Two distinct secretory vesicle-priming steps in adrenal chromaffin cells.

Abstract

Priming of large dense-core vesicles (LDCVs) is a Ca(2+)-dependent step by which LDCVs enter a release-ready pool, involving the formation of the soluble N-ethyl-maleimide sensitive fusion protein attachment protein (SNAP) receptor complex consisting of syntaxin, SNAP-25, and synaptobrevin. Using mice lacking both isoforms of the calcium-dependent activator protein for secretion (CAPS), we show that LDCV priming in adrenal chromaffin cells entails two distinct steps. CAPS is required for priming of the readily releasable LDCV pool and sustained secretion in the continued presence of high Ca(2+) concentrations. Either CAPS1 or CAPS2 can rescue secretion in cells lacking both CAPS isoforms. Furthermore, the deficit in the readily releasable LDCV pool resulting from CAPS deletion is reversed by a constitutively open form of syntaxin but not by Munc13-1, a priming protein that facilitates the conversion of syntaxin to the open conformation. Our data indicate that CAPS functions downstream of Munc13s but also interacts functionally with Munc13s in the LDCV-priming process.

Full text

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JCB: Article
The Rockefeller University Press $30.00
J. Cell Biol. Vol. 190 No. 6 1067–1077
www.jcb.org/cgi/doi/10.1083/jcb.201001164
JCB1067
Correspondence to Jens Rettig: jrettig@uks.eu
Abbreviations used in this paper: CAPS, calcium-dependent activator protein
for secretion; DKO, double KO; KO, knockout; LDCV, large dense-core vesicle;
MHD, Munc13 homology domain; RRP, readily releasable pool; SRP, slowly
releasable pool; SV, synaptic vesicle; UPP, unprimed pool.
Introduction
In neuroendocrine cells and synaptic terminals, only a fraction of
the secretory vesicles that are docked at the plasma membrane
can be released upon stimulation, indicating that a maturation or
priming step, which renders secretory vesicles fusion competent,
must take place after docking (Parsons et al., 1995; Plattner et al.,
1997). Interestingly, the pool of primed and fusion-competent
vesicles is heterogeneous in many secretory systems (Bittner and
Holz, 1992). In chromaffin cells, for example, high time resolu-
tion experiments revealed two populations of vesicles with dif-
ferent release rates, a readily releasable pool (RRP) and a slowly
releasable pool (SRP), which produce two phases of release
(Voets et al., 1999).
Several recent experiments showed that calcium-
dependent activator protein for secretion (CAPS) proteins play
a key role in the priming of large dense-core vesicles (LDCVs)
and synaptic vesicles (SVs; Stevens and Rettig, 2009). CAPS1
was discovered as a cytosolic factor that is required for
regulated fusion of LDCVs in PC12 cells (Walent et al., 1992).
Subsequently, it was shown that CAPS1 is a homologue of the
Caenorhabditis elegans protein UNC-31, whose mutation
leads to an uncoordinated phenotype with motor deficits (Ann
et al., 1997) and depresses release of SVs (Jockusch et al.,
2007) and LDCVs (Elhamdani et al., 1999). Deletion of CAPS
proteins causes a strong reduction in the size of the releasable
pool of vesicles in several organisms (Renden et al., 2001;
Speidel et al., 2005; Jockusch et al., 2007; Speese et al., 2007;
Liu et al., 2008).
In mouse and human, there are two CAPS genes encoding
isoforms that both contain a Munc13 homology domain (MHD;
Speidel et al., 2003). This domain is part of the minimal struc-
ture required for the function of Munc13-1 (Basu et al., 2005;
Madison et al., 2005; Stevens et al., 2005), a vesicle-priming
protein in neurons (Tokumaru and Augustine, 1999) and neuro-
endocrine cells (Ashery et al., 2000). The sequence required for
priming activity by Munc13-1 consists of a stretch of 672 amino
acids, including both MHDs (Basu et al., 2005; Madison et al.,
2005; Stevens et al., 2005), that has also been termed the MUN
P
soluble N-ethyl-maleimide sensitive fusion protein attach-
ment protein (SNAP) receptor complex consisting of syn-
taxin, SNAP-25, and synaptobrevin. Using mice lacking
both isoforms of the calcium-dependent activator protein
for secretion (CAPS), we show that LDCV priming in ad-
renal chromaffin cells entails two distinct steps. CAPS is re-
quired for priming of the readily releasable LDCV pool and
riming of large dense-core vesicles (LDCVs) is
a Ca2+-dependent step by which LDCVs enter a
release-ready pool, involving the formation of the
sustained secretion in the continued presence of high Ca2+
concentrations. Either CAPS1 or CAPS2 can rescue se-
cretion in cells lacking both CAPS isoforms. Furthermore,
the deficit in the readily releasable LDCV pool resulting
from CAPS deletion is reversed by a constitutively open
form of syntaxin but not by Munc13-1, a priming protein
that facilitates the conversion of syntaxin to the open con-
formation. Our data indicate that CAPS functions down-
stream of Munc13s but also interacts functionally with
Munc13s in the LDCV-priming process.
Two distinct secretory vesicle–priming steps in
adrenal chromaffin cells
Yuanyuan Liu,1 Claudia Schirra,1 Ludwig Edelmann,2 Ulf Matti,1 JeongSeop Rhee,4 Detlef Hof,1 Dieter Bruns,1
Nils Brose,4 Heiko Rieger,3 David R. Stevens,1 and Jens Rettig1
1Institut für Physiologie and 2Institut für Molekulare Zellbiologie, Universität des Saarlandes, 66421 Homburg, Germany
3Institut für Theoretische Physik, Universität des Saarlandes, 66123 Saarbrücken, Germany
4Max-Planck-Institut für Experimentelle Medizin, Abteilung Molekulare Neurobiologie, 37075 Göttingen, Germany
© 2010 Liu et al. This article is distributed under the terms of an Attribution–Noncommercial–
Share Alike–No Mirror Sites license for the first six months after the publication date (see
http://www.rupress.org/terms). After six months it is available under a Creative Commons
License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at
http://creativecommons.org/licenses/by-nc-sa/3.0/).
T H E J O U R N A L O F C E L L B I O L O G Y

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JCB • VOLUME 190 • NUMBER 6 • 2010 1068
loss of CAPSs, indicating that CAPS function may indeed be
similar to that of Munc13s. However, expression of Munc13-1
does not enhance priming in the absence of CAPS1, although its
expression in the presence of CAPS1 leads to the expected en-
hancement of secretion. These results indicate that LDCV priming
in mouse chromaffin cells involves the opening of the protein
syntaxin and that opening of syntaxin is facilitated by CAPS.
Both CAPS isoforms enhance priming activity and preferen-
tially prime LDCVs of the RRP.
Results
CAPS2 restores secretion to wild-type
levels in CAPS DKO chromaffin cells
Deletion of both CAPS genes in mouse chromaffin cells (CAPS
DKO) causes an 50% decrease in exocytosis as a result of a
strong (>50%) decrease in the size of the RRP and almost com-
plete block of sustained release (Liu et al., 2008). We stimulated
catecholamine secretion from mouse chromaffin cells using flash
photolysis of caged calcium to examine the ability of virally ex-
pressed CAPS2 protein to restore secretion in mouse chromaffin
cells lacking both CAPS1 and CAPS2. After UV flash illumina-
tion, secretion (as measured by a membrane capacitance change)
domain (Basu et al., 2005). Interestingly, an MUN domain–like
structure has also been identified in CAPS (Koch et al., 2000;
Hammarlund et al., 2008). In light of the role of the MUN do-
main in priming by Munc13s and the accumulating evidence
that CAPSs also promote priming (Stevens and Rettig, 2009), an
attractive hypothesis is that CAPSs carry out their priming func-
tion in a fashion similar to that of Munc-13s. Munc13-1 functions
by binding to syntaxin (Betz et al., 1997; Richmond et al., 2001),
promoting a conformational change in syntaxin that allows it to
engage in SNARE complex formation (Dulubova et al., 1999).
In mouse chromaffin cells, deletion of CAPSs (double
knockout [KO; DKO]) causes a reduction of the releasable LDCV
pool and of sustained release (Liu et al., 2008), the latter of which
occurs in the continued presence of elevated Ca2+ because vesi-
cles that are primed are immediately released and, thus, is an in-
dicator of vesicle priming. In addition, the loss of CAPS1 results
in reduced transmitter loading into chromaffin granules (Speidel
et al., 2005). Expression of CAPS1 in the KO background re-
stores normal transmitter loading (Speidel et al., 2005) and secre-
tion, increases the readily releasable LDCV pool, and enhances
sustained release (Liu et al., 2008).
In this study, we show that both CAPS1 and CAPS2 facili-
tate LDCV priming and that open syntaxin can overcome the
Figure 1. CAPS2 restores secretion to wild-type levels in CAPS DKO cells. (A) Responses to flash photolysis of caged calcium in CAPS DKO cells (n = 19)
and CAPS DKO cells expressing CAPS2 protein (n = 24). Upon elevation of intracellular calcium (top), the resulting change in membrane capacitance
(middle) shows a clear enhancement in those cells expressing CAPS2. Carbon fiber amperometry verifies that the observed increase in capacitance is
caused by an increase in (bottom) catecholamine release. (B) Analyses of the kinetics of the capacitance traces yield estimates of the releasable pools and
the sustained release rate. The RRP was strongly enhanced by CAPS2 expression (***, P < 0.001), whereas the SRP was unaffected. The rate of sustained
release in the period in which calcium remained elevated was also enhanced after CAPS2 expression (P < 0.001). (C) CAPS1 enhances secretion in wild-
type (WT) cells. Overexpression of CAPS1 in wild-type chromaffin cells (n = 26) results in a modest enhancement of secretion as compared with untreated
cells (n = 24). (D) A modest strengthening of the RRP of vesicles did not reach statistical significance. (E) CAPS2 expression enhances the exocytotic burst
in wild-type mouse chromaffin cells. Responses to a calcium stimulus induced by flash photolysis of caged calcium (top) in CAPS2-expressing cells (n = 31)
and wild-type chromaffin cells (n = 33) indicate that secretion is enhanced (middle), and this increase is mirrored by an increase in catecholamine release
as indicated by amperometric detection (bottom). (F) There was a significant increase in the RRP (**, P < 0.01) as compared with that of untreated wild-type
cells, with no difference in the SRP and a reduction in the sustained rate of release (P < 0.01). Error bars indicate mean ± SEM.

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1069CAPS primes to the readily releasable pool • Liu et al.
60.8 fF and 60.7 fF in CAPS1-expressing wild-type cells and
control cells, respectively. The sustained release rate was slightly
greater in CAPS1-expressing cells (26.1 ± 5.2 fF) than in con-
trol cells (19.1 ± 2.4 fF; Fig. 1 D).
We also tested whether overexpression of CAPS2 in wild-
type cells would have a similar effect as CAPS1 on secretion.
As shown in Fig. 1 E, introduction of CAPS2 in wild-type chromaf-
fin cells resulted in a comparable, selective increase in the RRP.
Although the RRP was enhanced (CAPS2 expression, 147.3 ±
18.4 fF [n = 31]; vs. control cells, 85.1 ± 8.1fF [n = 33]), the
SRP was unchanged (76.2 ± 8.3 vs. 76.4 ± 10.0 fF), and the sus-
tained release slightly decreased (27.1 ± 3.0 vs. 16.5 ± 2.0 fF;
Fig.1, E and F). These results indicate that both CAPS1 and
CAPS2 overexpression promote priming into the RRP in wild-
type chromaffin cells.
Open syntaxin restores RRP in CAPS DKO
cells to wild-type levels
In subsequent experiments, we tested whether expression of an
open form of syntaxin (syntaxin1A L165A/E166A; Dulubova
et al., 1999) can reverse the secretion deficit in CAPS DKO chro-
maffin cells (Fig. 2 A). Expression of open syntaxin in chro-
maffin cells from CAPS DKO mice led to strongly enhanced
secretion (threefold increase) as compared with that of DKO
cells. This enhancement was accounted for by an approximately
threefold increase in the RRP size (open syntaxin–expressing
DKO cells, 129.2 ± 21.8 fF [n = 24]; vs. CAPS DKO cells, 43.5 ±
8.9 fF [n = 23]; Fig. 2 B). There was also a modest increase in the
SRP size in open syntaxin–expressing DKO cells (79.5 ± 11.3 fF)
relative to untreated CAPS DKO cells (DKO, 52.3 ± 8.9 fF),
but there was virtually no detectable sustained component in
either group of cells. Application of a second flash after a 2-min
occurred in an exocytotic burst consisting of an RRP and an
SRP followed by a sustained release phase (Fig. 1). Expression of
CAPS2 in chromaffin cells from CAPS DKO mice resulted in a
strong enhancement of the exocytotic burst and of sustained re-
lease as compared with DKO cells (Fig. 1 A). By fitting the exo-
cytotic burst as the sum of two exponentials and sustained release
as a linear phase, we estimated the size of the RRP and SRP,
their time constants, and the rate of sustained release. There was
a significant increase in the RRP of DKO cells expressing CAPS2
(125.3 ± 25.8 fF; n = 24) compared with that of untreated DKO
cells (42.8 ± 18.6 fF; n = 19; P < 0.001; Mann-Whitney U test)
but no change in the release time constant of the RRP (CAPS2,
23.5 ± 5.2 ms; vs. DKO, 23.2 ± 1.4 ms). The amplitude of the
SRP was not altered in CAPS2-expressing DKO cells (51.7 ±
12.2 fF; n = 24) when compared with DKO cells (51.8 ± 7.5 fF;
n = 19). Sustained release, which was not measurable in DKO
cells, was 19.2 ± 3.7 fF/s in the CAPS2-expressing DKO cells
(Fig. 1 B). Thus, as is the case for CAPS1, expression of CAPS2
in CAPS DKO cells restores secretion with a selective effect on
the RRP and the sustained release phase.
CAPS1 and CAPS2 overexpression
increase the RRP size in wild-type
chromaffin cells
We next tested how overexpression of CAPS1 in wild-type cells
affects secretion. The results of this experiment are shown in
Fig. 1 C. CAPS1 overexpression in wild-type chromaffin cells
led to a modest enhancement of the exocytotic burst and sus-
tained release after photolysis of caged Ca2+, neither of which
was statistically significant. The RRP after CAPS1 overexpres-
sion in wild-type cells (n = 24) was 105 ± 16.1 fF as compared
with 79 ± 13.8 fF in control cells (n = 26). The SRP sizes were
Figure 2. Open syntaxin restores secretion
in CAPS DKO cells to wild-type levels. (A) Re-
sponses to flash photolysis of caged calcium
in CAPS DKO cells (n = 23) and CAPS DKO
cells expressing open syntaxin (n = 24) show
that open syntaxin restores secretion. The burst
component of the capacitance response is
much greater in the open syntaxin–expressing
cells (middle), and the catecholamine release
in amperometric recordings is also strongly
enhanced (bottom). (B) Estimates of the releas-
able pools and the sustained rate indicate that
open syntaxin strongly enhances the RRP (***,
P < 0.001), although the SRP is also enhanced
(this difference was not significant). Note that
there is little sustained release after the burst.
(C) Examination of a second flash stimulation
to the same cells after a 2-min recovery period
shows that open syntaxin–treated CAPS DKO
cells recover poorly after flash stimulation.
(D) Pool analysis shows that open syntaxin–
expressing CAPS DKO cells recover more
poorly and do not exhibit a greater RRP after a
second flash. **, P < 0.01. Error bars indicate
mean ± SEM.

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JCB • VOLUME 190 • NUMBER 6 • 2010 1070
We tested the residual secretory capacity that remains
after the ramp stimulus by applying a flash 3 s after the end of
the ramp stimulus. The DKO cells secreted slightly more in re-
sponse to the postramp flash (131.0 ± 31.7 fF; n = 22) than did
the open syntaxin–expressing DKO cells (98.3 ± 24.9 fF; n = 23;
Fig. 3 B). The secretion induced by the flash was equivalent to
65% of the total secretion in the DKO cells and to 25% of
the total secretion in the open syntaxin–expressing DKO cells.
Thus, in the open syntaxin–expressing DKO cells, 75% of the
secretory capacity was released during the ramp stimulation,
whereas only 35% of the secretory capacity was released by the
ramp stimulation in the DKO cells. The kinetics of release after
the flash stimulation were slow and similar in both groups, indi-
cating that residual release originated from an SRP of vesicles.
Total secretion was much greater in the open syntaxin–expressing
DKO cells in these ramp stimulation experiments, as was the
case in the original flash experiments. Thus, open syntaxin by-
passes the requirement for CAPS and generates a large pool of
releasable vesicles.
Chromaffin cells from mutant mice that only express open
syntaxin are characterized by a large reduction in the number of
morphologically docked vesicles (Gerber et al., 2008). The lack
of sustained release and the apparent fast exhaustion of release
that we observed in chromaffin cells expressing open syntaxin
might therefore be because of a reduction in vesicle docking
rather than to effects on priming. To test this, we expressed open
syntaxin in wild-type cells (Fig. 4 A). Open syntaxin expression
led to an approximately twofold increase in the RRP size (209.3 ±
26.0 fF; n = 24) relative to untreated wild-type cells (95.8 ± 9.4 fF;
n = 22) and enhanced the SRP size, although this effect was not
statistically significant (open syntaxin–expressing wild-type
cells,146.6 ± 25.3 fF; vs. untreated wild-type cells, 101.7 ± 14 fF)
but reduced the sustained phase (open syntaxin–expressing wild-
type cells, 4.6 ± 2.1 fF/s; vs. untreated wild-type cells, 22.5 ±
3.8 fF; Fig. 4 B).
As in CAPS DKO cells, wild-type cells expressing open
syntaxin recovered from stimulation poorly, as illustrated by re-
ductions of all phases of release in the responses to a second flash
stimulation (Fig. 4 C). In the responses to a second flash, the RRP
size was 104.6 ± 18.9 fF in open syntaxin–expressing wild-type
cells (n = 20) versus 136.8 ± 21.5 fF in untreated wild-type cells
(n = 18). The SRP was 56.3 ± 10.5 fF in open syntaxin–expressing
wild-type cells versus 99.8 ± 13.0 fF in untreated wild-type cells.
Sustained release was 1.6 ± 1.0 fF/s in open syntaxin–expressing
cells and 11.4 ± 2.5 fF/s in untreated cells (Fig. 4 D). The reduc-
tion of sustained release is thus independent of CAPS function.
These experiments indicate that open syntaxin promotes priming
into the RRP but simultaneously reduces sustained release and
pool recovery, possibly because of a reduction in the number of
docked vesicles.
Open syntaxin expression leads to reduced
LDCV docking
We next analyzed the distribution of LDCVs in chromaffin cells
to determine whether open syntaxin causes a docking defect
such as the one reported for mutant mice expressing only open
syntaxin (Gerber et al., 2008). We compared the distributions of
recovery period resulted in a reduced open syntaxin response
with a small RRP and no sustained component, whereas in the
CAPS DKO cells, the response to the second flash was equiva-
lent to the first response (Fig. 2, C and D). These findings are
compatible with the view that the supply of primable vesicles is
limited in the open syntaxin–expressing cells.
Open syntaxin expression in CAPS DKO
cells leads to rapid exhaustion of release
Stimulation of mouse chromaffin cells with a slowly rising cal-
cium concentration (calcium ramp) leads to biphasic secretion,
the late phase of which is likely the result of priming during
the stimulus, i.e., analogous to the sustained release during
flash photolysis (Sørensen et al., 2002). In CAPS DKO cells,
the late phase of secretion is either very small or absent (Liu
et al., 2008). We examined the effects of ramp stimulation in
DKO cells in which open syntaxin was expressed. In agree-
ment with the data obtained by flash stimulation (Fig. 2), open
syntaxin–expressing DKO cells showed strongly enhanced se-
cretion (295.5 ± 38.9 fF; n = 29) as compared with untreated
DKO cells (72.2 ± 13.2 fF; n = 27; Fig. 3 A). Secretion during
ramp stimulation started slowly, so we used the second deriva-
tive of the capacitance trace to more accurately determine the
increase in slope at the beginning of the secretory phase.
The Ca2+ value at the time of a peak in the second derivative of the
smoothed capacitance trace (Schonn et al., 2008), taken as the
threshold Ca2+ concentration required for secretion, was 850 nM
in both DKO and open syntaxin–expressing DKO cells. Both
the response of DKO cells and that of the open syntaxin–
expressing DKO cells were sigmoid. In spite of the greater se-
cretion in the open syntaxin–expressing DKO cells (or perhaps
as a result of this), secretion of the open syntaxin–expressing
cells reached a plateau before the end of the stimulation. This
was not the case in the DKO cells, which secreted throughout
the stimulation.
Figure 3. The releasable pools in CAPS DKO cells expressing open syn-
taxin are rapidly exhausted. (A) The free calcium concentration (top)
and the capacitance change (bottom) are shown. Those cells expressing
open syntaxin exhibit very strong secretion (n = 29) compared with CAPS
DKO cells not expressing open syntaxin (n = 27). (B) To determine the
amount of secretion remaining after the calcium ramp stimulation, a flash
was applied 3 s after the ramp ended to increase calcium to high levels.
The residual secretion in the CAPS DKO cells was larger than that of the
DKO cells expressing open syntaxin and accounted for 65% of the total
secretion, whereas the flash response in the open syntaxin–expressing
CAPS DKO cells accounted for 25% of the total secretion. Error bars
indicate mean ± SEM.

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1071CAPS primes to the readily releasable pool • Liu et al.
expression of a full-length Munc13-1–GFP construct in CAPS
DKO cells failed to restore secretion (Fig. 6 A). Unexpectedly, all
phases of flash-induced secretion in DKO cells expressing
Munc13-1 (RRP, 31.1 ± 8.5 fF; SRP, 24.7 ± 5.6 fF; sustained,
1.9 ± 1.0 fF/s; n = 23) were reduced compared with untreated
DKO cells (RRP, 48.4 ± 12.8 fF; SRP, 58.1 ± 10.0 fF; sustained,
2.9 ± 1.6 fF/s; n = 22; Fig. 6 B). We also tested whether expres-
sion of a truncated construct of Munc13-1 containing the mini-
mal priming domain (Stevens et al., 2005) can rescue secretion in
CAPS DKO cells, but expression of this construct also failed to
restore secretion in CAPS DKO cells, although it enhanced secre-
tion in wild-type mouse chromaffin cells (unpublished data).
Munc13-1 enhances secretion only in the
presence of CAPS1
Munc13-1 expression strongly enhances secretion in wild-type
mouse chromaffin cells (Stevens et al., 2005) but reduces secre-
tion in CAPS DKO cells (Fig. 6, A and B). In view of this dis-
crepancy, we tested whether the lack of a positive Munc13-1
effect on secretion seen in CAPS DKO cells might be directly
related to the lack of CAPS. We first examined the ability
of Munc13-1 to enhance secretion in cells from CAPS1+/
CAPS2/ mice (Fig. 6 C). In these cells, Munc13-1 expression
(n = 25) enhanced all phases of secretion significantly when
compared with untreated cells (n = 23). For CAPS1+/ CAPS2/
cells expressing Munc13-1, the RRP was 281.5 ± 39.9 fF, the
SRP was 278.3 ± 44.0 fF, and the sustained release was 50.7 ±
7.2 fF/s. In untreated CAPS1+/ CAPS2/ cells, the RRP was
145.8 ± 19 fF, the SRP was 82.5 ± 10.0 fF, and the sustained re-
lease was 20.0 ± 3.3 fF/s (Fig. 6 D).
LDCVs in untreated chromaffin cells from wild-type (n = 21)
and CAPS DKO cells (n = 16) to those in wild-type cells after
expression of open syntaxin (n = 10) and in CAPS DKO after
expression of open syntaxin (n = 7). We determined the shortest
distance from the plasma membrane of all identifiable LDCVs
in chromaffin cells derived from wild-type or CAPS DKO em-
bryonic day (E) 18/postnatal day (P) 0 mice either with or with-
out expression of open syntaxin. Representative micrographs
are shown in Fig. 5 (A–D). The distributions of measured dis-
tances (Fig. 5 E) showed a clear reduction in the fraction of
LDCVs in close apposition to the membrane in open syntaxin–
overexpressing CAPS DKO (62% reduction) and wild-type cells
(77% reduction), as compared with untreated cells of CAPS
DKO and wild-type mice. We conclude from these data that the
reduction in sustained release by expression of open syntaxin is
the result of a reduction in the transport of LDCVs to the plasma
membrane, i.e., docking.
Munc13-1 does not restore secretion
in CAPS DKO cells
The results of expressing open syntaxin indicate that LDCV prim-
ing is facilitated by opening of syntaxin, thus enabling syntaxin
to engage in SNARE complex formation. The MHD domain of
CAPS may be involved in the conformational change of syntaxin
required for priming, as is believed to be the case for the MHD
domains of Munc13s (Basu et al., 2005; Madison et al., 2005;
Stevens et al., 2005). If this were indeed the case, secretion in
chromaffin cells from CAPS DKO mice should also be restored
by expression of Munc13-1. Surprisingly, but in agreement with
data in cultured hippocampal neurons (Jockusch et al., 2007),
Figure 4. Expression of open syntaxin in wild-
type cells enhances the RRP selectively. (A) Flash
photolysis of caged calcium produces a
larger burst of secretion in open syntaxin–
expressing wild-type (WT) cells (n = 24) when
compared with cells not expressing open syn-
taxin (n = 22). This is mirrored in an increase
in catecholamine release. (B) Analysis of the
kinetics of the releasable pools and the rate of
sustained release show that the RRP is strongly
enhanced (**, P < 0.01), whereas the SRP is
unaltered. The sustained release is reduced
(***, P < 0.001). (C) Examination of the
second flash response after a 2-min recovery
period shows a deficit in refilling pools emp-
tied by a flash stimulation in open syntaxin–
expressing wild-type cells. (D) Analysis of pools
in the responses to the second stimulation.
Error bars indicate mean ± SEM.