From The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, United Kingdom
Received for publication, October 30, 2002, and in revised form, December 3, 2002
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ABSTRACT |
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Protein phosphorylation by protein kinase C (PKC)
has been implicated in the control of neurotransmitter release and
various forms of synaptic plasticity. The PKC substrates responsible
for phosphorylation-dependent changes in regulated exocytosis
in vivo have not been identified. Munc18a is essential for
neurotransmitter release by exocytosis and can be phosphorylated by PKC
in vitro on Ser-306 and Ser-313. We demonstrate that it is
phosphorylated on Ser-313 in response to phorbol ester treatment in
adrenal chromaffin cells. Mutation of both phosphorylation sites to
glutamate reduces its affinity for syntaxin and so acts as a
phosphomimetic mutation. Unlike phorbol ester treatment, expression of
Munc18 with this phosphomimetic mutation in PKC phosphorylation sites
did not affect the number of exocytotic events. The mutant did,
however, produce changes in single vesicle release kinetics, assayed by
amperometry, which were identical to those caused by phorbol ester
treatment. Furthermore, the effects of phorbol ester treatment on
release kinetics were occluded in cells expressing phosphomimetic
Munc18. These results suggest that the dynamics of vesicle release
events during exocytosis are controlled by PKC directly through
phosphorylation of Munc18 on Ser-313. Phosphorylation of Munc18
by PKC may provide a mechanism for the control of exocytosis and
thereby synaptic plasticity.
Protein phosphorylation has been long known as an important
mechanism for the regulation of exocytosis although, with only a few
exceptions such as the synapsins (1), the targets for regulation by
phosphorylation in vivo are unknown. Treatment with phorbol
esters modifies regulated exocytosis in many different neuronal and
non-neuronal (2, 3) cell types leading to increased vesicle recruitment
into the ready releasable pool (4-6), acceleration of fusion pore
expansion (7), or changes in the kinetics of exocytosis (8, 9). PKC
also has a key role in synaptic plasticity (10). The effects of phorbol
ester were originally attributed to activation of
PKC1 although the PKC
substrates responsible had not been identified, and it is not known if
the same target regulates all of the parameters modified by phorbol
esters. The SNARE proteins, syntaxin 1, SNAP-25, and VAMP play key
roles in exocytosis (11-13), and formation of the SNARE complex has
been suggested to be a driving force for membrane fusion (14). The
syntaxin-binding protein Munc18a (15) (also known as nSec1, Ref. 16) is
also essential for neurotransmitter release (17-19). Other key
proteins in regulated exocytosis include rab3 and its effectors (20)
and synaptotagmin the likely Ca2+ sensor (21, 22). Among
these proteins, SNAP-25 (23), VAMP (24), synaptotagmin I (25), rab3
(26), and Munc18 (27, 28) have been shown to be PKC substrates in
vitro. PKC phosphorylation of specific residues in intact cells
has only been demonstrated for SNAP-25 (29) and synaptotagmin I (25).
In no case has the functional consequences of these phosphorylation
events for exocytosis been established. Indeed, the phosphorylation of
SNAP-25 by PKC in PC12 cells lagged well behind the effects of phorbol ester on the extent of exocytosis (29). In that study, it was also
shown that the phorbol ester effects had both a
PKC-dependent and a PKC-independent component. The synaptic
protein Munc13 has been identified as an alternative phorbol
ester-binding protein (30, 31), and recently it has been suggested that
the effects of phorbol ester on synaptic transmission are mediated
entirely by Munc13 (32). A PKC-dependent component of
phorbol ester stimulation of exocytosis was shown to involve vesicle
recruitment to the plasma membrane in PC12 cells (33). Phorbol
ester-stimulated recruitment of vesicles into the ready releasable pool
in adrenal chromaffin cells was inhibited by bisindolylmaleimide in
chromaffin cells (4, 34) consistent with a role for PKC. In addition, we have shown that another aspect of exocytosis, the kinetics of single
vesicle release events, is modified by phorbol esters in a
PKC-dependent (bisindolylmaleimide-sensitive) fashion in adrenal chromaffin cells (9). It is clear that alternative approaches
are needed to establish whether PKC-mediated phosphorylation of any
identified substrate directly regulates one or more aspect of exocytosis.
Munc18 binds tightly to syntaxin holding it in a closed conformation
that prevents its assembly into a SNARE complex (35-37). In some as
yet uncharacterized way, Munc18 is required to donate syntaxin ready
for its interaction with the other SNARE proteins and in its absence
membrane fusion is abolished. Modification of the interaction between
Munc18 and syntaxin could, therefore, be an important mechanism for the
regulation of membrane fusion, and we have shown that mutations in
Munc18, which modify the affinity of this interaction, change the
kinetics of single vesicle release events in chromaffin cells (38).
Interestingly, Munc18a is phosphorylated by PKC on Ser-306 and Ser-313
in vitro, and this reduces the amount of Munc18 that binds
to syntaxin (28). We have investigated whether phosphorylation on these
sites occurs in intact cells and demonstrate that phosphorylation of
Ser-313 of Munc18 in response to PKC activation leads to changes in the
kinetics of vesicle fusion and release.
Phosphospecific Antisera--
Rabbit polyclonal phosphospecific
antibodies were produced by AbCam Ltd (Cambridge, UK) using the
peptides CQEVTRpSLKDFS for Ser-306 and CDFSSpSKRMNTG for Ser-313.
Cysteine was included at the N terminus of each peptide for conjugation
to carrier proteins and for use in affinity purification. Rabbit
polyclonal antibodies were raised to these peptides, affinity-purified
using a Sulfolink kit (Pierce), and characterized by Western blotting
of phosphorylated and non-phosphorylated Munc18. For phosphorylation,
His-tagged proteins were incubated in MES buffer, pH 6.9 (50 mM MES, 10 mM MgCl2, 1 mM dithiothreitol, 0.5 mM EDTA) with 100 µM ATP, 1.25 mg/ml phosphatidylserine, 2.5 µg/ml PMA,
and 1.2 mM CaCl2. The final reaction volume was
50 µl containing 2 µg of Munc18 protein and 70 milliunits of PKC.
Reactions were incubated for 3 h at 30 °C. For mock
phosphorylation, PKC was omitted.
Binding Assays with Bacterially Expressed Munc18--
Munc18a
and mutants were expressed and purified as full-length GST fusion or
His-tagged proteins. The cytoplasmic domain of syntaxin was expressed
as GST fusion or His-tagged protein. Binding reactions (39) were
performed in binding buffer (20 mM HEPES, 150 mM NaCl, 2 mM MgCl2, 1 mM dithiothreitol, 0.5% (v/v) Triton X-100, pH 7.4).
Munc18-GST proteins at a concentration of 200 nM were
incubated with His-tagged syntaxin at various concentrations, and then
20 µl of glutathione-Sepharose beads (Amersham Biosciences) and
binding buffer were added to make the total reaction volume 100 µl.
Reactions were incubated for 3 h at 4 °C, the supernatants were
removed by passing through spin filter columns (Affinity Research
Products Ltd, Exeter, UK), and the glutathione-Sepharose beads were
washed four times in binding buffer. Proteins were eluted with 60 µl
of SDS gel buffer and boiled for 5 min, after which 20 µl of each
assay was run on SDS-PAGE. Western blotting was then performed to assay
bound syntaxin using monoclonal anti-syntaxin (HPC-1, Sigma) and
125I-labeled secondary antibodies (Amersham Biosciences) to
allow quantification. In control incubations with GST instead of
GST-Munc18 or GST-syntaxin, nonspecific binding was not detected.
Binding Assays with in Vitro Translated Munc18--
Wild-type
and mutant Munc18 proteins were produced in radiolabeled form by
in vitro transcription/translation with the TNT T7 PCR DNA system (Promega) using PCR template amplified from plasmids
encoding Munc18 wild type or Munc18 mutants. The
35S-labeled proteins were bound to syntaxin in the absence
or presence of unlabeled wild-type Munc18 protein to act as a binding
competitor as follows: purified GST-syntaxin (5 µg) was incubated
with 20 µl of glutathione-Sepharose (Amersham Biosciences) in 200 µl of PBS for 1 h at 4 °C. The glutathione-Sepharose pellet
was washed twice with 500 µl of PBS and twice with 500 µl of
binding buffer (20 mM HEPES, 150 mM NaCl, 1 mM dithiothreitol, 2 mM MgCl2,
0.5% (v/v) Triton X-100, pH 7.5) and resuspended in binding buffer to
a final volume of 195 µl giving a final GST-syntaxin concentration of
0.4 µM. 5 µl of the radiolabeled Munc18 (giving final
concentrations of ~5 pM) was incubated with the
GST-syntaxin for 2 h at 4 °C in the presence of various
concentrations of purified wild-type His-tagged Munc18. After 2 h,
the supernatant from the reaction was removed, precipitated with an
equal volume of methanol at Cell Culture and Transfection of Chromaffin Cells--
Isolated
bovine adrenal chromaffin cells (40) were plated on non-tissue
culture-treated 10-cm Petri dishes and left overnight at 37 °C.
Non-attached cells were resuspended in growth medium at a density of
1 × 107/ml. A plasmid encoding wild-type Munc18a (in
pcDNA3) (41) was mutated at Ser-306 and Ser-313 using the
QuickChange system (Stratagene). Plasmids (encoding EGFP and Munc18 or
mutants) were mixed and added at 2 µg/1 × 106
cells, and cells were electroporated using a Bio-Rad Gene Pulser II.
The cells were then rapidly diluted to 1 × 106/ml
with fresh growth medium and maintained in culture for 3-5 days. For
immunofluorescence, transfections were performed as described above,
and cells plated onto glass coverslips. After washing twice with PBS,
cells were fixed in 4% formaldehyde in PBS for 30 min at room
temperature, processed for detection of overexpressed Munc18 as
described (38), and viewed using appropriate filters to visualize
EGFP and immunofluorescence.
Expression of Munc18 in HeLa Cells--
The cells were
transfected with 1 µg of plasmid (pcDNA3, or the plasmids
encoding Munc18 and mutants) using 3 µl of FuGENE 6 transfection
reagent (Roche Diagnostics). After an additional 72 h, the cells
were lysed in 200 µl of SDS dissociation buffer. Samples were
separated by SDS-PAGE, transferred to nitrocellulose, and probed with
monoclonal antibody against Munc18 (BD Biosciences).
Amperometric Recording--
Electrophysiological recording
conditions were as described previously (9, 38). Briefly, cells were
incubated in bath buffer (139 mM potassium glutamate, 0.2 mM EGTA, 20 mM PIPES, 2 mM ATP, and
2 mM MgCl2, pH 6.5) and a 5-µm diameter
carbon fiber electrode was positioned in contact with a cell. For
stimulation, a cell permeabilization/stimulation buffer (139 mM potassium glutamate, 20 mM PIPES, 5 mM EGTA, 2 mM ATP, 2 mM
MgCl2, 20 µM digitonin, and 10 µM free Ca2+, pH 6.5) was pressure-ejected
from a glass pipette on the opposite side of the cell, and amperometric
responses were monitored with a VA-10 amplifier (NPI Electronic, Tamm,
Germany). For examination of the effects of phorbol ester treatment,
recordings were taken of untreated control cells, and then 100 nM PMA added to the bath for at least 10 min before
recording from treated cells. For the comparison of control- and
mutant-expressing cells, analysis of untransfected and transfected
cells before and after PMA treatment was carried out in parallel on the
same batch of cells. To rule out any variability between cell batches
and carbon fibers, transfected cells and untransfected cells as
controls were recorded alternately in the same dishes and with the same
carbon fibers. Data from transfected cells was always compared with the
respective control cells. For all treatments, cells were derived from
multiple cell preparations. The data were subsequently analyzed using
Origin (9). All of the data are shown as mean ± S.E., and
statistical differences were assessed using the non-parametric Mann
Whitney test.
Analysis of Catecholamine Release from Cell
Populations--
Chromaffin cells in culture were washed in a
Krebs-Ringer buffer (145 mM NaCl, 5 mM KCl, 1.3 mM MgCl2, 1.2 mM
NaH2PO4, 10 mM glucose, and 20 mM HEPES, pH 7.4), and then incubated for 10 min in the
presence or absence of 100 nM PMA. The cells were then stimulated and permeabilized by incubation in 139 mM
potassium glutamate, 20 mM PIPES, 0.2 mM EGTA,
2 mM ATP, 2 mM MgCl2, 20 µM digitonin, pH 6.5 with either no added
CaCl2 or CaCl2 added to give a free
Ca2+ concentration of 10 µM. After 10 min,
the supernatant and the cells were taken for analysis of catecholamine
using a fluorimetric assay (40).
Use of Phosphospecific Antisera to Demonstrate Phosphorylation of
Ser-306 and Ser-313 of Munc18 in Intact Cells--
To test whether PKC
phosphorylated residues Ser-306 and Ser-313 of Munc18, phosphospecific
antisera were generated using phosphorylated peptides surrounding each
phosphorylation site. These antisera specifically recognized
recombinant Munc18a phosphorylated by PKC in vitro but not
the non-phosphorylated protein (Fig.
1A). In these experiments the
bacterially expressed Munc18 often ran as a doublet, presumably due to
some limited proteolysis, but the two forms behaved essentially
identically in all assays. Recombinant His6-tagged Munc18
was also prepared with double mutations to either alanine (dAla) or
glutamate (dGlu) at Ser-306 and Ser-313. Specificity of the antisera
was further shown by the fact that neither antisera detected the
proteins mutated at Ser-306 and Ser-313 after the proteins had been
incubated with PKC (Fig. 1B). Adrenal chromaffin cells are
widely used in the study of regulated exocytosis, and phorbol ester
treatment modifies the extent (4, 6) and kinetics of exocytosis (8, 9)
from them. Phosphorylation of Munc18, therefore, was examined in these
cells using the phosphospecific antisera. A specific signal from the
antiphospho-Ser-306 antiserum was not obtained from either control or
PMA-treated cells and therefore, it is unclear if this residue is
phosphorylated in vivo. In contrast, a specific signal for
Ser-313 phosphorylation was obtained based on competition with the
immunizing peptide (Fig. 1, C and D).
Phosphorylation of Ser-313 was low in control cells but was markedly
increased by PMA treatment, showing that this residue is phosphorylated
following PKC activation in intact cells (Fig. 1C).
Effect of Mutations in Ser-306 and Ser-313 of Munc18 on Binding to
Syntaxin--
It has been shown that phosphorylation of Munc18 by PKC
reduced the amount of the protein recovered with syntaxin in an
in vitro binding assay (28). As shown in Fig.
2A, in vitro
phosphorylation of Munc18 by PKC significantly reduced the affinity of
its binding to syntaxin 1A (39) from a KD of 56 to
390 nM. In the study identifying Ser-306 and Ser-313 as
phosphorylation sites, two additional phosphorylated peptides were not
characterized. In order to determine whether phosphorylation of only
the two identified residues was responsible for the effect on syntaxin binding, they were both mutated to the potentially phosphomimetic residue glutamate. The dGlu mutant also showed a reduced affinity (from
a KD of 10.3-840 nM) as seen for
phosphorylated wild-type Munc18 (Fig. 2B). This effect was
specific for phosphomimetic residues, because mutation of Ser-306 and
Ser-313 to alanine (dAla) did not affect syntaxin binding
(KD of 17.2 nM). In order to confirm the
reduced affinity of the dGlu mutant for binding to syntaxin and to rule
out any potential problems resulting from bacterial expression of the
mutant, binding assays were carried out using radiolabeled Munc18s
prepared by in vitro transcription and translation. Binding
of 35S-labeled proteins to syntaxin was carried out in the
absence or presence of competing unlabeled wild-type Munc18. Binding of the dGlu form of Munc18 was much more efficiently competed than that of
wild-type protein, showing the reduced affinity of the dGlu mutant
(Fig. 2C).
Effect of Phorbol Ester or the Phosphomimetic Mutation of Ser-306
and Ser-313 of Munc18 on the Extent of Vesicle Fusion--
In order to
test the effect of phosphorylation in vivo we expressed
Munc18 with Ser-306 and Ser-313 mutated to glutamate as this mutant was
phosphomimetic in the syntaxin binding assay. Use was made of a well
characterized assay (8, 9, 42, 43) based on the direct stimulation of
exocytosis with local application of digitonin and Ca2+ to
permeabilize the cells and allow Ca2+ entry, and use of
carbon-fiber amperometry to analyze the extent and kinetics of single
vesicle release events from adrenal chromaffin cells (44, 45).
Treatment of chromaffin cells with phorbol esters results in an
increase in catecholamine release when measured in populations of
intact or permeabilized cells (6, 46, 47) most likely due to an
increase in the recruitment of secretory vesicles (6). The extent of
this overall increase in catecholamine release for a cell population
experiment (around 40%) is shown by the example in Fig.
3A for digitonin-permeabilized
cells challenged with 0 and 10 µM Ca2+. We
examined whether phorbol ester treatment or expression of the dGlu
Munc18 mutant would increase the number of exocytotic events detected
by amperometry. In a series of independent experiments, PMA-treatment
increased the number of amperometric spikes to 166.6 + 18.1% of
control values (n = 8, p < 0.01, data
from a total of 137 control and 139 PMA-treated cells). The effect of
PMA on the time course of release is shown for one experiment in Fig. 3B. In contrast, expression of the Munc18 dGlu mutant had no
effect on either the overall number of amperometric spikes per cell
(21.9 + 5.0 for control cells, n = 32 and 19.5 + 5.2 for transfected cells, n = 35) or the time course of
the cell responses (Fig. 3C).
Effect of Expression of the dGlu Munc18 Mutant on Single Vesicle
Release Kinetics--
From examination of the kinetics of release from
single vesicles using amperometry we have previously shown that
treatment with the phorbol ester PMA reduced the amount of release per
vesicle (total charge) and the spike half-width and reduced both the
rise and fall time of the amperometric spikes (8, 9). These effects were blocked by the PKC inhibitor bisindolylmaleimide (9) implicating PKC as the target for PMA. Identical effects were seen following expression of a Munc18 mutant (R39C) that had a reduced affinity for
syntaxin (38) because Arg-39 makes an important contact with syntaxin
(35). Overexpression of wild-type Munc18 had no detectable effects on
either the extent or kinetics of exocytosis (38, 41). In the current
series of experiments, changes in release kinetics seen as alterations
in amperometric spike parameters were again observed in six of six
independent experiments on different cell batches. The pooled data are
shown in Fig. 4A. The
importance of Munc18 phosphorylation for the PKC effects was tested by
expressing the dGlu form of Munc18. Expression of the dGlu mutant,
resulted in a reduction in total charge released, in the half-width of the spikes and in rise and fall times of the spikes (Fig.
4B). These effects were essentially identical to those
following PMA treatment (Fig. 4A).
In order to determine whether phosphomimetic mutations at both of the
Munc18 phosphorylation sites was required for the functional effects,
Munc18 with a single mutation, S306E or S313E, were also tested.
Expression of either of these had no detectable effect on any spike
parameters (Fig. 5, A and
B). At the same time that the effect of
expression of the single mutants was examined, parallel experiments on the same batch of cells demonstrated that the expected changes in spike parameters following PMA treatment did occur. To
ensure that the difference between double and single mutants was not
due to differences in expression levels the extent of expression was
examined. All three proteins could be detected by immunofluorescence in
EGFP-positive transfected chromaffin cells using a concentration of
antiserum too low to detect the endogenous Munc18 (Fig.
6A). In this and in previous
studies (8, 9), we have established that close to 95% of cells
coexpress proteins from both plasmids used in the transfection. Since
the low efficiency of transfection of these cells (1-5%) precludes analysis by Western blotting we also used transfection of HeLa cells
that is efficient enough to allow Western blotting analysis. No
differences were observed in expression in HeLa cells (Fig. 6B). These results suggest therefore, that single mutations
to Ser-306 or Ser-313 are indeed ineffective in modifying exocytosis kinetics and that both sites may need to be phosphorylated to affect
exocytosis.
Comparison of the increase in the rate of rise of the amperometric
spikes showed that the double glutamate Munc18 had an effect of similar
magnitude to that due to PMA (Fig.
7A) We then tested whether the
effect of PMA was occluded in cells expressing the double mutant as
expected if the effects involved the same pathway. Expression of dGlu
in this additional experiment again modified the release kinetics and
increased the rate of rise of the spikes. PMA had no statistically
significant effect in the dGlu mutant-expressing cells over that from
the dGlu mutant alone (Fig. 7A) showing that the PMA effect
was indeed occluded. We also examined the effect of PMA on cells
expressing the single S306E Munc18 mutant, which did not increase the
rate of rise. In this case, PMA treatment resulted in a significantly
increased rate of rise of the spikes in the cells expressing this
mutant.
Previous studies using phorbol esters and PKC inhibitors or
following correlation of phosphorylation and secretion have failed to
convincingly identify a PKC substrate linked to the regulation of a
defined aspect of exocytosis. We have used, therefore, a more direct
functional approach with phosphomimetic mutations to examine the role
of PKC phosphorylation of Munc18 in the regulation of vesicle release
kinetics. We have demonstrated that Munc18 is phosphorylated in cells
on Ser-313 and that phosphorylation on Ser-313 is increased by phorbol
ester treatment. The physiological significance of Munc18
phosphorylation was demonstrated by expression of Munc18 with double
mutations in Ser-306 and Ser-313. With phosphomimetic mutations to
glutamate the double mutant modified vesicle release kinetics in an
identical manner to PMA treatment. The PKC phosphorylation sites in
Munc18a are not conserved in related Munc18/sec1 proteins involved in
other intracellular membrane fusion steps (48) suggesting that PKC
phosphorylation of Ser-313 is related to specific control of regulated exocytosis.
Regulation of exocytosis by protein phosphorylation has been
extensively investigated (3). In particular, phosphorylation of
synapsin via calmodulin-dependent kinase II has been
established as a mechanism for controlling vesicle recruitment (1). It is clear, however, that protein phosphorylation has diverse effects on
regulated exocytosis including direct effects of PKC activation on
membrane fusion. Many of the proteins that form the machinery for
exocytosis have been shown to be substrates for PKC or other kinases
(49, 50) in vitro. However, few in vivo PKC
substrates have been demonstrated, and the physiological regulation of
exocytosis by PKC has not been functionally linked to the
phosphorylation of any particular protein. Considerable work over many
years has implicated protein kinase C in controlling neurotransmitter
release based on the use of phorbol esters (2, 3). Very recently, however, it has been controversially suggested that all of the effects
of phorbol esters are due instead to an alternative phorbol ester
target, Munc13 (32). In contrast, we have now provided evidence that
Munc18 is a key substrate for the regulation of one aspect of
exocytosis by PKC. We have shown that it is phosphorylated on Ser-313
in response to phorbol ester in intact cells. In adrenal chromaffin
cells, phorbol ester treatment has at least two effects that appear to
be mediated by PKC. One is to increase the number of vesicles in the
ready-releasable pool (4). The second is to modify release kinetics
monitored at the level of single vesicles as described by us (8, 9) and
more recently confirmed by others (51). We show here that
phosphorylation of Munc18 is responsible for the regulation of
exocytosis kinetics of dense-core granules seen in response to phorbol
ester. It does not appear, however, to be linked to the increase in
exocytotic events following phorbol ester treatment and so this must
involve a distinct PKC substrate.
In using amperometry we have taken care to maximize the consistency of
this assay by parallel analysis of transfected and untransfected cells
in the same dishes and with the same carbon fibers. In addition, in
parallel experiments we have confirmed our previously reported effects
of phorbol esters on release kinetics. The robustness of our assay is
demonstrated by the closeness of the control values for spike
parameters from different batches of cells (Figs. 4 and 5) and by the
reproducible effect of PMA treatment seen in two previous studies (8,
9) and again in the current work. In addition, the effect of the dGlu
mutant of Munc18 was seen in two distinct series of experiments (Figs. 4 and 7), whereas the single Munc18 mutants had no detectable effect on
any of the spike parameters. The similar effects on spike parameters of
the dGlu and R39C (38) mutants of Munc18 is also notable and is
consistent with both mutations reducing the affinity of Munc18 for
syntaxin. Another study that examined the characteristics of
amperometric spikes in chromaffin cells of the Munc18a-null mouse did
not show any differences compared with control cells (52). Given the
essential nature of Munc18a for neurotransmitter release (19) and Sec1
family members for other fusion steps (53), it is possible that the
observed exocytotic events in the chromaffin cells from null mice were
supported by low levels of other Munc18 isoforms.
Phorbol ester treatment was demonstrated to increase catecholamine
release in cell population measurements on intact or permeabilized cells via activation of PKC (6, 46, 47, 54). Later work examining the
rapid kinetics of exocytosis using patch clamp capacitance measurement
revealed a specific increase in the size of the ready releasable pool
leading to an increase in a fast component of the secretory response.
This was seen as 2-3-fold increase in exocytosis in response to
depolarization or flash photolysis of caged Ca2+ (4) over a
subsecond time course. In other cases, the increase was more modest
(around 50%) (55). We have characterized here two effects of phorbol
ester, an increase in the number of exocytotic events and also changes
in single vesicle release kinetics that result in a reduction in
release per vesicle. Are these two effects compatible with the earlier
data on cell populations? Phorbol ester treatment results in an overall
increase in catecholamine release of about 40%, based on our data and
that of others (6). Despite a 20% reduction in catecholamine released
per spike, the overall increase in spike number by close to 70% due to
PMA would be entirely compatible with an overall increase in
catecholamine release due to PKC activation. We have shown, however,
that the two effects of phorbol ester can be dissociated as the effect on release kinetics, but not the spike number, appears to
involve PKC-mediated phosphorylation of Munc-18.
The kinetics of amperometric spikes are determined by multiple factors.
During initial fusion pore formation little release of vesicle contents
is detected, and the rising phase of the spike occurs as fusion pore
expansion initiates (56, 57). This rising phase could be affected by
the rate of fusion pore expansion but also by the rate of catecholamine
dissociation from the granule core, diffusion to the carbon fiber, and
consumption of the catecholamine by oxidation (44, 58). It is difficult
to imagine how expression of a cytoplasmic protein such as a Munc18
mutant could modify diffusion and oxidation of catecholamine or even
release from the granule core. As Munc18 interacts directly with known
components of the fusion machinery, we hypothesize that the increased
rate of rise of the amperometric spikes due to expression of the dGlu Munc18 or PKC activation is likely to be a consequence of an increased rate of expansion of the fusion pore. This would be consistent with the
direct demonstration that PKC activation increases the rate of fusion
pore expansion in eosinophils (7). Similarly, the decrease in the
amount and time of release per spike could be most easily be
interpreted as being caused by faster re-closure of the expanded fusion
pore, as it has been shown previously that even an expanded fusion pore
can re-close abruptly (59, 60). We cannot, however, formally rule out
other explanations for the data.
The importance of R39 Munc18 is predicted from the crystal structure of
the Munc18-syntaxin complex (35). In this structure Ser-313 and Ser-306
of Munc18 are close to a region of syntaxin containing acidic residues
(Asp-140 and Glu-143) (Fig. 7B), and it is likely that an
acidic phosphate (or glutamate in the mutants) would disrupt the
complex due to electrostatic repulsion. The increased rate of rise of
the amperometric spikes could be explained by an increase in the
efficiency of dissociation of Munc18 from syntaxin allowing more rapid
assembly of the SNARE complex and thereby driving fusion pore formation
and more rapid pore expansion. Alternatively, this may be due to
increased interaction of released Munc18 with other binding proteins
(61). We hypothesize that the reduced half-width and decreased fall
time of the spikes, suggests a second effect on accelerating vesicle
retrieval by closure of the expanded fusion pore (as in kiss-and-run
exocytosis, Refs. 62 and 63) and that this may have a distinct
mechanistic explanation. We and others (8, 64) have obtained a variety of independent evidence that changes in amperometric spike parameters represent changes in membrane fusion dynamics and retrieval consistent with changes in the extent of kiss-and-run exocytosis. Rapid
kiss-and-run exocytosis was originally proposed for synaptic exocytosis
(65) but first demonstrated for dense-core granule exocytosis (60). It
was suggested to occur in hippocampal neurons (66) and its existence in
a CNS synapse has recently been confirmed (67). Overall, our data point
to Munc18 as a target for the regulation of neurotransmission through
PKC-mediated phosphorylation that would allow both faster release
kinetics and more rapid vesicle recycling. Changes in vesicle release
kinetics have been suggested to cause the presynaptic changes that lead
to long term depression in the hippocampus (68), and an increase in the
kinetics of release have been linked to long term potentiation (69).
Munc18 phosphorylation is therefore, a candidate mechanism for changes in synaptic efficacy underlying synaptic plasticity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
20 °C for 30 min, resuspended in 40 µl of SDS-PAGE sample buffer, and boiled for 5 min. The
glutathione-Sepharose pellet was washed four times with 500 µl of
binding buffer, resuspended in 20 µl of SDS-PAGE sample buffer, and
boiled for 5 min. Samples of the binding reactions were analyzed by
SDS-PAGE and gels air-dried in cellulose sheets before exposing to
Biomax MR film (Kodak) for 16 h.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Characterization of phosphospecific antisera
for Ser-306 and Ser-313 of Munc18 and demonstration of phosphorylation
in intact chromaffin cells. A, recombinant His-tagged
Munc18a, which runs as a doublet on SDS-PAGE, was incubated under
phosphorylating conditions in the presence (phospho) or
absence (mock) of PKC. After SDS-PAGE the proteins were
probed with an anti-Munc18 monoclonal antibody (Munc18) or
affinity-purified antisera raised against peptides incorporating
phospho-Ser-306 (pS306-2) or phospho-Ser-313
(pS313-1). B, wild-type recombinant Munc18 or
Munc18 with both Ser-306 and Ser-313 mutated to alanines (dAla) or
glutamate (dGlu) were incubated under phosphorylating conditions with
PKC, and then the separated proteins were probed with anti-Munc18 or
the phosphospecific antisera. C, adrenal chromaffin cells in
culture were treated under control conditions or with PMA for 15 min.
Separated proteins were then probed with anti-Munc18 or the
phosphospecific antisera as indicated. D, the same blots
were reprobed with the phosphospecific antisera but in the presence of
excess of the immunizing peptide.
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Fig. 2.
Phosphorylation of Munc18 or mutation of
Ser-306 and Ser-316 to glutamate reduces its affinity for
syntaxin. A, recombinant His-tagged wild-type Munc18
was incubated with (phospho, open circles) or
without (mock, closed circles) PKC under
phosphorylating conditions, and then binding to GST-syntaxin (200 nM) was assayed at the indicated Munc18 concentrations.
Binding is shown as a percentage if the maximum observed with wild-type
protein. B, wild-type Munc18 or recombinant proteins with
Ser-306 or Ser-313 mutated to alanine or glutamate were expressed as
GST fusion proteins, incubated with syntaxin at the indicated
concentrations, and syntaxin binding assayed. Data from wild-type
protein are indicated by closed circles, dAla by open
circles, and dGlu by triangles. C, binding
of 35S-labeled Munc18 and the dGlu mutant to syntaxin and
competition by the indicated concentration of unlabeled His-tagged
wild-type Munc18.
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Fig. 3.
PMA treatment but not expression of a double
glutamate mutant of Munc18 increases the extent of exocytosis.
A, effect of PMA on release of catecholamine in a cell
population experiment. The cells were pretreated with PMA and then
permeabilized with digitonin in the absence or presence of 10 µM Ca2+ as indicated. Catecholamine released
over a 20-min period was assayed and expressed as a percentage of total
cellular content. B, time course of amperometric spikes
after stimulation with digitonin/Ca2+ in control
(n = 20) and PMA-treated (n = 23)
cells. C, time course of amperometric spikes after control
(n = 32 cells) and dGlu Munc18-expressing
(n = 35 cells) from the same plates.
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Fig. 4.
Expression of a double glutamate mutant of
Munc18 modifies the kinetics of vesicle release events in a similar
manner to PMA treatment. A, typical amperometric
responses from adrenal chromaffin cells, following addition of
digitonin and Ca2+, before and after PMA treatment are
shown. Amperometric spikes were analyzed from 93 control
(n = 1636 spikes) and 84 PMA-treated cells
(n = 1755 spikes) pooled from six experiments on
different batches of cells. Average values for total charge,
half-width, rise time to peak, and fall time of the spikes are shown.
B, chromaffin cells were transfected with plasmids encoding
EGFP and encoding Munc18 with both Ser-306 and Ser-313 mutated to
glutamate (dGlu). Recordings were made from EGFP-positive cells.
EGFP-negative cells in the same dishes were recorded with the same
carbon fiber to act as controls in each experiment. Typical traces are
shown for control and Munc18 dGlu-expressing cells. Amperometric spikes
were analyzed from 12 control (n = 466 spikes) and 15 transfected (n = 349 spikes) cells.
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Fig. 5.
Amperometric recordings and analysis of spike
parameters from cells expressing Munc18 with the single mutations S306E
or S313E. A, amperometric recordings and analysis of spike
parameters from cells expressing Munc18(S306E), (n = 8 cells, 197 spikes) and the respective control cells (n = 8 cells, 215 spikes). B, amperometric recordings and
analysis of spike parameters from cells expressing Munc18(S313E)
(n = 20 cells, 460 spikes) and the respective control
cells (n = 24 cells, 435 spikes).
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Fig. 6.
Double and single Munc18 mutants are
expressed to the same extent. A, expression of Munc18
mutants in EGFP-expressing chromaffin cells. The cells were processed
for immunofluorescence using a low concentration of antiserum antiserum
(1:200) that was suboptimal for the detection of endogenous Munc18 to
reveal those cells expressing Munc18 above endogenous levels after
transfection. The scale bar represents 10 µm.
B, levels of expression of mutant Munc18s in HeLa cells
detected by Western blotting with anti-Munc18. Control cells were
transfected with empty vector.
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Fig. 7.
Modification of release kinetics by Munc18
mutants and phorbol ester. A, comparison of the effect
of PMA treatment, expression of Munc18 mutants or PMA treatment with
Munc18 mutants on the rate of rise of the amperometric spikes. In each
case the values derived from treated cells were expressed as the
percentage difference compared with the appropriate control values from
the same experiment. The effects of PMA were seen in parallel
experiments on untransfected cells for each mutant expressed. The data
are derived from the following number of cells: PMA, 20 cells (412 spikes) and control 12 cells (179 spikes); dGlu, 20 cells (333 spikes),
dGlu plus PMA 21 cells (332 spikes) and control 20 cells (235 spikes);
S306E, 8 cells (197 spikes), S306E plus PMA 12 cells (330 spikes) and control 8 cells (215 spikes). B, position
of Ser-306 and Ser-313 in the structure of the syntaxin/Munc18a
complex.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by grants from the Wellcome Trust, the Biotechnology and Biological Science Research Council, and the Medical Research Council.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.
These authors contributed equally to this work.
§ Supported by a Natural Science and Engineering Research Council of Canada Postdoctoral Fellowship.
¶ Supported by a Wellcome Trust Prize Studentship.
Supported by a Wellcome Trust Prize Fellowship. Present
address: Dept. of Physiology, University of Oxford, Parks Road, Oxford OX1 3PT, UK.
** To whom correspondence should be addressed. Tel.: 44-151-794-5305; Fax: 44-151-794-5337; E-mail: burgoyne@liverpool.ac.uk.
Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M211114200
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ABBREVIATIONS |
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The abbreviations used are: PKC, protein kinase C; SNARE, soluble NSF attachment protein receptors; MES, 4-morpholineethanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; PMA, phorbol 12-myristate 13-acetate; GST, glutathione S-transferase; PBS, phosphate-buffered saline; EGFP, enhanced green fluorescent protein; SNAP, soluble NSF attachment protein; dGlu, double mutations to Glu; dAla, double mutations to Ala.
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REFERENCES |
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