N] third instars, synaptic transmission at the NMJ might have been modified during development as the result of defective synaptic transmission and may not quantitatively reflect the effect of mutation per se. 3) Synaptic transmission was assessed as membrane potential changes, thus impairing quantitative comparisons with previous results from voltage-clamp experiments. To cover these shortfalls, in this study we examined synaptic transmission in P[syt IB-D3,4N] and P[syt IB-D1,2N] embryos in the absence of wild-type syt I using patch-clamp techniques and found that synaptic transmission at NMJs was severely reduced in the former and virtually abolished in the latter. We concluded that both Ca2+-binding sites in the C2B domain are essential for synchronous synaptic transmission. We then examined the negative regulatory function of Syt I on high K+-induced vesicle fusion in these transformants and found in P[syt IB-D1,2N] that the frequency of high K+-induced quantal events was Ca2+-dependently depressed. In addition, by substituting Sr2+ for Ca2+ we found that Sr2+ partially substitutes for Ca2+ in synchronous release but does not support the negative regulatory function of Syt I. Thus the negative regulatory function is unique to Ca2+.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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There are five aspartate residues forming two Ca2+-binding sites in the C2B domain of Syt I (Fernandez et al. 2001). The following transformants that have amino acid substitutions in those residues were used in this study (Mackler et al. 2002). In one transformant the third and fourth aspartate residues were mutated to asparagines (D416,418N), and the mutant transgene was expressed in the syt I–null background (syt IAD4; DiAntonio and Schwarz 1994). This line is referred to as P[syt IB-D3,4N]. In the second transformant the first and second aspartate residues were mutated to asparagines (D356,362N) and expressed in the syt I–null background. This line is referred to as P[syt IB-D1,2N]. As a control, wild-type syt I was expressed in the syt I–null background (referred to as P[syt IWT]). Genotypes of the transgenic lines used were: P[syt Iwt] = yw; syt IAD4/syt IAD4P[elav Gal4]; P[UAS syt Iwt]/+, P[syt IB-D3,4N] = yw; syt IAD4/syt IAD4P[elav Gal4]; P[UAS syt IB-D3,4N]/+, and P[syt IB-D1,2N] = yw; syt IAD4/syt IAD4P[elav Gal4]; P [UAS syt I B-D1,2N]/+.
Solutions
The ionic composition of Ca2+-free saline used for dissection of embryos was (in mM): NaCl, 140; KCl, 2; MgCl2, 6; and HEPES-NaOH, 5 (pH 7.1). For nerve stimulation to evoke synaptic currents and for experiments with Ca2+ ionophores, HL3 solution was used and the Ca2+ concentration was changed by substituting the same amount of Mg2+. The ionic composition of HL3 solution was as follows (in mM): NaCl, 70; KCl, 5; CaCl2, 1.5; MgCl2, 20; NaHCO3, 10; Trehalose, 5; sucrose, 115; and HEPES-NaOH, 5 (pH, 7.1) (Stewart et al. 1994). The ionic composition of high K+ solution was (in mM): NaCl, 80; KCl, 62; MgCl2, 6; and HEPES-NaOH, 5 (pH, 7.1). To study the effect of Ca2+, CaCl2 (0.05–0.20 mM) was added by replacing the same amount of MgCl2. In high K+ solutions the Mg2+ concentration was kept relatively high compared with changes in [Ca2+] so that the effect on surface negative charges of the presynaptic membrane was minimal (Hagiwara and Takahashi 1967). The Ca2+ concentration in the nominally zero Ca2+ high K+ solution was about 0.6 µM, which should not affect our conclusions. The internal solution for the patch pipette had the following ionic composition (in mM): CsCl, 158; EGTA, 5; HEPES-NaOH, 10; and ATP, 2 (pH 7.1).
Preparations and recording conditions
Embryos (19–21 h after fertilization) of transformants and controls were used in this study. Dissecting procedures were the same as described previously (Kidokoro and Nishikawa 1994; Nishikawa and Kidokoro 1995) and carried out in Ca2+-free saline. After treating the dissected preparation with collagenase (1 mg/ml) for 30 s to 2 min, synaptic currents were recorded with patch-clamp techniques in the whole cell configuration from abdominal longitudinal muscle #6. The series resistance of the recording electrode, which varied between 5 and 30 M
, was compensated at an 80% level. The membrane potential was held at –60 mV. The internal solution contained Cs+ and the junction potential of electrodes filled with the Cs+ internal solution was –5 mV in normal saline (HL3 solution). Thus the true holding potential was –65 mV.
Nerve stimulation and calculation of the quantal content
For nerve stimulation, the tip of a microelectrode, which has a resistance of 10 to 20 M after being filled with 4 M K-acetate, was placed in the ventral nerve cord near the exit of the segmental nerve, and rectangular pulses of 1-ms duration and about 2-µA intensity were delivered at 0.3 Hz. In the case of syt I mutants, each stimulus did not necessarily produce a synaptic current, which made it difficult to judge whether the stimulation was effective. However, even in those cases, tetanic stimulation (10 Hz for 2 s) invariably increased asynchronous release, thus indicating its effectiveness. Then the stimulus frequency was switched to 0.3 Hz to collect data. To determine the quantal content, we used the failure method assuming the Poisson process for synaptic transmission (Katz 1969), that is, the quantal content: m = –ln (n0/N), where ln is the natural logarithm, n0 is the number of failures, and N is the total number of stimuli. We adopted this method because in developing synapses the amplitudes of minis were not normally distributed and varied widely (Zhang et al. 1999). Thus it is not certain whether the mean amplitude of minis is equal to the quantal size.
Hypertonicity and Ca2+ ionophore responses
A hypertonic solution was prepared by adding 420 mM sucrose to the Ca2+-free external solution. A Ca2+ ionophore, A23187 [GenBank] (20 µM), was dissolved in HL3 solution containing 0.5 mM Ca2+. These solutions were applied to the NMJ by the puff method with a gas pressure of 0.5 kg/cm2 for 11 s. The puff pipette had a tip diameter of 3–5 µm and the tip was placed within about 20 µm of the junctional area. The quantal synaptic events were counted individually every 0.5 s. The total number of events during each response was counted during a period of 30 s starting at the onset of puff pulse. For application of A23187 [GenBank] the bath solution was the Ca2+-free HL3 solution containing 20 µM A23187 [GenBank] .
All experiments were carried out at room temperature (18–27°C).
CHEMICALS. Tetrodotoxin (TTX) and collagenase were purchased from Sigma (St. Louis, MO). A23187 [GenBank] was obtained from Alomone Labs (Jerusalem, Israel). A23187 [GenBank] was dissolved in DMSO at 5 mM and stock solutions were stored at –20°C.
STATISTICAL ANALYSES. For comparison among multiple groups, ANOVA was used with the Tukey test. For comparison of two groups, Student’s t-test was used.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). This transformant is referred to as P[syt IB-D1,2N]. In the other transformant, two aspartates in position 416 (D3) and 418 (D4) were mutated to asparagines and expressed in the syt I–null background, which is referred to as P[syt IB-D3,4N]. As a control, we used a strain in which wild-type syt I is expressed in the syt I–null background, which is referred to as P[syt IWT] (Mackler et al. 2002). Nerve-evoked synchronous synaptic transmission at the NMJ in syt I transformant embryos
The nerve was stimulated at 0.3 Hz with a microelectrode, the tip of which was placed in the CNS at the corresponding segment and nerve-evoked synaptic currents were recorded as described previously (Deitcher et al. 1998). In control embryos, P[syt IWT], bathed in HL3 containing 1 mM Ca2+, the majority of nerve stimulations produced synaptic currents (Fig. 1A2). The synaptic events occurred predominantly between 2 and 10 ms after the onset of the stimulation pulse as shown in the event frequency histogram in Fig. 1A1 (99 events in the first bin, between 2 and 10 ms, responding to 102 stimuli). Some cells also exhibited delayed synaptic events (three of eight cells examined at 1 mM Ca2+). The average of entries in eight bins after stimulation (excluding the first bin) was 0.88 ± 1.13 (mean ± SD; data in the text are expressed in this format hereafter, unless otherwise stated.). Some of the spontaneous and delayed quantal events may occur during the first bin. To correct for a contribution of these events in the first bin, we subtracted the average number of entry of the following eight bins (0.88/bin) from the entry number in the first bin (99) to estimate the number of events synchronous to nerve stimulation (98.1). (This correction procedure is based on the assumption that the mechanism underlying the delayed release is also operating during the period covered by the first bin; Goda and Stevens 1994.) The quantal content was calculated by the failure method assuming Poisson statistics (Katz 1969). Accordingly, the quantal content of the P[syt IWT] cell shown in Fig. 1A is 1.75 (=–ln[(102 – 98.1)/102]). The mean quantal content of P[syt IWT] embryos at 1 mM Ca2+ was 1.65 ± 0.47 (mean ± SD, n = 8). The quantal content in this transgenic control line was smaller than that in the control line where wild-type syt I was expressed from the native syt I gene used in a previous study under the same experimental condition (2.5 ± 0.6; Okamoto et al. 2005). This finding is in accord with the report by Mackler and Reist (2001) that a significantly smaller mean evoked synaptic potential (67% of the native wild-type control) was observed in third instar P[syt IWT] larvae.
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In contrast, P[syt IB-D1,2N] embryos exhibited neither synchronous synaptic transmission nor delayed asynchronous release when stimulated at 0.3 Hz in 5 mM Ca2+ (n = 6). Figure 1, C1 and C2 shows this lack of response observed in one cell after delivery of 94 stimuli. Furthermore, these negative results were confirmed in eight cells in 1.5 mM external Ca2+ concentration ([Ca2+]e) (data not shown). This total lack of synaptic transmission in P[syt IB-D1,2N] is in contrast to rare but significant synchronous synaptic transmission in syt Inull embryos (Broadie et al. 1994; Kidokoro 2003; Okamoto et al. 2005). Thus the presence of mutated Syt I in P[syt IB-D1,2N] suppresses the function of another Ca2+ sensor (a second, non-Syt I, Ca2+ sensor) for synchronous synaptic transmission that operates in the absence of Syt I.
The mutated Syt I in P[syt IB-D1,2N] also depresses the function of wild-type Syt I for synchronous synaptic transmission because in heterozygous third instar larvae expressing both syt IWT and P[syt IB-D1,2N], nerve-evoked synchronous synaptic potentials were reduced to <10% of control, whereas in larvae expressing wild-type syt I and P[syt IB-D3,4N] the mean amplitude of synaptic potentials was reduced to roughly 50% of the control (Mackler et al. 2002).
However, in one single cell (out of five examined) when the nerve was stimulated at 10 Hz in 5 mM Ca2+, a very few evoked quantal synaptic events with appropriate timing occurred (Fig. 1, D1 and D2, marked with asterisks). This finding indicates that synchronous synaptic transmission in P[syt IB-D1,2N] can be evoked, although extremely rarely. This finding suggests that Ca2+ binds to the mutated Syt I in P[syt IB-D1,2N] and facilitates vesicle fusion.
Responses to a Ca2+ ionophore in P[syt IB-D1,2N] embryos
So far we found that in P[syt IB-D1,2N] synchronous transmitter release to nerve stimulation was virtually abolished and attributed this defect to a disability of mutated Syt I to sense Ca2+. However, Syt I interacts with presynaptic Ca2+ channels by the synprint motif of the channel (Catterall 1999). It is then possible that the observed defect in synaptic transmission arises from a lack of Ca2+ influx during depolarization in this transformant. Although this motif in the Ca2+ channel is missing in Drosophila (Littleton and Ganetzky 2000), it is still possible that another motif substitutes for it in this animal.
To test this possibility we bypassed voltage-gated Ca2+ channels with a Ca2+ ionophore and directly measured the Ca2+ sensitivity of the vesicle fusion machinery in P[syt IB-D1,2N] embryos. As shown in Fig. 2, the response to puff-applied ionophore (20 µM A23187
[GenBank]
with 0.5 mM Ca2+) in P[syt IB-D1,2N] was reduced to roughly 16% of the control in the peak frequency. Thus the Ca2+ sensitivity of the Ca2+ sensor for vesicle fusion was dramatically reduced in this transformant, but the elevation of Ca2+ level was detected, although poorly. Because similar residual responses, 9–14% of the control, to Ca2+ ionophores were observed even in syt IAD4, a syt I–null allele (Okamoto et al. 2005), the increased Ca2+ level may be detected in P[syt IB-D1,2N] by either the mutated Syt I or the second Ca2+ sensor.
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Next we examined the external Ca2+ dependency of synchronous synaptic currents in P[syt IB-D3,4N] embryos. The quantal content increased with external Ca2+ between 1 and 5 mM, and a straight line was fitted to these points on a double-logarithmic plot using the least-squares method (Fig. 3). At 1 mM [Ca2+]e the quantal content in P[syt IB-D3,4N] embryos was 1/100 of that in P[syt IWT]. The slope N was 1.86 ± 0.40 (mean ± SE of estimate, n = 32), which is significantly smaller than that in the control (2.88 ± 0.43, n = 34, P < 0.05). The value for N in P[syt IWT] is not different from that in control embryos with wild-type syt I (3.01 ± 0.36; Okamoto et al. 2005). Thus the P[syt IB-D3,4N] mutation reduced the value of N, suggesting that the Ca2+-binding sites in the C2B domain sense Ca2+ for synchronous synaptic transmission. It should be noted that the apparent cooperativity N for P[syt IB-D3,4N] is significantly larger than that for syt Inull (0.95 ± 0.36, P < 0.05; Okamoto et al. 2005), indicating that the mutated Syt I in P[syt IB-D3,4N] is sensing Ca2+ for synchronized synaptic transmission. [The data presented in this study were obtained under identical experimental conditions during the period mostly overlapping as those described in Okamoto et al. (2005). Thus a direct comparison of the data should be meaningful.]
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In the HL3 solution containing 1.5 mM Ca2+ and 3 µM TTX, spontaneous synaptic currents (miniature synaptic currents, or minis) occurred infrequently. The mean frequencies were 1.2 ± 1.0/min (n = 9) and 2.0 ± 2.0/min (n = 10) in P[syt IB-D1,2N] and P[syt IB-D3,4N], respectively. These values are not significantly different from those in P[syt IWT], 2.6 ± 2.9/min (n = 15; P > 0.05). This finding indicates that the negative regulatory function of Syt I is operating in these transformants. In contrast, Mackler et al. (2002) reported that the mini frequency in P[syt IB-D3,4N] third instar larvae was twofold higher than that in P[syt IWT]. This might be the result of developmental modification of NMJs during the period from embryos to third instar larvae in this transformant.
[Ca2+]e dependency of the frequency of high K+-induced quantal synaptic events
In high K+ solutions, the presynaptic terminal membrane is continuously depolarized and voltage-gated Ca2+ channels open asynchronously, unlike the synchronized opening induced by nerve stimulation. In this situation the elevated cytosolic Ca2+ in the terminal is most likely to be detected by a high-affinity, second Ca2+ sensor that has been postulated to explain delayed asynchronous release after nerve stimulation (Geppert et al. 1994; Goda and Stevens 1994). We assume, for the sake of simplicity, that synchronous as well as asynchronous release observed in syt Inull mutants is mediated by this second Ca2+ sensor (Okamoto et al. 2005). Nevertheless, Syt I is also involved in regulation of these quantal synaptic events because mutations in syt I profoundly affect the frequency of high K+-induced synaptic events (Okamoto et al. 2005; this study).
Synaptic events were induced in high K+ solutions (62 mM K+ and 3 µM TTX) containing Ca2+ between 0 and 0.20 mM in the control, P[syt IWT], and the two syt I transformants. In P[syt IWT], the frequency of high K+-induced quantal events was low in 0 mM [Ca2+]e but increased progressively 
0.15 mM and abruptly at 0.20 mM (Fig. 4A, diamonds). In P[syt IB-D3,4N] embryos a similar dependency of the quantal event frequency was observed as [Ca2+]e was increased (Fig. 4A, triangles). In contrast, in P[syt IB-D1,2N] embryos, the frequency was not different from that in the control at 0 and 0.05 mMCa2+ but significantly lower at 0.10 and 0.15 mM than those in the control (P < 0.05) (Fig. 4A, circles). Furthermore, the frequency in P[syt IB-D1,2N] at 0.15 mM was significantly lower than that at 0.05 mM [Ca2+]e (Fig. 4B), but increased abruptly at 0.20 mM (Fig. 4A, circle). These results indicate that in the Ca2+ range around 0.15 mM the mutated Syt I in P[syt IB-D1,2N] is inhibiting high K+-induced vesicle fusion in a Ca2+-dependent manner.
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). This finding suggests that mutated Syt I in P[syt IB-D1,2N] binds Ca2+ and facilitates vesicle fusion. Hypertonicity responses in P[syt IB-D1,2N] and P[syt IB-D3,4N] embryos
The lower frequencies of high K+-induced quantal events in P[syt IB-D1,2N] compared with those in P[syt IWT] could be a result of fewer release-ready synaptic vesicles at the NMJ. To assess the population of release-ready vesicles, we next examined the hypertonicity response. The quantal event frequency increases with puff application of hypertonic solutions at embryonic NMJs (Suzuki et al. 2002a). Because Ca2+ is not required for this response (Rosenmund and Stevens 1996), the population of release-ready vesicles can be assessed regardless of Ca2+ sensitivity of the release mechanism.
The hypertonicity response evoked with 420 mM sucrose added to Ca2+-free HL3 in P[syt IWT] (Fig. 5A; total number of events, 291 ± 40, Fig. 5D, left column; peak frequency, 66 ± 20/s, n = 5, Fig. 5E, left column) was not different from that previously reported for wild-type embryos (Okamoto et al. 2005; Suzuki et al. 2002a,b). Furthermore, the response in P[syt IB-D3,4N] (Fig. 5B; total number of events, 286 ± 35; Fig. 5D, right column; peak frequency, 73 ± 21/s, n = 5, Fig. 5E, right column) was not different from that in P[syt IWT]. This finding is in accord with the findings in the third instars where neither the hypertonicity response (Mackler et al. 2002) nor the abundance of synaptic vesicles (Loewen et al. 2006) was disrupted in P[syt IB-D3,4N] larvae. However, the hypertonicity response in P[syt IB-D1,2N] embryos (Fig. 5C) was significantly smaller than that in P[syt IWT] (total number of events, 201 ± 57, Fig. 5D, middle column; peak frequency, 45 ± 10/s, n = 6, Fig. 5E, middle column). This smaller response (roughly 70% of controls), however, does not explain the severe defect in nerve-evoked synchronous synaptic transmission (Fig. 1) nor the lower frequency of high K+-induced quantal events in P[syt IB-D1,2N] at 0.1 and 0.15 mM [Ca2+] (Fig. 4).
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), suggesting that the mutated Syt I in P[syt IB-D1,2N] predominantly depresses the second Ca2+ sensor that operates in syt IAD4. Sr2+ decreases nerve-evoked synchronous synaptic transmission but enhances asynchronous delayed release and high K+-induced quantal synaptic events
First, we tested the effect of Sr2+ on nerve-evoked release in P[syt IWT] embryos. In HL3 solution containing 1.0 mM Sr2+, substituting for Ca2+, nerve stimulation evoked synchronous synaptic currents as well as delayed asynchronous events (Fig. 6, A1 and A2). As in HL3 solution containing Ca2+, the synaptic events occurred predominantly between 2 and 10 ms after the onset of the stimulation pulse as shown in the event frequency histogram in Fig. 6A1 (17 events in the first bin responding to 100 stimuli). The average of the entries in eight bins after stimulation, excluding the first bin, was 5.4 ± 0.7, which was significantly higher than that in five bins before stimulation 1.4 ± 0.5 (P < 0.01). We then subtracted the average number of entry of the following eight bins (5.4/bin) from the number of entries in the first bin (17) to estimate the number of events synchronous to nerve stimulation (11.6) as we did for nerve-evoked synaptic events in Ca2+. Thus the quantal content in this cell was 0.12. The average quantal content in six cells was 0.09 ± 0.05, which is significantly smaller than that in HL3 solution containing 1.0 mM Ca2+ (1.65 ± 0.47, n = 8). However, in the Sr2+ solution a few initial stimuli at 0.3 Hz often induced bursts of asynchronous release, which did not subside for many minutes. In those cells we could not measure synchronous release and those data were not used for further analysis. Even in cells in which bursting did not occur, the delayed asynchronous release was prominent (Fig. 6A1). The average entry in the eight bins after stimulation, excluding the first bin, was 0.054 ± 0.018 per stimulus, which was significantly higher than the average entry in the five bins preceding stimulus, 0.014 ± 0.011 per stimulus (P < 0.01). These properties of synaptic transmission in Sr2+ in P[syt IWT] embryos are similar to those reported in cultured mammalian synapses (Goda and Stevens 1994) and in mouse cerebellar slices (Xu-Friedman and Regehr 2000).
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). When Sr2+ substitutes for Ca2+ in high K+ solutions, the frequency of quantal events was significantly higher in P[syt IWT] than that in Ca2+. For example, at 0.1 mM Sr2+ the frequency of high K+-induced quantal events was 2,070 ± 420/min (n = 8); this value was much larger than that in 0.1 mM Ca2+ (132 ± 34/min, n = 15). The frequency of high K+-induced quantal events increased with the Sr2+ concentration (Fig. 6C). It was also higher in P[syt IB-D3,4N] and P[syt IB-D1,2N] embryos compared with corresponding values in Ca2+ and increased with the Sr2+ concentration (compare Fig. 4A for Ca2+ with Fig. 6C for Sr2+). The decline of quantal event frequency detected in P[syt IB-D1,2N] embryos (Fig. 4, A, broken line and B) was not observed in high K+ solutions containing Sr2+ between 0.02 and 0.15 mM. This observation suggests that Sr2+ does not support the negative regulatory function of Syt I.
If the negative regulation of spontaneous vesicle fusion by Syt I is operating in normal saline with Ca2+ at the resting state, we expect higher mini frequencies in P[syt IWT] embryos in Sr2+ than in Ca2+. Indeed the mini frequency was 7.3 ± 7.6/min (n = 10) in HL3 solution with 1.5 mM Sr2+ and 3 µM TTX, which is significantly higher than that in HL3 with 1.5 mM Ca2+ and 3 µM TTX (2.6 ± 2.9/min, n = 15).
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DISCUSSION |
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Each of those mutations is likely to affect both Ca1 and Ca2 binding sites. It is known that the D2N mutation strongly impairs Ca2+ binding at both binding sites (Fernandez et al. 2001), which is in accord with our finding that synaptic transmission was more severely depressed in P[syt IB-D1,2N] than in P[syt IB-D3,4N] (Fig. 1). Does Ca2+ still bind at C2B in these mutants? Because [Ca2+]e dependency of synchronous synaptic transmission in P[syt IB-D3,4N] (n = 1.86 ± 0.4) is significantly different from that in syt Inull (0.95 ± 0.36; Okamoto et al. 2005), it is likely that Ca2+ binds to the mutated Syt I in this mutant. Also in P[syt IB-D1,2N], synchronized transmitter release was observed in a single cell (Fig. 1D). In addition, the frequency of high K+-induced quantal events at 0.05 mM [Ca2+]e in P[syt IB-D1,2N] (53.8 ± 27.3 events/min) was significantly higher than that in syt Inull (11.7 ± 3.1 events/min; Okamoto et al. 2005). These findings suggest that Ca2+ binds to C2B in both transformants.
Nerve-evoked synchronous synaptic transmission in syt I transformants
The effects of these mutations on synaptic transmission were previously studied in third instar larvae (Mackler et al. 2002). In P[syt IB-D3,4N], nerve-evoked synaptic transmission at NMJs was dramatically depressed compared with that in the control, P[syt IWT]. Neither immunostaining of nerve terminals with an antibody against Syt I (Mackler et al. 2002) nor the distribution of synaptic vesicles in the presynaptic terminal revealed by electron-microscopic (EM) analysis (Loewen et al. 2006) was different from that in the control. These findings support the idea that the Ca2+-binding sites in C2B are sensing Ca2+ for synchronous synaptic transmission. The P[syt IB-D1,2N] mutation was lethal and not studied in the syt I–null background. Instead, synaptic transmission was examined in third instars expressing P[syt IB-D1,2N] in syt IWT heterozygotes. The amplitude of evoked synaptic potentials was strongly depressed to <10% of the control, whereas that in syt IWT heterozygotes expressing P[syt IB-D3,4N] was about 50%. These findings suggest that the mutated Syt I in P[syt IB-D1,2N] predominantly depresses the function of wild-type Syt I. In P[syt IB-D1,2N] we found that synchronous synaptic transmission was more severely depressed than that in syt Inull, syt IAD4, whereas the hypertonicity response in the former is much greater than that in the latter (Okamoto et al. 2005). Thus this indicates that the mutated Syt I in P[syt IB-D1,2N] also suppresses the function of the second Ca2+ sensor.
Nishiki and Augustine (2004b) studied the mutations in which each of the aspartates (D) in C2B was individually mutated to asparagines (N) in cultured mouse hippocampal neurons. When mutated Syt I (D2N or D3N) was expressed in syt I knockout synapses synchronous synaptic currents were virtually abolished. However, these mutations still suppressed asynchronous release seen in syt I knockout synapses. Other substitutions (D1, D4, D5) had relatively minor effects on synaptic transmission. Our results in P[syt IB-D1,2N] and P[syt IB-D3,4N] are consistent with theirs, indicating that D2 and D3 are critical for synchronous release.
If Ca1 and Ca2 in C2B are only Ca2+-binding sites in Syt I involved in synchronous synaptic transmission, it is difficult to explain the apparent cooperativity (2.88) in syt IWT (Fig. 3), which indicates more than three binding sites are involved for Ca2+ detection. This conundrum can be resolved by invoking oligomerization of Syt I.
Ca2+-dependent oligomerization of Syt I
Ca2+-dependent oligomerization occurs at the C2B domain and may be involved in synchronous vesicle fusion (Chapman et al. 1996). A Drosophila syt I mutant, syt IAD3, has one amino acid substitution in C2B and synchronous synaptic transmission in syt IAD3 embryos is severely impaired (Okamoto et al. 2005; Yoshihara and Littleton 2002). A homologous mutation in mouse Syt II, which is virtually identical to Syt I (Fernandez et al. 2001), blocks Ca2+-dependent self-oligomerization (Fukuda et al. 2000), and the AD3 mutation impairs Ca2+-dependent oligomerization in Drosophila (Littleton et al. 2001). Thus the depressed synaptic transmission in syt IAD3 may be explained, at least partly, by a defect in Ca2+-dependent oligomerization. Supporting this view, the apparent cooperativity was smaller (N = 1.54) in syt IAD3 compared with 3.01 in a control (Okamoto et al. 2005), which suggests that the binding sites are decreased in this mutant in the absence of oligomerization. However, it is possible that the decreased N in syt IAD3 is the result of other changes induced by this mutation.
Because Sr2+ does not induce oligomerization of Syt I (Chapman et al. 1996), we expect synchronous synaptic transmission to be depressed and N to be decreased in Sr2+. We found that synchronous synaptic transmission in Sr2+ was 5.5% of that in Ca2+, and N in Sr2+ was 2.01, in contrast to 2.88 in Ca2+. Similarly in mouse cerebellar slices synchronous synaptic transmission in Sr2+ is reduced to 7.9% of that in Ca2+ and N in Sr2+ was 1.7, in contrast to 3.2 in Ca2+ (Xu-Friedman and Regehr 2000). These data support an involvement of Syt I oligomerization in synchronous transmission. However, a recent crystallographic study showed that the C2B domain binds only one Sr2+ (Cheng et al. 2004). The N = 1.7 in mice or N = 2.01 in Drosophila in Sr2+ could not be explained with only one binding site without self-oligomerization of Syt I. Alternatively, multiple Syt I molecules without oligomerization might be involved in synchronous release.
In spite of the above arguments that support an involvement of self-oligomerization of Syt I for synchronous release, other reports indicate otherwise. Mackler and Reist (2001) substituted three lysines to glutamines in the C2B domain of Syt I and expressed the mutated Syt I in the syt I–null background. This substitution was expected to block Ca2+-dependent oligomerization (Chapman et al. 1998). Nerve-evoked synaptic potentials were depressed by only roughly 36% in the third instar mutant larvae compared with a control. However, this relatively minor effect of the mutation could be a result of developmental compensation for defective synaptic transmission. Borden et al. (2005) recently examined synaptic transmission in cultured mouse neurons that lack endogenous syt I but overexpress exogenous mutant syt I(Y311N), equivalent to Drosophila syt IAD3, or other mutant syt Is that are impaired in self-oligomerization. Synaptic transmission was depressed in these synapses, which they explained by a decrease in the Ca2+-binding affinity. They thus concluded that Syt I self-oligomerization plays no role in synaptic transmission. However, to estimate apparent Ca2+ dissociation constants they assumed that the maximum response and the cooperativity constant N are the same in the mutants as in the control. These assumptions may not be valid because it was previously shown in syt IAD3 that N is significantly smaller (Okamoto et al. 2005).
The frequency of miniature synaptic currents (minis)
The mini frequency is often higher in syt I–deficient synapses (DiAntonio and Schwarz 1994; Littleton et al. 1993; Mackler et al. 2002; Pang et al. 2006). In syt IAD4 embryos, the frequency was similar to that in the control, but because the number of vesicles adjacent to the presynaptic membrane is considerably reduced (Reist et al. 1998) and the hypertonicity response was roughly 10% of the controls, the release probability of release-ready vesicles must be higher in this mutant compared with that in controls (Okamoto et al. 2005). These findings suggest the negative regulatory function of Syt I on spontaneous release. In this study, we found that the mini frequencies in P[syt IB-D1,2N] and P[syt IB-D3,4N] were similar to those in control, and the hypertonicity responses were not greatly different in these mutants. Thus the negative regulatory function of Syt I remains operating in these transformants in spite of severe defects in synchronous release.
High K+-induced quantal release
The frequency of high K+-induced synaptic events is lower in syt IAD3, compared with that in syt IAD4, a syt I–null allele (Okamoto et al. 2005), suggesting that Syt I(AD3) inhibits high K+-induced vesicle fusion. This inhibitory effect was revealed only in syt IAD3 and not in wild-type, probably because the counteracting enhancing effect of Syt I on vesicle fusion is impaired in this mutant. In P[syt IB-D1,2N], in which the enhancing effect is further depressed, the frequency of high K+-induced quantal events was significantly lower at 0.15 mM [Ca2+]e than at 0.05 mM, suggesting that mutated Syt I in P[syt IB-D1,2N] Ca2+-dependently inhibits vesicle fusion. The frequency increased again at 0.2 mM Ca2+ (Fig. 4A), which is probably attributable to facilitatory effects of the mutated Syt I and the second Ca2+ sensor. We did not observe this inhibitory effect in P[syt IB-D3,4N], which probably arises from a stronger remaining facilitatory function of Syt I in this transformant. A Ca2+-dependent decrease of frequency of high K+-induced quantal synaptic potentials in wild-type animals was previously reported at NMJs (Cooke and Quastel 1973; Ohta and Kuba 1980). Thus the inhibitory effect that we observed in P[syt IB-D1,2N] is not specific to this mutation. Although we did not observe a similar inhibitory effect in control Drosophila embryos, an appropriate combination of K+ and Ca2+ concentrations might reveal such an effect.
The effects of syt I mutations on nerve-induced synchronous and asynchronous release and on high K+-induced quantal release are mediated by the facilitatory functions of Syt I and the second Ca2+ sensor as well as by the negative regulatory function of Syt I. Nishiki and Augustine (2004b) observed that D2N and D3N mutations strongly decreased nerve-evoked synchronous release but did not enhance asynchronous release as in syt I knockout synapses. In this study we found a clear Ca2+-dependent decrease of the frequency of high K+-induced quantal events in P[syt IB-D1,2N], whereas the Ca2+ sensing function is greatly reduced. These findings support the idea of dual roles of Syt I (Nishiki and Augustine 2004).
Sr2+ does not support the negative regulatory function of Syt I
Nerve-induced synchronous synaptic transmission was strongly depressed but asynchronous release was enhanced in normal Ca2+ saline at the NMJ of a Drosophila syt I–null mutant (syt IAD4; Okamoto et al. 2005; Yoshihara and Littleton 2002) and at synapses formed in culture among neurons derived from syt I knockout mice (Geppert et al.,1994; Nishiki and Augustine 2002a,b). The delayed asynchronous release is postulated to be mediated by the high-affinity second Ca2+ sensor. It is then possible that wild-type Syt I is negatively regulating the second Ca2+ sensor and reducing the delayed release. When Sr2+ substitutes for Ca2+ synchronous release is reduced, whereas asynchronous release is enhanced. The enhanced asynchronous release in Sr2+ is interpreted to be the result of a more efficient activation of the second Ca2+ sensor by Sr2+ than by Ca2+ (Goda and Stevens 1996). Alternatively, in Sr2+ solutions Syt I might not effectively regulate asynchronous vesicle fusion. Because we clearly observed a negative regulatory effect of Syt I in P[syt IB-D1,2N] in high K+-induced quantal events, we next tested this idea in a solution where Sr2+ replaced Ca2+.
We found in control embryos that synchronous synaptic currents in Sr2+ were depressed but asynchronous release after nerve stimulation was enhanced. In Sr2+ the high K+-induced synaptic release was more frequent in all strains than in Ca2+ and, unlike in Ca2+, was not inhibited concentration dependently in P[syt IB-D1,2N]. These findings support the idea that Sr2+ does not support the negative regulatory function of Syt I, resulting in the enhanced asynchronous release after nerve stimulation as well as higher frequencies of high K+-induced quantal events and minis in Sr2+. In fact, in syt IAD4 embryos the high K+-induced quantal events are not more frequent in Sr2+ than in Ca2+, suggesting that Sr2+ is not efficient in activating the second Ca2+ sensor (Tamura and Kidokoro, unpublished observation). Thus it appears that the negative regulatory function of Syt I is supported by Ca2+ but not by Sr2+.
In addition to roles in vesicle fusion Syt I is also implicated for vesicle recycling (Poskanzer et al. 2003; Zhang et al. 1994). Clearly Syt I has multiple functions. Further detailed analyses are required for their elucidation.
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Address for reprint requests and other correspondence: Y. Kidokoro, Department of Physiology, David Geffen School of Medicine at UCLA, 10833 Le Conte Avenue, Los Angeles, CA 90095-1751 (E-mail; ykidokoro{at}mednet.ucla.edu)
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