Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Medical Faculty, 1000 Ljubljana; and Celica Biomedical Science Center, 1000 Ljubljana, Slovenia
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ABSTRACT |
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Synaptotagmin I (Syt I), a low-affinity Ca2+-binding protein, is thought to serve as the Ca2+ sensor in the release of neurotransmitter. However, functional studies on the calyx of Held synapse revealed that the rapid release of neurotransmitter requires only approximately micromolar [Ca2+], suggesting that Syt I may play a more complex role in determining the high-affinity Ca2+ dependence of exocytosis. Here we tested this hypothesis by studying pituitary cells, which possess high- and low-affinity Ca2+-dependent exocytic pathways and express Syt I. Using patch-clamp capacitance measurements to monitor secretion and the acute antisense deletion of Syt I from differentiated cells, we have shown that the rapid and the most Ca2+-sensitive pathway of exocytosis in rat melanotrophs requires Syt I. Furthermore, stimulation of the Ca2+-dependent exocytosis by cytosol dialysis with solutions containing 1 µM [Ca2+] was completely abolished in the absence of Syt I. Similar results were obtained by the preinjection of antibodies against the CAPS (Ca2+-dependent activator protein for secretion) protein. These results indicate that synaptotagmin I and CAPS proteins increase the probability of vesicle fusion at low cytosolic [Ca2+].
rat melanotrophs; exocytic module; membrane capacitance; calcium sensor
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INTRODUCTION |
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SYNAPTOTAGMINS constitute a family of vesicle membrane proteins (35) that play a role in various aspects of vesicular trafficking, such as endocytosis (19, 58), but most notably in regulated exocytosis. Genetic studies in Drosophila, Caenorhabditis elegans, and mice have implicated synaptotagmin (Syt) I as an essential component of rapid Ca2+-dependent neurotransmitter release (11, 14, 15, 24, 25, 34). Biochemical properties of Syt I, a low-affinity Ca2+-binding protein (10), are consistent with the view that Syt I may serve as a major Ca2+ sensor for exocytosis (5, 10, 14, 15, 23, 24). However, physiological studies of the Ca2+ requirement in transmitter release from the calyx of Held revealed that the apparent Ca2+ affinity of the exocytic apparatus is much higher than that of Syt I (4, 42). This may indicate that Syt I is not the only Ca2+ sensor but may play a part in a more complex mechanism of high-affinity Ca2+ sensing, for example, by interacting with other molecular partners (14, 23, 45). If this is the case, then in the absence of Syt I it should be possible to record a secretory response devoid of the high-affinity Ca2+-dependent exocytosis. Moreover, one needs to show that the presence of Syt I is required to elicit secretory activity even at very low [Ca2+], at which binding to Syt I in vitro appears to be negligible (10).
An ideal approach to study such a hypothesis would be to use a secretory cell type in which multiple Ca2+-dependent pathways of exocytosis are present, such as the pituitary melanotrophs, which secrete pro-opiomelanocortin-derived peptides via dense-core vesicle exocytosis (27). Previous work has shown that melanotrophs possess a high- and a low-affinity Ca2+-dependent exocytic pathway (38). Here we used an acute strategy to block the expression of Syt I by transfection of an antisense construct into differentiated neuroendocrine cells to study the impact of selective deletion of Syt I on the Ca2+-triggered vesicle exocytosis. Secretory activity was monitored by membrane capacitance (Cm) measurements (31) combined with flash photolysis to deliver rapid and spatially homogeneous steps in cytosolic [Ca2+] ([Ca2+]i), (32), which elicit multiple kinetic components in secretory activity (17, 20, 32, 33, 38, 46, 49, 50, 57). We report that the rapid but not the slow component of exocytosis (38) is blocked in the absence of Syt I, indicating a role of Syt I in increasing the probability of vesicle fusion at low [Ca2+]i. Furthermore, secretory activity stimulated by dialysis of pipette solutions containing 1 µM [Ca2+] was abolished in Syt I-depleted cells and by preinjection of the CAPS (Ca2+-dependent activator protein for secretion) antibody. These data show directly that Syt I and CAPS increase the probability of vesicle fusion at low [Ca2+]i in pituitary cells.
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MATERIALS AND METHODS |
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Isolation of mRNA, cell culture, tissue preparation, and RT-PCR.
To isolate mRNAs of Syt isoforms, cell cultures or tissue of Wistar
rats (200-300 g, male) were used. Tissue mRNA from hippocampus, cerebellum (one hemisphere), and spinal cord (thoracic region) were
prepared from homogenized samples (~0.1 g). Cell cultures from the
pars intermedia and anterior lobe were prepared as described (39). Cells (~107) were washed in Earl's
balanced salt solution and sedimented before RNA extraction. The
QuickPrep micro mRNA purification kit (Pharmacia Biotech) was used to
isolate polyadenylated RNA. For the synthesis of first-strand cDNA,
6-12 ng of poly(A) mRNA were incubated for 10 min at 25°C and
then for 60 min at 42°C in the presence of 5 µM random hexamers
(Pharmacia Biotech), 500 µM mixed deoxynucleotide triphosphates
(Pharmacia Biotech), 3 U/µl RNase inhibitor (RNasin; Promega), 10 mM
dithiothreitol (Sigma), 10 U/µl reverse transcriptase (SuperScript
II; GIBCO), and 1× First Strand buffer (GIBCO) in a reaction volume of
10 µl. PCR was carried out in Eppendorf Mastercycler Gradient
(Eppendorf, Hamburg, Germany) thermal cyclers. The 100-µl
amplification mixture consisted of 1 µl of the sample from the RT, 2 mM MgCl2, 1× GeneAmp PCR buffer II and 2.5 U AmpliTaq Gold
polymerase (Perkin Elmer), 200 µM mixed deoxynucleotides (Pharmacia
Biotech), and 2 µM primers (Table 1). A
9-min pre-PCR heat step at 95°C was performed. PCR cycling times and
temperatures were as follows: 1 min at 94°C, 1 min at 57.5°C, and 1 min at 72°C; 42-48 cycles were used. The final elongation step
was at 72°C for 5 min. The RT-PCR products were electrophoresed on
ethidium bromide-stained (1.4%) agarose gels.
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Introduction of oligonucleotides and plasmid DNA into mammalian cells. An antisense Syt I oligonucleotide (5'-TCA-GGA-TGA-CTG-GCA-CTC-ACC-3') and a sense Syt I oligonucleotide (5'-GGT-GAG-TGC-CAG-TCA-TCC-TGA-3') were introduced into 1- to 2-day-old cells with the plasmid DNA encoding EGFP (pEGFP-C1; Clontech) by cationic lipids (Lipofectamine Plus reagent; Life Technologies), according to the manufacturer's instructions. We used 0.5 µg of EGFP plasmid and 0.5 µg of oligonucleotides dissolved in 100 µl of culture medium without serum (transfection medium). To this solution, we added 6 µl of Plus reagent and incubated for 15 min at room temperature. Lipofectamine reagent (4 µl) was then added to the transfection medium and incubated for another 15 min. This mixture was then added to the cells for 3-5 h at 37°C, after which the procedure was stopped by the addition of 30 µl of serum. After 24 h, cells were observed under the microscope and patch-clamp examined or prepared for immunocytochemistry. These conditions yielded 2-6% of cells (around 60 cells per coverslip) fluorescing in green. Immunocytochemistry using an anti-Syt I antibody was used to verify that the transfection of cells with antisense oligonucleotides affected the level of Syt I protein levels in a cell. When the antisense oligonucleotide was used together with the EGFP plasmid, the level of anti-Syt I signal was significantly reduced in all green-fluorescing cells (see Fig. 2), indicating the inhibition of expression of Syt I in these cells by the antisense procedure.
Immunocytochemistry and confocal microscopy. Melanotrophs were washed with PBS and then fixed for 15 min in 4% paraformaldehyde in PBS. For the following 10 min, cells were kept in fixative containing 0.1% Triton X-100 and then washed four times with PBS. Nonspecific staining was reduced by incubation in 3% bovine serum albumin (BSA) and 10% normal goat serum in PBS. Cells were then incubated with anti-Syt I primary antibodies for 2 h at 37°C (21, 46), diluted 1:2,000 in PBS containing 3% BSA. Cells were then washed and incubated in PBS containing Cy3-labeled anti-mouse secondary antibodies (Molecular Probes, Leiden, The Netherlands) and 3% BSA for 45 min, washed, and mounted in Light Antifade (Molecular Probes). Stained cells were observed by the confocal microscope (Zeiss LSM 510; oil-immersion lens ×63, NA 1.4). EGFP was excited with the argon laser (488 nm) and Cy3 with the helium-neon laser (543 nm). The EGFP emission signal was filtered at 505-530 nm, and the Cy3 emission signal was long-pass filtered at 560 nm.
Electrophysiology, flash photolysis, and [Ca
2+]i measurements.
A SWAM IIB patch-clamp/lock-in amplifier (Celica, Ljubljana, Slovenia)
was used for Cm measurements (31,
59). Uncompensated Cm measurements were
used to monitor slow changes in Cm, as described previously (39), by applying a sine wave voltage [1 mV
(RMS), 1.6 kHz] to the holding potential. Direct current (DC), holding potential, and real and imaginary admittance signals (low pass 10 Hz,
3 dB, 6-pole Bessel) were digitized by a CED 1381 analog-to-digital converter (Cambridge, UK) and analyzed with the CAP3 software (J. Dempster, Univ. of Strathclyde, Strathclyde, UK), which computed Cm, access (Ga), and
membrane conductance (Gm). The recording bath
solution consisted of (in mM) 131.8 NaCl, 1.8 CaCl2, 5 KCl, 2 MgCl2, 10 HEPES/NaOH, 10 D-glucose, 0.5 NaH2PO4 · 2H2O,
and 5 NaHCO3 (pH 7.2). The pipette solution consisted of
(in mM) 150 KCl, 2 MgCl2, 10 HEPES/KOH, 0.65 EGTA, 4.35 Ca2+-saturated EGTA ([Ca2+]i = 1 µM), 2 Na2ATP (pH 7.2).
[Ca2+]i was calculated by assuming an
apparent dissociation constant (Kd) for the
Ca-EGTA complex of 150 nM (16) and assuming that the
cytosol equilibrates with the pipette solution upon the establishment of the whole cell recording. Total EGTA concentration was 5 mM, which
exceeds the buffering capacity of melanotrophs (48). Cells were voltage clamped at a holding potential of
70 mV. The reversal potential of the whole cell current was
50 mV and did not change during recordings. Fast changes in Cm were
measured by using the compensated method [111 mV (RMS), 1.6 kHz]
(38). Upon establishment of the whole cell configuration,
Cm and Ga were
compensated by Cslow and
Ga controls. The phase angle setting was
determined by a 1-pF pulse and by monitoring the projection of the
pulse from the C (proportional to Cm) to the G
output of the lock-in amplifier. These two signals were stored
unfiltered (C-DAT4; Cygnus) for off-line analysis. Simultaneously, we
recorded filtered (300 Hz, 4-pole Bessel) C and G signals, the
fluorescence intensity from a C660 photon counter (Thorn EMI), and DC
(0-10 Hz, low pass). The PhoCal program (LSR, Cambridge, UK) was
used to acquire signals every 5 ms. For high temporal resolution
measurements of Cm, the records on DAT were
played back and a 10-s epoch of the signal enveloping each flash was
digitized at 50 kHz using the CDR program (J. Dempster). Signals were
digitally filtered (1 kHz, 2-way 150th-order FIR filter; MATLAB) and
resampled at 10 kHz. The pipette solution contained (in mM) 110 KCl, 10 TEA-Cl, 38 KOH/HEPES, 2 Na2ATP, 2 MgCl2, 4 K4-nitrophenyl-EGTA, 3.6 CaCl2, and 0.5 furaptra (pH 7.2). The bath contained (in mM) 131.8 NaCl, 1.8 CaCl2, 5 KCl, 2 MgCl2, 10 HEPES/NaOH, 10 D-glucose, 0.5 NaH2PO4 · 2H2O,
and 5 NaHCO3 (pH 7.2). All salts were obtained from Sigma.
Recordings were made at room temperature with pipette resistance
between 1 and 4 M
(in KCl-rich solution), giving
Ga of >80 nS. The pipette and bath solutions
were of similar osmolarity (within 5%) measured by freezing point
depression (Camlab, Cambridge, UK). We used nitrophenyl-EGTA (Molecular
Probes, Eugene, OR) to manipulate [Ca2+ ]i
(13). A UV flash from a xenon arc flash lamp
(38) was delivered to cells through a ×38 fluor
oil-immersion objective of a Nikon Diaphot microscope. The same optical
pathway as in flash photolysis was used to illuminate the fluorescent
[Ca2+ ]i indicator furaptra (Molecular
Probes) as described previously (7, 38).
Microinjection. Transjector 4657 with a micromanipulator (Eppendorf, Germany) was used. Pulses (8-12 hPa, 0.3-1 s) were applied with a compensation pressure (1 hPa). Microinjection solution consisted of (in mM) 150 K-gluconate, 2 MgCl2, 10 HEPES, 6 mg/ml RITC, and 2-20 mg/ml antibody. The anti-CAPS antibody was affinity purified, and the control antibodies (preimmune antibody) were purified by using protein A-Sepharose, to prepare immunoglobulin fractions (29, 55). Pipettes were prepared with a horizontal puller (P-87; Sutter Instruments, city, CA). Microinjected cells were identified with coinjection of rhodamine-labeled dextran (25 kDa; Sigma). Injection of dextran marker alone did not affect secretory responses per se. Botulinum neurotoxin A, B, or E (a gift from Dr. Das Gupta, Madison, WI) was microinjected into cells (20 ng/µl) together with the dextran marker. Before the patch-clamp experiments were continued, the cells were allowed to rest for 1-2 h at 37°C.
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RESULTS |
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Syt I, III, IV, V, and VII are expressed in rat melanotrophs.
Previous work indicated that Syt I transcript is present in the rat
pituitary (28), but it is not clear which of the other Syt
isoforms are expressed. We used RT-PCR to examine the presence of Syt
transcripts (I-VII) in the pituitary pars intermedia and anterior
lobe, hippocampus, cerebellum, and spinal cord. Poly(A) mRNA was
isolated from the tissues, and then cDNAs for Syt isoforms were
amplified by specific primer sets (Table 1). Figure
1 depicts the expression analyses of the
Syt transcripts from the pituitary pars intermedia. Contamination of
cDNA with genomic DNA was tested by performing parallel amplifications
on mRNA; contamination from extraneous sources was checked by replacing
cDNA templates with water (not shown). Two to three separately isolated
mRNA samples were analyzed for the presence of Syt isoforms, and the
fragments obtained by RT-PCR were verified by nucleotide sequencing.
Figure 1 shows that the transcripts for Syt I, III (a/b; see Table 1), IV, V (IX; see Ref. 44), and VII are present in the
pituitary pars intermedia and all other tissues tested (not shown). Syt II was present in cerebellum and spinal cord, and Syt VI was present in
cerebellum, spinal cord, and the anterior lobe of pituitary (not
shown). In conclusion, the Syt isoforms I, III, IV, V, and VII are
expressed in the pituitary pars intermedia.
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Antisense strategy to block the expression of Syt I. To block the expression of Syt I from differentiated melanotrophs, we introduced an antisense oligonucleotide complementary to the sequence at the 5'-end of Syt I. The transfected cells were labeled by cotransfection of the antisense or sense oligonucleotides with the expression plasmid for EGFP. The content of Syt I was checked by immunocytochemistry, using monoclonal antibodies against Syt I with a Cy3-labeled secondary antibodies.
When the sense probe and EGFP plasmid were introduced into cells, the successfully transfected cells were fluorescing green and were immunocytochemically positive for Syt I (Fig. 2A). The detected amounts of Syt I were comparable in transfected and untransfected cells. Transfected cells had therefore retained Syt I in the presence of EGFP. In contrast, when the cells were transfected with the antisense probe alongside the EGFP plasmid, the green-fluorescing cells contained greatly reduced amounts of Syt I, because the signal reporting the presence of Syt I was faint or negative (Fig. 2B). This finding demonstrates a significant reduction of the Syt I levels in the antisense-treated cells.
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Rapid exocytosis is diminished in the absence of Syt I.
With the use of Syt I null mutant mice, it was shown that in chromaffin
cells, Syt I expression is required for the rapid exocytosis, whereas
the slow exocytosis was not affected (54). If many
isoforms of a protein exist in a cell, the deletion of one member of
the family may be compensated during embryogenesis by other isoforms in
the knockout animals (8, 41). The family of synaptotagmins
consists of at least 13 members (43), therefore one has to
consider that the physiology of neuroendocrine cells in Syt I null
animals may be a result of a complex embryogenetic corrective response
induced by the loss of Syt I. To this end, we performed an acute block
of the expression of Syt I in differentiated cells. Figure
3 (middle traces) shows the
exocytic response of a control cell (sense probe) and of a cell without
Syt I (antisense probe) to a flash-induced rise in
[Ca2+]i to ~50 µM (top
traces). In control cells, the increase in
Cm over the first 800 ms consisted of two
components, a rapid and a slow sustained rise in
Cm, whereas with the antisense probe the rise in
Cm appeared to be devoid of the rapid component
(Fig. 3, bottom traces). The two kinetic components of
Cm increase in control cells represent two
populations of vesicles that enter distinct exocytic pathways
(36, 38). Figure 4 shows the
results of measurements of the amplitude of the
Cm increase at two time points. The amplitude A
(rapid exocytosis) was measured as the positive peak value in
Cm (relative to the Cm
value preceding the flash) during the first 100 ms after the flash,
whereas the amplitude B of the slow exocytosis was determined as the
positive peak value in Cm after the time point
of 100 ms. The control cells, pretreated with the sense probe,
exhibited a biphasic response in Cm as described
previously (38). In these cells, the amplitude A of the
rapid component was indistinguishable from responses measured in
untreated cells (not shown). In contrast, cells treated with the
antisense probe exhibited a significantly reduced amplitude A,
indicating the reduction of the rapid component of exocytosis (Fig. 3,
bottom; Fig. 4C). The amplitude of the slow
component was similar in cells treated with the sense and the antisense probe (Fig. 4B). Although the inspection of Fig. 3
(middle traces) appears to indicate that the treatment of
cells by the antisense probe did affect endocytosis measured as the
reduction in Cm 20 s after the peak
positive value (Fig. 3, amplitude C), statistical analysis shows that
this is unlikely because of relatively large variability (Fig.
4A). These results indicate that Syt I is required for the
rapid exocytosis in neuroendocrine cells and is not affecting endocytosis, as reported previously (54).
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Syt I and the CAPS protein are required for secretory activity at
low [Ca2+]i.
The requirement of Syt I in the rapid secretory component (Figs. 3 and
4) may indicate that Syt I increases the probability of vesicle fusion
at low [Ca2+]i. Therefore, we stimulated
secretory activity by cytosol dialysis with pipette solutions
containing 1 µM [Ca2+]. The increase in
Cm, after 200 s from the start of
recording, was in control cells around 15% relative to the resting
Cm (Fig. 5),
determined at the start of the whole cell recording. Responses in
Cm were completely abolished in antisense
probe-treated cells (Fig. 5, A and D). This
effect was specific, because responses in Cm
were not affected by the transfection of EGFP only (not shown) or by
the pretreatment with the sense probe and the EGFP plasmid (Fig.
5D) compared with untreated cells (Fig. 5, C and D). These results support the view that Syt I augments the
probability of vesicle fusion at low [Ca2+] in rat
melanotrophs. Moreover, cytosol dialysis with solutions containing low
[Ca2+] activate a pathway that corresponds to the rapid,
Syt I-dependent response recorded after flash photolysis.
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Slow endocytosis requires Syt I.
Syt I binds also to the clathrin assembly protein complex AP-2 and may
serve as a receptor (58) or regulator (18) in
endocytosis. Although rapid endocytosis after the delivery of a UV
flash was not affected by the absence of Syt I (Figs. 3 and 4), we
examined whether slow endocytosis (time scale of minutes) may require
Syt I. If melanotrophs are dialyzed by practically zero
[Ca2+]i, Cm is
characterized by a slow, steady decline that is dominated by
endocytosis (Fig. 6B)
(39, 40). In the antisense-treated cells, the rate of
decline in Cm is not negative but becomes
positive. The slopes of regression lines in Fig. 6B are
significantly different, indicating that endocytosis is blocked in the
absence of Syt I. This response is specific, because changes in
Cm are not affected in EGFP or in the sense
probe- and EGFP-treated cells (Fig. 6C). These results show
that some, but not all, endocytic pathways require Syt I.
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DISCUSSION |
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The distribution of Syt isoforms (I-VII) in pituitary pars intermedia (Fig. 1), hippocampus, cerebellum, and spinal cord (not shown) is consistent with previous reports (22, 28, 30, 51, 52). In anterior lobe, the presence of Syt isoforms is similar to that in the pars intermedia except that Syt VI transcript was also detected (not shown). With in situ hybridization, Syt I was detected in abundance in pars intermedia and at a lower level in the anterior lobe, but Syt II was not detected (28), as found in this work (Fig. 1). We detected Syt III in both examined parts of the pituitary, which contrasts with a previous report (28). However, Syt III was detected in the whole pituitary (30). Different techniques used and/or animals of different developmental stages may account for the discrepancies. In addition to Syt I and III, rat pars intermedia also expresses Syt IV, V, and VII. Syt IV was previously reported to be present in the whole pituitary (53). Note that two different Syt sequences have been named Syt V. In our work, the Syt V isoform is that reported by Craxton and Goedert (9), corresponding to Syt IX as listed by Südhof and Rizo (44).
In transgenic mice, the Syt I-insensitive component of secretory response that remained in the absence of Syt I displayed a Ca2+ dependence similar to that of the Syt I-dependent exocytosis (54). Similar Ca2+ dependence of secretion in the Syt I null mutants and in controls may be due to a compensatory mechanism that occurs during embryogenesis, where proteins other than Syt I, such as CAPS (55), may partially correct for the loss of Syt I function (see Ref. 37). Here we used an acute deletion of Syt I to test the hypothesis that Syt I plays a role in determining the high-affinity Ca2+ sensing by the exocytic apparatus. We blocked the expression of a protein by introducing an antisense DNA probe into cells (26). The transfection of EGFP, a marker of lipofection, did not alter the properties of recorded functional parameters (Figs. 4 and 6; see Ref. 3). In the presence of the antisense probe, green-fluorescing cells were devoid of Syt I (Fig. 2). Compared with the control sense probe-treated cells, antisense probe-treated cells revealed that the flash-induced rapid component of Cm was significantly diminished (Figs. 3 and 4). This finding indicates a role of Syt I in the rapid exocytosis of neuroendocrine cells, as reported previously (54). The rapid exocytosis, characterized by a high sensitivity to Ca2+, was also diminished by the CAPS-neutralizing antibodies (38). Thus Syt I and CAPS may increase the probability of vesicle fusion at low [Ca2+]i. Therefore, we stimulated secretory responses by cytosol dialysis with solutions containing low [Ca2+] and found that secretory responses were completely blocked in the antisense-treated cells (Fig. 5). Furthermore, changes in Cm were also completely blocked by the CAPS antibody (1, 47), whereas responses in cells treated with the sense Syt I probe or preinjected with the preimmune serum were not significantly different from the controls (Fig. 5D). These results show that Syt I and CAPS act in the high-affinity Ca2+-dependent exocytosis, probably by increasing the probability of vesicle fusion at low [Ca2+]i.
Syt I was proposed to act as the receptor for AP-2 in endocytosis (58). The decline in Cm following the flash-induced peak in Cm was insensitive to the absence of Syt I (Fig. 4A). In contrast, slow endocytosis that dominates the time course of Cm for several minutes in the cytosol dialysis experiments is sensitive to the absence of Syt I (Fig. 6). These results indicate that certain types of endocytosis in melanotrophs require Syt I, which is consistent with the existence of multiple pathways of endocytosis (2). Furthermore, these results support the proposal that in melanotrophs there are distinct cycles of exo-endocytosis (36). Multiple exo-endocytosis cycles appear to have distinct biochemical characteristics. Exocytosis that is sensitive to Syt I appears to be coupled to Syt I-sensitive endocytosis (Fig. 6). On the other hand, Syt I-insensitive exocytosis is coupled to the Syt I-insensitive endocytosis (Fig. 4). Distinct exo-endocytosis mechanisms appear to cluster into distinct functional modules, termed exocytic modules.
It was proposed that functional roles of multiple Syt isoforms are based on their in vitro biochemical properties (44, 45), grouped into three classes. Class A, represented by Syt I and II, requires high [Ca2+] for syntaxin binding. Class B, represented by Syt III and VII, requires low [Ca2+] for syntaxin binding. Class C includes synaptotagmins that do not bind syntaxin as a function of [Ca2+] [Syt IV, V (IX), and VI]. Although all of these classes are expressed in melanotrophs (Fig. 1), our results on single cells show that Syt I may play a role in the high-affinity Ca2+-dependent exocytosis, whereas the low-affinity Ca2+-dependent mechanism of secretion appears to be Syt I insensitive (Fig. 3). We propose that Syt I plays a role in Ca2+ sensing by increasing the probability of vesicle fusion at low [Ca2+]i. CAPS may function in a similar way, because functional neutralization of this protein results in the attenuation of the rapid, high-affinity Ca2+-dependent exocytosis in neuroendocrine cells (38) (see also Fig. 5D). Moreover, the genetic deletion of CAPS homolog resulted in a reduced Ca2+ sensitivity of the exocytic apparatus in both Drosophila neurons (37) and neuroendocrine cells (38). Finally, CAPS and Syt I both appear to affect the stability of the fusion pore dwell time (12, 56). We propose that CAPS and the low-affinity Ca2+-binding protein Syt I are essential elements of the exocytic apparatus and act by augmenting the probability of vesicle fusion at low [Ca2+]i. This mechanism may involve interactions with other molecular partners in the exocytic module.
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ACKNOWLEDGEMENTS |
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We thank Dr. M. Seagar (Univ. de la Méditerranée, Marseille, France) for the antibody against Syt I and Dr. T. F. J. Martin (Univ. of Wisconsin, Madison, WI) for the antibody against CAPS.
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FOOTNOTES |
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This work was supported by the Ministry of Education, Sciences, and Sports of the R. Slovenia Fund P3 521 0381.
Present address of M. Rupnik: European Neuroscience Institute, 37073 Göttingen, Germany.
Address for reprint requests and other correspondence: R. Zorec, Medical Faculty, Institute of Pathophysiology, Zaloska 4, 1000 Ljubljana, Slovenia (E-mail: Robert.Zorec{at}mf.uni-lj.si).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published October 16, 2002;10.1152/ajpcell.00333.2002
Received 17 July 2002; accepted in final form 15 October 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ann, K,
Kowalchyk JA,
Loyet KM,
and
Martin TF.
Novel Ca2+-binding protein (CAPS) related to UNC-31 required for Ca2+-activated exocytosis.
J Biol Chem
272:
19637-19638,
1997
2.
Apodaca, G.
Endocytic traffic in polarized epithelial cells: role of the actin and microtubule cytoskeleton.
Traffic
2:
149-159,
2001[ISI][Medline].
3.
Ashery, U,
Betz A,
Xu T,
Brose N,
and
Rettig J.
An efficient method for infection of adrenal chromaffin cells using the Semliki Forest virus gene expression system.
Eur J Cell Biol
78:
525-532,
1999[ISI][Medline].
4.
Bollmann, JH,
Sakmann B,
and
Borst JGG
Calcium sensitivity of glutamate release in a calyx-type terminal.
Science
289:
953-957,
2000
5.
Brose, N,
Petrenko AG,
Südhof TC,
and
Jahn R.
Synaptotagmin: a calcium sensor on the synaptic vesicle surface.
Science
256:
1021-1025,
1992[ISI][Medline].
6.
Burgoyne, RD,
and
Morgan A.
Analysis of regulated exocytosis in adrenal chromaffin cells: insights into NSF/SNAP/SNARE function.
Bioessays
20:
328-335,
1998[ISI][Medline].
7.
Carter, TD,
and
Ogden D.
Acetylcholine-stimulated changes of membrane potential and intracellular Ca2+ concentration recorded in endothelial cells in situ in the isolated rat aorta.
Pflügers Arch
428:
476-484,
1994[ISI][Medline].
8.
Castillo, PE,
Janz R,
Südhof TC,
Tzounopoulos T,
Malenka RC,
and
Nicoll RA.
Rab3A is essential for mossy fibre long-term potentiation in the hippocampus.
Nature
388:
590-592,
1997[ISI][Medline].
9.
Craxton, M,
and
Goedert M.
Synaptotagmin V: a novel synaptotagmin isoform expressed in rat brain.
FEBS Lett
361:
196-200,
1995[ISI][Medline].
10.
Davis, AF,
Bai J,
Fasshauer D,
Wolowick MJ,
Lewis JL,
and
Chapman ER.
Kinetics of synaptotagmin responses to Ca2+ and assembly with the core SNARE complex onto membranes.
Neuron
24:
363-376,
1999[ISI][Medline].
11.
DiAntonio, A,
and
Schwarz TL.
The effect on synaptic physiology of synaptotagmin mutations in Drosophila.
Neuron
12:
909-920,
1994[ISI][Medline].
12.
Elhamdani, A,
Martin TF,
Kowalchyk JA,
and
Artalejo CR.
Ca2+-dependent activator protein for secretion is critical for the fusion of dense-core vesicles with the membrane in calf adrenal chromaffin cells.
J Neurosci
19:
7375-7383,
1999
13.
Ellis-Davies, GC,
and
Kaplan JH.
Nitrophenyl-EGTA, a photolabile chelator that selectively binds Ca2+ with high affinity and releases it rapidly upon photolysis.
Proc Natl Acad Sci USA
91:
187-191,
1994[Abstract].
14.
Fernandez-Chacon, R,
Konigstorfer A,
Gerber SH,
Garcia J,
Matos MF,
Stevens CF,
Brose N,
Rizo J,
Rosenmund C,
and
Südhof TC.
Synaptotagmin I functions as a calcium regulator of release probability.
Nature
410:
41-49,
2001[ISI][Medline].
15.
Geppert, M,
Goda Y,
Hammer RE,
Li C,
Rosahl TW,
Stevens CF,
and
Südhof TC.
Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse.
Cell
79:
717-727,
1994[ISI][Medline].
16.
Grynkiewicz, G,
Poenie M,
and
Tsien RY.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J Biol Chem
260:
3438-3450,
1985.
17.
Heinemann, C,
Chow RH,
Neher E,
and
Zucker RS.
Kinetics of the secretory response in bovine chromaffin cells following flash photolysis of caged Ca2+.
Biophys J
67:
2546-2557,
1994[Abstract].
18.
Jarousse, N,
and
Kelly RB.
The AP2 binding site of synaptotagmin 1 is not an internalization signal but a regulator of endocytosis.
J Cell Biol
154:
857-866,
2001
19.
Jorgensen, EM,
Hartwieg E,
Schuske K,
Nonet ML,
Jin Y,
and
Horvitz HR.
Defective recycling of synaptic vesicles in synaptotagmin mutants of Caenorhabditis elegans.
Nature
378:
196-199,
1995[ISI][Medline].
20.
Kasai, H,
Takagi H,
Ninomiya Y,
Kishimoto T,
Ito K,
Yoshida A,
Yoshioka T,
and
Miyashita Y.
Two components of exocytosis and endocytosis in phaeochromocytoma cells studied using caged Ca2+ compounds.
J Physiol
494:
53-65,
1996[Abstract].
21.
Leveque, C,
Hoshino T,
David P,
Shoji-Kasai Y,
Leys K,
Omori A,
Lang B,
el Far O,
Sato K,
Martin-Moutot N,
The synaptic vesicle protein synaptotagmin associates with calcium channels and is a putative Lambert-Eaton myasthenic syndrome antigen.
Proc Natl Acad Sci USA
89:
3625-3629,
1992[Abstract].
22.
Li, C,
Ullrich B,
Zhang JZ,
Anderson RGW,
Brose N,
and
Südhof TC.
Ca2+-dependent and -independent activities of neural and non-neural synaptotagmins.
Nature
375:
594-599,
1995[ISI][Medline].
23.
Littleton, JT,
Bai J,
Vyas B,
Desai R,
Baltus AE,
Garment MB,
Carlson SD,
Ganetzky B,
and
Chapman ER.
Synaptotagmin mutants reveal essential functions for the C2B domain in Ca2+-triggered fusion and recycling of synaptic vesicles in vivo.
J Neurosci
21:
1421-1433,
2001
24.
Littleton, JT,
Stern M,
Perin M,
and
Bellen HJ.
Calcium dependence of neurotransmitter release and rate of spontaneous vesicle fusions are altered in Drosophila synaptotagmin mutants.
Proc Natl Acad Sci USA
91:
10888-10892,
1994
25.
Littleton, JT,
Stern M,
Schulze K,
Perin M,
and
Bellen HJ.
Mutational analysis of Drosophila synaptotagmin demonstrates its essential role in Ca2+-activated neurotransmitter release.
Cell
74:
1125-1134,
1993[ISI][Medline].
26.
Lledo, PM,
Vernier P,
Vincent JD,
Mason WT,
and
Zorec R.
Inhibition of Rab3B expression attenuates Ca2+-dependent exocytosis in rat anterior pituitary cells.
Nature
364:
538-544,
1993.
27.
Mains, RE,
and
Eipper BA.
Synthesis and secretion of corticotropins, melanotropins and endorphins by rat intermediate pituitary cells.
J Biol Chem
254:
7885-7894,
1979[Abstract].
28.
Marqueze, B,
Boudier JA,
Mizuta M,
Iganaki N,
Seino S,
and
Seagar M.
Cellular localization of synaptotagmin I, II, and III mRNAs in the central nervous system and pituitary and adrenal glands of the rat.
J Neurosci
15:
4906-4917,
1995[Abstract].
29.
Martin, TF,
and
Kowalchyk JA.
Docked secretory vesicles undergo Ca2+-activated exocytosis in a cell-free system.
J Biol Chem
272:
14447-14453,
1997
30.
Mizuta, M,
Iganaki N,
Nemoto Y,
Matsukura S,
Takahashi M,
and
Seino S.
Synaptotagmin III is a novel isoform of rat synaptotagmin expressed in endocrine and neuronal cells.
J Biol Chem
269:
11675-11678,
1994
31.
Neher, E,
and
Marty A.
Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin cells.
Proc Natl Acad Sci USA
79:
6712-6716,
1982[Abstract].
32.
Neher, E,
and
Zucker RS.
Multiple calcium-dependent processes related to secretion in bovine chromaffin cells.
Neuron
10:
21-30,
1993[ISI][Medline].
33.
Ninomiya, Y,
Kishimoto T,
Yamazawa T,
Ikeda H,
Miyashita Y,
and
Kasai H.
Kinetic diversity in the fusion of exocytotic vesicles.
EMBO J
16:
929-934,
1997
34.
Nonet, ML,
Grundahl K,
Meyer BJ,
and
Rand JB.
Synaptic function is impaired but not eliminated in C. elegans mutants lacking synaptotagmin.
Cell
73:
1291-1305,
1993[ISI][Medline].
35.
Perin, MS,
Fried VA,
Mingnery GA,
Jahn R,
and
Südhof TC.
Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C.
Nature
345:
260-261,
1990[ISI][Medline].
36.
Poberaj, I,
Rupnik M,
Kreft M,
Sikdar SK,
and
Zorec R.
Modeling excess retrieval in rat melanotroph membrane capacitance records.
Biophys J
82:
226-232,
2002
37.
Renden, R,
Berwin B,
Davis W,
Ann K,
Chin CT,
Kreber R,
Ganetzky B,
Martin TF,
and
Broadie K.
Drosophila CAPS is an essential gene that regulates dense-core vesicle release and synaptic vesicle fusion.
Neuron
31:
421-437,
2001[ISI][Medline].
38.
Rupnik, M,
Kreft M,
Sikdar SK,
Grilc S,
Romih R,
Zupani
G,
Martin TF,
and
Zorec R.
Rapid regulated dense-core vesicle exocytosis requires the CAPS protein.
Proc Natl Acad Sci USA
97:
5627-5632,
2000
39.
Rupnik, M,
and
Zorec R.
Cytosolic chloride ions stimulate Ca2+-induced exocytosis in melanotrophs.
FEBS Lett
303:
221-223,
1992[ISI][Medline].
40.
Rupnik, M,
and
Zorec R.
Intracellular Cl modulates Ca2+-induced exocytosis from rat melanotrophs through GTP-binding proteins.
Pflügers Arch
431:
76-83,
1995[ISI][Medline].
41.
Schlüter OM, Khvotchev MV, Jahn R, and Südhof TC.
Localization versus function of Rab3 proteins: evidence for a common
regulatory role in controlling fusion. J Biol Chem
(ahead of print), 2002.
42.
Schneggenburger, R,
and
Neher E.
Intracellular calcium dependence of transmitter release rates at a fast central synapse.
Nature
386:
889-893,
2000.
43.
Südhof, TC.
Synaptotagmins: why so many?
J Biol Chem
277:
7629-7632,
2002
44.
Südhof, TC,
and
Rizo J.
Synaptotagmins: C2-domain proteins that regulate membrane traffic.
Neuron
17:
379-388,
1996[ISI][Medline].
45.
Sugita, S,
Shin OH,
Han W,
Lao Y,
and
Südhof TC.
Synaptotagmins form a hierarchy of exocytotic Ca2+ sensors with distinct Ca2+ affinities.
EMBO J
21:
270-280,
2002
46.
Takahashi, M,
Arimatsu Y,
Fujita S,
Fujimoto Y,
Kondo S,
Hama T,
and
Miyamoto E.
Protein kinase C and Ca2+/calmodulin-dependent protein kinase II phosphorylate a novel 58-kDa protein in synaptic vesicles.
Brain Res
551:
279-292,
1991[ISI][Medline].
47.
Tandon, A,
Bannykh S,
Kowalchyk JA,
Banerjee A,
Martin TF,
and
Balch WE.
Differential regulation of exocytosis by calcium and CAPS in semi-intact synaptosomes.
Neuron
21:
147-154,
1998[ISI][Medline].
48.
Thomas, P,
Surprenant A,
and
Almers W.
Cytosolic Ca2+, exocytosis, and endocytosis in single melanotrophs of the rat pituitary.
Neuron
5:
723-733,
1990[ISI][Medline].
49.
Thomas, P,
Wong JG,
and
Almers W.
Millisecond studies of secretion in single rat pituitary cells stimulated by flash photolysis of caged Ca2+.
EMBO J
12:
303-306,
1993b[Abstract].
50.
Thomas, P,
Wong JG,
Lee AK,
and
Almers W.
A low affinity Ca2+ receptor controls the final steps in peptide secretion from pituitary melanotrophs.
Neuron
11:
93-104,
1993[ISI][Medline].
51.
Ullrich, B,
and
Südhof TC.
Differential distributions of novel synaptotagmins: comparison to synapsins.
Neuropharmacology
34:
1371-1377,
1995[ISI][Medline].
52.
Ullrich, B,
Li C,
Zhang JZ,
McMahon H,
Anderson RG,
Geppert M,
and
Südhof TC.
Functional properties of multiple synaptotagmins in brain.
Neuron
13:
1281-1291,
1994[ISI][Medline].
53.
Vician, L,
Lim IK,
Ferguson G,
Tocco G,
Baudry M,
and
Herschman HR.
Synaptotagmin IV is an immediate early gene induced by depolarization in PC12 cells and in brain.
Proc Natl Acad Sci USA
92:
2164-2168,
1995[Abstract].
54.
Voets, T,
Moser T,
Lund PE,
Chow RH,
Geppert M,
Südhof TC,
and
Neher E.
Intracellular calcium dependence of large dense-core vesicle exocytosis in the absence of synaptotagmin I.
Proc Natl Acad Sci USA
98:
11680-11685,
2001
55.
Walent, JH,
Porter BW,
and
Martin TF.
A novel 145 kd brain cytosolic protein reconstitutes Ca2+-regulated secretion in permeable neuroendocrine cells.
Cell
70:
765-775,
1992[ISI][Medline].
56.
Wang, CT,
Grishanin R,
Earles CA,
Chang PY,
Martin TF,
Chapman ER,
and
Jackson MB.
Synaptotagmin modulation of fusion pore kinetics in regulated exocytosis of dense-core vesicles.
Science
294:
1111-1115,
2001
57.
Xu, T,
Binz T,
Niemann H,
and
Neher E.
Multiple kinetic components of exocytosis distinguished by neurotoxin sensitivity.
Nat Neurosci
1:
192-200,
1998[ISI][Medline].
58.
Zhang, JZ,
Davletov BA,
Südhof TC,
and
Anderson RG.
Synaptotagmin I is a high affinity receptor for clathrin AP-2: implications for membrane recycling.
Cell
78:
751-760,
1994[ISI][Medline].
59.
Zorec, R,
Henigman F,
Mason WT,
and
Korda M.
Electrophysiological study of hormone secretion by single adenohypophyseal cells.
Methods Neurosci
4:
194-210,
1991.