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INTRODUCTION |
Neuronal calcium sensor-1
(NCS-1),1 the mammalian
homologue of frequenin, belongs to the EF-hand family of
Ca2+-binding proteins (1, 2), which includes
recoverin/S-modulin, visinin, visinin-like proteins, neurocalcin, and
hippocalcin. Some members of this family, such as recoverin/S-modulin
and visinin, are expressed only in photoreceptor cells and are thought
to function in the control of visual transduction pathways, whereas the
functional roles of the other members are largely unknown. Most family
members are solely expressed in neuronal and neuroendocrine cells, but a frequenin homologue has been identified in yeast, suggesting a more
general role for this protein in mediating Ca2+ responses.
All members of the NCS family share four EF-hand motifs and a
myristoylated N terminus. For NCS-1, three of the four EF-hands are
functional Ca2+-binding motifs, whereas the EF-hand closest
to the N terminus (EF1) is non-functional (2). Biochemical and
structural analysis of recoverin have led to the calcium/myristoyl
switch model in which Ca2+ binding to the NCS
proteins may trigger their translocation from the cytosol to
intracellular membranes (3, 4). This model suggests that
Ca2+ binding to the NCS proteins induces a large
conformational change, resulting in the exposure of the myristoyl group
believed to allow membrane attachment. In addition, the movement of the
myristoyl group is thought to expose a hydrophobic pocket within the
protein that could then interact with target proteins (4). However, recent studies suggest that the calcium/myristoyl switch is not a
general feature of all members of this family, because the localization of NCS-1 was found to be independent of Ca2+, indicating
that the myristoyl group may be freely accessible in the absence of
Ca2+ binding to NCS-1 (5).
Overexpression of frequenin in Drosophila facilitates evoked
neurotransmission at the neuromuscular junction (6), and injection of
frequenin into Xenopus spinal neurons enhances both
spontaneous and evoked neurotransmission (7). NCS-1 is also present in adrenal chromaffin and PC12 neuroendocrine cells, where its
overexpression has been shown to increase Ca2+-regulated
exocytosis from dense-core granules (8), analogous to the effect
reported for Drosophila frequenin in enhancing synaptic vesicle exocytosis. However, the mechanism(s) by which NCS-1/frequenin regulates vesicular release is not known.
A wide array of binding partners has been identified for
NCS-1/frequenin, only some of which may be of physiological relevance. NCS-1/frequenin has been shown to activate membrane-bound guanylate cyclase and to inhibit rhodopsin kinase (6, 9). However, these in
vitro effects on photoreceptor proteins are unlikely to be
relevant to the function of NCS-1/frequenin in vivo in
neurons and neuroendocrine cells. NCS-1 also seems to activate various calmodulin targets, such as cyclic nucleotide phosphodiesterase, calcineurin, and nitric-oxide synthase (10, 11), and to modulate a
variety of ion channels such as voltage-gated Ca2+ channels
(12-15) and A-type K+ channels (16). Despite these
interactions, the binding partners for NCS-1 in vivo are not
known for certain, and thus the molecular downstream targets
mediating NCS-1 function remain to be determined.
Recently, yeast frequenin was demonstrated to genetically and
biochemically interact with, and regulate the activity of, the yeast
phosphatidylinositol 4-OH kinase (PI4K) Pik1 (17), which itself has
been shown to be essential for Golgi-to-cell surface vesicular
trafficking (18, 19). Similarly, the mammalian Pik1 homologue PI4K
(20) has been shown to interact with NCS-1 in vitro and if
immunoprecipitated upon co-expression in non-neuronal cells (21, 22),
suggesting that PI4K
may be an evolutionarily conserved and
functionally important downstream target of NCS-1/frequenin.
Inositol phospholipids have recently emerged as important regulators of
exo- and endocytosis (23, 24). Regulated exocytosis of dense core
vesicles has been shown to require a phosphatidylinositol transfer
protein (PITP
) (25) and a PI(4)P-5-kinase (26). In addition,
the presence of a dense core vesicle-associated PI4K activity essential
for exocytosis (27) has led to the hypothesis that generation of
PtdIns(4,5)P2 at the plasma membrane (28) is an
important step in secretion. PtdIns(4,5)P2 is a substrate for phospholipase C (PLC) activated by G-protein-coupled receptors, and
changes in PtdIns(4,5)P2 levels are thus anticipated to
lead to changes in secretion evoked by G-protein-coupled receptor
agonists. However, in both neuronal and neuroendocrine cells,
PtdIns(4,5)P2 has been shown to play an important role in
exocytosis independent from its PLC-mediated cleavage (29, 30), and
PtdIns(4,5)P2 is likely to act directly on a specific
target (31).
In the present study, we provide evidence that NCS-1 enhances secretion
from PC12 cells through regulating the activity of PI4K. Overexpression
of NCS-1, but not of its myristoylation mutant, led to an enhancement
of ATP- and ionomycin-evoked release. NCS-1 expression also decreased
the sensitivity of the cells to phenylarsine oxide (PAO), an
inhibitor of PI4K, and increased the levels of PtdIns(4,5)P2 at the plasma membrane. Finally, three
mutations in NCS-1 equivalent to those that interfere with the binding
of recoverin to rhodopsin kinase had lost their enhancing effect on
exocytosis, suggesting that NCS-1 acts through activating PI4K, leading
to enhanced PtdIns(4,5)P2 levels and a downstream
enhancement of regulated secretion.
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EXPERIMENTAL PROCEDURES |
Construction of Expression Vectors--
The NCS-1 construct was
generated as described previously (32). The PI4K
, PI4K
(D656A),
and PI4K
N terminus-GFP constructs were kindly provided by Rachel
Meyers and have been described previously (33). The INP51-GFP and
PH-EGFP/PH(R40L)-EGFP constructs were donated by John York and Tamas
Balla, respectively.
PCMV5-hGH was generated by subcloning the coding region of hGH,
amplified by PCR and incorporating 5' EcoRI and 3'
XbaI sites, into the equivalent sites of pCMV5. NCS-1
mutants (G2A, E120Q, F22A, E26A, F55A, T92A) were generated using a
QuikChange site-directed mutagenesis kit (Stratagene) according to the
manufacturer's instructions. The identity of all constructs was
verified using automated DNA sequencing.
PC12 Cell Culture, Transfection, and Secretion
Experiments--
Cell culture, transfection, and secretion experiments
were performed as described (34) with some modifications. PC12 cells (Riken Cell Bank) were cultured on 100-mm dishes coated with
collagen (rat tail collagen type I; BD Biosciences). Cells were
grown at 37 °C in 5% CO2 in full medium (RPMI 1640 with
heat-inactivated 10% horse serum and 5% fetal bovine serum,
penicillin (50 units/ml), and streptomycin (50 units/ml)). Confluent
cells (passage 6 throughout all experiments) were harvested using
0.125% trypsin, 0.5 mM EDTA in PBS without
Ca2+ and Mg2+ and plated onto collagen-coated
12-well dishes at 80% confluency. The next day (90-95% confluency),
0.6 µg of pCMV5-hGH and 0.6 µg of plasmid of interest were
co-transfected using 4 µl of LipofectAMINE 2000 (Invitrogen)
according to the manufacturer's specification in Dulbecco's modified
Eagle's medium without serum and antibiotics to increase transfection
efficiency. Each LipofectAMINE 2000 tube was used only once, as
transfection and co-transfection efficiencies decreased when tubes were
re-used multiple times. After 4 h, the transfection mixture was
replaced by full medium. PC12 cells from two wells transfected with the
same plasmids were harvested and pooled the next day to compensate for
possible slight differences in transfection and co-transfection
efficiencies. Cells were re-plated into 24-well plates at a ratio of
two 12-wells for four 24-wells. Two days after replating (90-95%
confluency), secretion experiments were performed with all test and
control conditions carried out on the same pool of transfected cells.
Controls were treated with 0.5 ml of physiological saline solution
(PSS; 145 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl2, 0.5 mM MgCl2,
5.6 mM glucose, 15 mM Hepes-NaOH, pH 7.4).
ATP-evoked secretion was stimulated by a 10 min incubation with ATP
buffer (PSS containing 300 µM ATP), KCl-triggered
secretion was stimulated by a 10-min incubation with high
K+ saline solution (PSS containing 95 mM NaCl
and 56 mM KCl), and ionomycin-triggered secretion was
stimulated by a 10-min incubation with ionomycin (5 µM
ionomycin (Sigma) in PSS, final Me2SO concentration 0.5% (v/v)). Cells were incubated with
-latrotoxin (final 1.5 nM) in PSS for 10 min. At the end of the experiment, dishes
were transferred to ice, and the supernatant was removed and
centrifuged for 10 min in an Eppendorf centrifuge. hGH in the
supernatant from this centrifugation was taken as secreted hGH. The
cells in the dishes were resuspended in 0.5 ml of ice-cold
phosphate-buffered saline containing 1 mM EDTA and added to
the pellet of the Eppendorf centrifugation of the medium. Cells were
then lysed by five freeze-thaw cycles (in a dry ice/ethanol bath and a
37 °C heating block), and insoluble material was pelleted in an
Eppendorf centrifuge. hGH in the supernatant from this centrifugation
was taken as the cellular hGH that was not secreted. hGH levels in the
various samples were measured using an enzyme-linked immunosorbent
assay kit (Roche Molecular Biochemicals) according to the
manufacturer's instructions. All experiments were performed in
duplicates or triplicates, and the average percent of total hGH
released was calculated. Statistical analyses were performed with the
paired Student's t test.
Western Blot Analysis of Overexpressed Proteins in PC12 and COS-7
Cells--
PC12 cells were plated onto collagen-coated 6-well tissue
culture plates at 80% confluency as described above. COS-7 cells were
grown at 37 °C in 5% CO2 in COS cell medium
(Dulbecco's modified Eagle's medium with 10% fetal bovine serum,
penicillin (50 units/ml), and streptomycin (50 units/ml)) on 100-mm
dishes. The next day (90-95% confluency), cells were transfected with
the appropriate vector using 2.4 µg of vector/well of a 6-well plate
for PC12 cells and 10 µg of vector/100-mm dish for COS-7 cells,
respectively, using LipofectAMINE 2000 (Invitrogen) as described above.
One day later, cells were lysed using 500 µl of hot 1% SDS, and cell lysates were centrifuged at 12,000 × g for 10 min.
Protein concentration in the supernatant was determined by BCA assay
(Pierce). Equal amounts of total protein were separated by SDS-PAGE and
transferred to Hybond-N membranes (Amersham Biosciences).
Membranes were blocked with 2% non-fat dry milk in TTBS (0.05% Tween
20, 150 mM NaCl, 20 mM Tris, pH 7.5) for 1 h at room temperature and hybridized with primary antibody (either
anti-NCS-1 rabbit polyclonal or anti-PI4K
rabbit polyclonal,
respectively, at 1:1000 dilution) overnight at 4 °C. After washing
in TTBS, blots were incubated with a horseradish peroxidase-tagged
anti-rabbit antibody (DAKO A/S) at 1:2000 dilution for 1 h
at room temperature, washed, and developed using an enhanced
chemiluminescence system (Amersham Biosciences).
Inhibition of PI4K Activity--
Cells were co-transfected with
either pCMV5-hGH and pcDNA3.1 or with pCMV5-hGH and
pcDNA3.1-NCS-1, as described above. Cells were incubated in RPMI
1640 medium containing various concentrations of phenylarsine oxide
(Sigma) (final Me2SO concentration in all conditions
maintained at 0.5% (v/v)) for 15 min at 37 °C in 5% CO2, followed by secretion assays as described above.
Immunofluorescence--
To visualize localization of endogenous
NCS-1 and PI4K
, non-transfected cells were plated onto coverslips
coated with poly-L-lysine (coated for 1 h with 50 µg/ml poly-L-lysine, Mr
30,000-70,000 (Sigma)) and grown for 3 days in either full medium
(non-differentiated) or in serum-reduced (1%) medium containing 50 ng/ml NGF 2.5 S (Invitrogen). Cells were fixed for 20 min at 37 °C
with 4% (w/v) paraformaldehyde in PBS containing 4% (w/v)
sucrose. Cells were permeabilized in 0.5% Triton X-100 in PBS for 30 min and preincubated in blocking buffer (10% goat serum (Vector
Laboratories) in 0.5% Triton X-100 in PBS) for 1 h at room
temperature, followed by exposure to primary antibody (diluted in
blocking buffer) for 1 h at room temperature. Cells were washed in
0.5% Triton X-100 in PBS for 30 min and incubated with secondary
antibodies for 1 h at room temperature.
To determine the amount of overexpression of NCS-1, as well as the
amount of co-expression of NCS-1 and hGH, 50% of cells were processed
for secretion assays, whereas the remainder were replated onto
poly-L-lysine-coated coverslips and fixed and processed for
immunocytochemistry 2 days after replating. The amount of overexpressed
protein was qualitatively assessed using antibody dilutions that barely
visualized endogenous protein levels but easily detected overexpressed
proteins. The amount of co-expression was calculated by counting the
number of cells expressing both hGH and the protein of interest, as
compared with cells expressing either one or the other protein only.
Transfection efficiencies were calculated by counting the number of
cells expressing either proteins, and 700-1200 cells were counted for
each construct. Transfection efficiencies regularly amounted to around
15%, and all NCS-1 constructs reached co-transfection efficiencies of
90%.
The following antibodies were used: anti-NCS-1 rabbit polyclonal
(44162) (35) or chicken polyclonal (22), anti-PI4K
rabbit polyclonal
(Upstate Biotechnology), anti-synaptobrevin/VAMP2 mouse monoclonal (Cl 69.1; Synaptic Systems), anti-syntaxin 1 mouse monoclonal (HPC-1; Sigma), and anti-hGH rabbit polyclonal (National Hormone and Peptide Program, NIDDK, National Institutes of Health). The
specificities of the polyclonal anti-NCS-1 and anti-PI4K
antibodies
have been described previously (15, 33). Secondary antibodies included
horse anti-mouse IgG conjugated to fluorescein isothiocyanate or Texas
red (TR), goat anti-rabbit IgG conjugated to fluorescein isothiocyanate
or TR (all from Vector Laboratories) and rabbit anti-chicken IgG
conjugated to fluorescein isothiocyanate (Sigma). Cells were either
examined on a confocal microscope (Leica TCS NT) under a ×40
oil-immersion objective, and data were acquired using Leica software or
on an upright microscope (Leica) under a ×40 or ×100 oil-immersion
objective, and data were acquired using IPLab.
PH Domain Imaging and Analysis--
Cells were co-transfected
with NCS-1 or NCS-1(G2A) and either phospholipase C-
PH domain-GFP
construct (PH-EGFP) or PH(R40L)-EGFP, a mutant unable to bind
PtdIns(4,5)P2 (36). Cells were either visualized live or
after fixation and permeabilization as described above, 2 days after
transfection, with identical results. For experiments measuring PH-EGFP
intensities upon PAO treatment, cells were fixed but not permeabilized.
To quantify intensities, rectangles of 80 × 110 pixels centered
on each GFP-positive, well separated cell were collected from each
random field acquired using a ×40 oil-immersion objective. Pixel
intensities within each rectangle were calculated in IPLab, and a total
of 100 cells were counted for each condition. Cells with the highest
PH-EGFP expression levels were excluded, as they often gained a rounded appearance and formed membrane blebs (37). Experiments were done three
times, and the data were analyzed using the paired Student's
t test.
Electroporation of Cells, Endogenous Lipid Phosphorylation, and
Thin Layer Chromatography--
PC12 cells were resuspended in RPMI
1640 medium (3 × 107 cells/ml). Cells (400 µl) were
incubated with 15 µg of pcDCNA3.1-NCS-1, 15 µg of pcDNA3.1, or
with 15 µg of pCMV5-GFP for 3 min and electroporated (330 V, 20 ms)
using an ECM 830 Square Wave Electroporator (BTX; Genetronics Inc., San
Diego, CA). Electroporated cells were immediately transferred into 10 ml of full medium, washed once, and counted. Under the indicated
electroporation conditions, cell survival usually amounted to around
10-15%, and electroporation efficiency, as measured by counting the
fraction of cells expressing GFP 2 days after electroporation, amounted
to around 50%. Cells were plated onto collagen-coated 100-mm dishes at
5 × 106 cells/dish. Two days later (95% confluency),
attached cells were harvested, washed into PBS, and counted, and
0.5 × 106 cells were pelleted in Eppendorf tubes.
Pellets were quick-frozen in liquid N2 until further use.
Cells were resuspended in 100 µl of kinase buffer (20 mM
Hepes/NaOH, pH 7.5, 100 mM NaCl, 10 mM
MgCl2) and disrupted by passing five times through a
17-gauge needle syringe. The aliquot of the prepared lysate was used
directly in an assay, or the resulting lysate was further fractionated
by centrifugation at 10,000 × g for 15 min at 4 °C.
The supernatant of this centrifugation was taken as cytosol fraction,
and the pellet was resuspended in kinase buffer and taken as membrane
fraction. PI4K activity was measured as described previously
(61, 62). Briefly, to initiate phospholipid biosynthesis, cell
lysate and membrane fractions were diluted with kinase buffer to 45 µl, and 5 µl of 0.5 mM ATP containing 10 µCi of
[
-32P]ATP (3000-6000 Ci/mmol; PerkinElmer Life
Sciences) was added. The reaction was incubated for 10 min at room
temperature and stopped by adding 60 µl of 1 N HCl. A
two-phase mixture was induced, and the phosphorylated lipids were
extracted by adding 160 µl of chloroform:methanol, 1:1 (v/v). The
extracted products were analyzed by thin layer chromatography (TLC)
(silica gel 60 thin layer plates, using chloroform/methanol/15
N ammonium hydroxide/water (90:90:7:22) (v/v/v/v) as the
solvent system). Radiolabeled PtdIns(4)P was identified by TLC mobility
in comparison with purified PtdIns(4)P standard.
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RESULTS |
Localization of Endogenous NCS-1 and PI4K
in PC12 Cells--
To
identify the subcellular localization of NCS-1 and PI4K
in PC12
cells, antibodies against NCS-1, PI4K
, VAMP2, and syntaxin 1 were
used to stain non-transfected PC12 cells. In both non-differentiated and NGF-differentiated cells, endogenous NCS-1 was mainly diffuse and
cytosolic, with some additional perinuclear staining (Fig. 1, A and B).
PI4K
staining was observed in the cytosol and in a perinuclear area,
as described for chromaffin cells (38), and upon NGF differentiation,
PI4K
staining was enriched in processes, consistent with its
reported localization on secretory vesicles (27, 39) (Fig. 1,
C and D). In comparison, VAMP2 staining was
primarily observed in a perinuclear area in non-differentiated cells
and was significantly enriched in processes in differentiated cells, in
accordance with its localization on secretory vesicles of PC12 cells
(40, 41) (Fig. 1, E and F). Syntaxin 1 was
localized to the plasma membrane, with more intracellular staining
observed upon NGF differentiation, suggesting that under conditions of intense membrane fusion, as occurs during neurite formation, syntaxin 1 undergoes recycling in differentiating PC12 cells (42) (Fig. 1,
G and H). In all cases, no immunostaining was
observed when incubation with primary antibodies was omitted (not
shown). The comparative subcellular localization data suggest similar
subcellular distributions for NCS-1 and PI4K
, with both proteins
being partially cytosolic and partially associated with perinuclear
structures.

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Fig. 1.
Localization of endogenous NCS-1 and
PI4K in PC12 cells. Non-differentiated
and NGF-differentiated cells were stained as follows: A and
B, NCS-1 (chicken anti-NCS-1, 1:50); C and
D, PI4K (rabbit anti-PI4K , 1:50); E and
F, VAMP2 (mouse anti-VAMP2, 1:1000); or G and
H, syntaxin 1 (mouse anti-syntaxin 1, 1:100). Scale
bars, 10 µm.
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NCS-1 Enhances ATP-evoked Exocytosis from PC12 Cells--
To
study the effects of NCS-1 and PI4K
on exocytosis, we employed an
assay developed by Holz and co-workers (43) in which plasmids encoding
hGH and a second protein of interest are transiently co-transfected
into PC12 cells. In transfected cells, hGH is packaged into vesicles of
the regulated secretory pathway and serves as a reporter for exocytosis
as a function of stimulation. A high probability of co-transfection of
two distinct plasmids into the same cells makes it possible to
investigate the effect of the protein of interest on hGH secretion. A
change in hGH secretion upon co-transfection of a particular protein is
taken as evidence for an involvement of the protein in exocytosis. This
assay has been successfully used to implicate a variety of proteins in
exocytosis (34, 44-46) and thus serves as a useful model system to
study the role of NCS-1 and PI4K
in secretion.
Extracellular ATP is thought to evoke secretion from PC12 cells by
binding to two purinergic receptors: a P2Y receptor, which leads to the
activation of PLC and concomitant inositol 1,4,5-trisphosphate-mediated release of Ca2+ from intracellular stores, and a P2X
non-selective cation channel, which leads to Ca2+ entry
across the plasma membrane (47, 48). ATP caused robust hGH secretion
from PC12 cells co-transfected with control vector, which was enhanced
when cells were co-transfected with NCS-1 (Fig. 2A), as reported previously
(8). In the absence of secretagogue, the amount of hGH in the PC12 cell
medium (~10%) did not increase upon NCS-1 transfection, indicating
that NCS-1 does not induce a significant amount of constitutive
secretion of hGH (Fig. 2A). Expression of NCS-1 did not
alter the expression levels of hGH (Fig. 2B), indicating
that the enhancement of exocytosis observed with NCS-1 is not because
of a change in the relative amounts of expressed hGH. NCS-1 enhanced
ATP-stimulated secretion by 31 ± 8% (n = 9, p < 0.01; see Fig. 2C). To test the
functional relevance of the myristoyl group in NCS-1, cells were
co-transfected with hGH and NCS-1(G2A), a point mutation that renders
the myristoylation consensus sequence non-functional. This mutant was
unable to enhance ATP-evoked exocytosis (3.6 ± 9.5% enhancement;
n = 6; see Fig. 2C). In addition, a mutant
in EF-hand EF3 (NCS-1(E120Q)) that has been shown to have impaired
Ca2+-binding activity (12) still enhanced ATP-evoked
secretion, albeit to a lesser extent as compared with wild-type NCS-1
(15.8 ± 1.9% enhancement; n = 5;
p < 0.01; see Fig. 2C).

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Fig. 2.
NCS-1 enhances ATP-evoked secretion from PC12
cells. Cells were co-transfected in parallel either with pCMV5-hGH
and empty control vector (pcDNA3) or with pCMV5-hGH and various
test plasmids. hGH secretion was stimulated for 10 min with either
control physiological solution (ctrl) or 300 µM ATP in physiological solution. The amount of hGH in
the medium and in the cells was determined by enzyme-linked
immunosorbent assay, and the percentage of secreted hGH and the total
amount of hGH were calculated against an hGH standard curve.
A and B, representative experiment comparing
effects of control and NCS-1 vectors on hGH secretion and total amounts
of hGH in cells co-transfected with pCMV5-hGH. C, to
standardize results from the same test plasmids from repeated
experiments, secretion observed in the control vector transfections was
set to 100% for all experiments, and relative enhancement or
inhibition of secretion of test plasmids was normalized to the control
(normalized enhancement of release). Values shown represent means ± S.E. from multiple (n = 2-9) experiments.
Statistically significant differences are marked by asterisks (*,
p < 0.15; **, p < 0.01).
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Double immunocytochemistry was performed on cells processed in parallel
for secretion assays, using an anti-hGH antibody and an anti-NCS-1
antibody at a dilution that barely visualized endogenous protein but
easily recognized overexpressed protein. For both mutant and wild-type
NCS-1 constructs, transfection efficiencies were around 15%, and
co-transfection efficiencies were comparable and
90% (data not
shown). In addition, the level of overexpression of the different NCS-1
constructs was analyzed using both Western blotting and
immunocytochemistry (Fig. 3, A
and B). As assessed by immunocytochemistry and antibody
concentrations that barely visualized endogenous protein, all three
NCS-1 constructs were heavily overexpressed as compared with endogenous
NCS-1 (8-10-fold), and no partial degradation of mutant NCS-1
constructs was detected by Western blotting (Fig. 3, A and
B), indicating that the relative or total lack of an effect
of the NCS-1 mutants on ATP-evoked secretion is not because of a
difference in co-transfection efficiencies, expression levels, or
partial degradation of expressed proteins. Overexpressed NCS-1 was
found partially cytosolic and partially bound to the plasma membrane
(Fig. 3, A and B). The myristoylation mutant
NCS-1(G2A) was largely cytosolic (Fig. 3B), supporting the
notion that the myristoyl anchor is required for membrane attachment.
The Ca2+-binding mutant NCS-1(E120Q) was
membrane-associated like wild-type NCS-1 (Fig. 3B), in
agreement with the observation that the localization of NCS-1 is
independent of Ca2+ (5).

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Fig. 3.
Localization and amounts of overexpressed
NCS-1 and PI4K . A, extracts (60 µg) of
PC12 cells transfected with various NCS-1 constructs were analyzed by
Western blotting. B, overexpression and localization of
NCS-1 constructs in NGF-differentiated cells was assessed by
immunocytochemistry using anti-NCS-1 chicken polyclonal (22) at a 1:500
dilution to preferentially visualize NCS-1-overexpressing cells.
C, extracts (60 µg) of PC12 or COS-7 cells transfected
with various PI4K constructs were analyzed by Western blotting.
D, expression of GFP-tagged N-terminal PI4K (PI4KN-term.)
and GFP-tagged, palmitoylated/myristoylated
5'-PtdIns(4,5)P2-phosphatase (INP51) was visualized in live
cells. Scale bar, 10 µm.
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Next, we tested whether co-expression of PI4K
, or mutants thereof,
affect ATP-evoked hGH secretion. Neither wild-type PI4K
, nor
PI4K
(D656A), a catalytically inactive mutant (33), nor a GFP-tagged
N-terminal fragment (PI4KN-term.) changed ATP-evoked secretion, as
compared with empty control vector (Fig. 2C).
Immunocytochemistry with an anti-PI4K
antibody (data not shown), as
well as Western blot analysis, revealed that none of the three PI4K
constructs were significantly expressed above endogenous protein
levels, even though they could be overexpressed in COS-7 cells (Fig.
3C). Although not detectable above endogenous PI4K
levels, expression of the GFP-tagged N-terminal fragment (PI4KN-term.)
could be demonstrated (Fig. 3D). Finally, decreasing
PtdIns(4,5)P2 levels presumably at the plasma membrane by
co-expression of a GFP-tagged 5'-PtdIns(4,5)P2-phosphatase (INP51) predominantly plasma membrane-targeted by an N-terminal myristoylation/palmitoylation sequence (37) (Fig. 3D)
significantly inhibited ATP-stimulated hGH secretion (24.9 ± 2.8% decrease; n = 2; p < 0.15; see
Fig. 2C), indicating that PtdIns(4,5)P2 levels are important for regulated secretion. Thus, the lack of an effect of
PI4K
on ATP-evoked secretion may either be because of the endogenous
enzyme not being present in rate-limiting concentrations or because of
a lack of efficient overexpression of exogenous PI4K
in this cell system.
Differential Effect of NCS-1 on hGH Secretion Evoked by Different
Secretagogues--
As another means to address whether NCS-1 acts to
enhance secretion by activating PI4K
, we tested its effects on
Ca2+-dependent secretion stimulated by
secretagogues with distinct mechanisms of action. KCl at high
concentrations induces Ca2+ influx by membrane
depolarization, ionomycin acts as a Ca2+ ionophore, leading
to global increases in [Ca]i, and
-latrotoxin triggers exocytosis by an unknown mechanism. Both KCl
and ionomycin evoke secretion in a PLC-independent manner in PC12 cells
(49). Although ATP-evoked hGH secretion was enhanced upon NCS-1
transfection (23.8 ± 2.7% increase, n = 5, p < 0.005; see Fig. 4,
A and D), there was no effect on secretion when
cells were stimulated with KCl (3.2 ± 4.4% increase,
n = 5; see Fig. 4B) or
-latrotoxin (data
not shown). There was a slight, variable NCS-1-dependent
stimulation of hGH secretion induced by ionomycin (10 ± 6.7%;
p < 0.25, n = 5; see Fig. 4,
C and D).

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Fig. 4.
NCS-1 differentially modulates secretion
evoked by different secretagogues. Cells were co-transfected in
parallel either with pCMV5-hGH and empty control vector (pcDNA3) or
with pCMV5-hGH and NCS-1. Representative experiments in which hGH
secretion was stimulated for 10 min with control physiological saline
(ctrl) and with 300 µM ATP in physiological
saline (A), high K+ (56 mM KCl)
solution (B), or with 5 µM ionomycin in
physiological saline (C). D, secretion observed
in cells co-transfected with pCMV5-hGH and empty vector was set to
100%, and relative enhancement of secretion with NCS-1 was normalized
to control empty vector (normalized enhancement of release). Values
shown represent means ± S.E. from five experiments. Statistically
significant differences are marked by asterisks (*, p < 0.25; **, p < 0.005).
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If NCS-1 enhances secretion through activating PI4K
and
concomitantly increasing PtdIns(4,5)P2 levels, the
enhancement of ionomycin-stimulated release suggests that
PtdIns(4,5)P2 levels may be directly relevant for
exocytosis downstream from Ca2+ entry in a manner
independent of PLC. In that case, it seems unclear why KCl-evoked
release is not equally potentiated upon the expression of NCS-1.
However, overexpressed NCS-1 was found enriched at the plasma membrane
(Fig. 3) and thus may, at least in part, buffer depolarization-induced
Ca2+ influx, which may mask its effect in enhancing
exocytosis. Indeed, upon overexpression of NCS-1(E120Q), KCl-evoked
secretion was slightly enhanced (10.3 ± 4.0; p < 0.1, n = 2), suggesting that decreasing the
Ca2+ buffering activity of NCS-1(E120Q) at the plasma
membrane unveils the stimulatory activity of this partially functional mutant.
Overexpression of NCS-1 Leads to a Shift in the Dose-response Curve
Using Phenylarsine Oxide--
In chromaffin and PC12 cells,
phosphorylation of phosphatidylinositols by PI4K is required for
Ca2+-triggered exocytosis (25-27). In these cells, PI4K
can be potently inhibited by PAO, resulting in a block of exocytosis
(27, 50). Indeed, incubation of the cells with 3 µM PAO
for 15 min prior to triggering Ca2+-dependent
secretion with ATP resulted in a robust inhibition of hGH release in
both vector and NCS-1-transfected cells (Fig. 5A). These PAO concentrations
are similar to those reported to inhibit granule secretion and
PtdIns(4,5)P2 production from synaptosomes (50). Treatment
of the cells with PAO did not affect the total levels of hGH (data not
shown), suggesting that it specifically blocks
Ca2+-dependent secretion evoked by ATP, rather
than interfering with hGH expression. Further, PAO blocked exocytosis
in a dose-dependent manner in both control and
NCS-1-transfected cells. When the levels of hGH release in the absence
of PAO were normalized to 100%, inhibition of release was less
pronounced in cells expressing NCS-1 as compared with cells expressing
control vector (Fig. 5B). These data are consistent with our
proposal that NCS-1 expression leads to enhanced levels of active
PI4K
and thus to a concomitant shift in the PAO dose-response curve
of hGH secretion.

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Fig. 5.
NCS-1 expression causes a shift in the
dose-response curve using phenylarsine oxide. Cells were
co-transfected in parallel either with pCMV5-hGH and empty control
vector (pcDNA3) or with pCMV5-hGH and NCS-1. A,
experiment in which cells were incubated in medium containing either
Me2SO (final 0.5% (v/v)) or 3 µM PAO in
Me2SO (final 0.5% (v/v)) for 15 min before secretion
assays were carried out using either control physiological saline
(ctrl) or 300 µM ATP in physiological saline.
B, experiment in which cells were incubated with 0, 0.3, 1, or 3 µM PAO for 15 min before secretion assays. The
amount of secretion in the absence of PAO was set to 100% for both
control vector and NCS-1-transfected cells. Results are shown from a
single experiment performed in duplicate. Although the amount of
inhibition with specific PAO concentrations was variable between
experiments, the shift in the dose-response curve upon NCS-1
transfection was reproducible (n = 3 experiments, each
performed in duplicate).
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Increase in Plasma Membrane PtdIns(4,5)P2 Levels upon
NCS-1 Transfection--
As another means to measure enhanced PI4K
activity upon NCS-1 expression, we visualized plasma membrane
PtdIns(4,5)P2 levels using a PLC
PH domain-GFP (PH-EGFP)
construct. The PH domain of PLC
specifically binds
PtdIns(4,5)P2 and has been shown to be predominantly
associated with the plasma membrane (28, 37, 51). Indeed, PH-EGFP
specifically labeled the plasma membrane, and to a lesser extent the
cytosol, in live PC12 cells (data not shown). When cells were fixed and
permeabilized, cytoplasmic staining was minimal, and PH-EGFP
predominantly localized to the plasma membrane (Fig.
6A). The PLC
PH domain
contains three critical basic residues that interact with
PtdIns(4,5)P2, including Arg-40 (52). A mutated PH
domain construct, PH(R40L)-EGFP (28), did not label the plasma membrane
but was entirely cytosolic (Fig. 6A). These two constructs
were used in co-transfection experiments with either NCS-1 or the
non-functional NCS-1(G2A). The intensity of PH-EGFP staining in cells
co-transfected with NCS-1 was counted from 300 individual cells well
separated from each other and compared with the intensity of PH-EGFP
staining in cells co-transfected with NCS-1(G2A) or cells
co-transfected with empty control vector (pcDNA3) (Fig.
6B). NCS-1 expression led to a 25 ± 9.6% increase in
PH-EGFP staining as compared with control vector and a 53 ± 7.4%
increase as compared with NCS-1(G2A) (Fig. 6B). To exclude that the differences in PH-EGFP staining were because of differences in
residual cytosolic PH-EGFP intensities upon NCS-1 transfection, we used
the cytosolic PH(R40L)-EGFP as a control. NCS-1 expression did not lead
to enhanced PH(R40L)-EGFP staining, suggesting that the observed
increase in PH-EGFP staining is specific for
PtdIns[4,5]P2 levels at the plasma membrane imaged by
PH-EGFP. Co-expression of PH(R40L)-EGFP with either NCS-1 or NCS-1(G2A)
led to a slight decrease in the averaged fluorescence intensities as
compared with control empty vector (pcDNA3) (Fig. 6B),
indicative of a competition for the transcriptional/translational
machinery of the two co-expressed constructs. This competition was also
visible upon co-expression of PH-EGFP and NCS-1(G2A), suggesting that the direct comparison between effects of NCS-1 and NCS-1(G2A) is most
appropriate.

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Fig. 6.
Increase in plasma membrane
PtdIns(4,5)P2 levels upon NCS-1 transfection as imaged with
PH-EGFP. Cells were co-transfected in parallel with PH-EGFP and
NCS-1, PH-EGFP and NCS-1(G2A), PH(R40L)-EGFP and NCS-1, or
PH(R40L)-EGFP and NCS-1(G2A). Two days after transfection, cells were
fixed and permeabilized, and EGFP fluorescence was imaged. Fluorescence
intensities were quantified using rectangles of identical pixel size
centered on 100 EGFP-positive cells for each condition. A,
representative cells expressing NCS-1 and PH-EGFP (left) or
NCS-1 and PH(R40L)-EGFP (right). Scale bar, 10 µm. B, averaged arbitrary fluorescence intensities
(means ± S.E., n = 3) from a total of 300 cells
each co-expressing empty control vector (pcDNA3) or NCS-1 or
NCS-1(G2A) and either PH-EGFP or PH(R40L)-EGFP, respectively. *,
p < 0.05. C, distribution of cells
expressing identical plasma membrane-associated (PH-EGFP) or cytosolic
(PH(R40L)-EGFP) GFP fluorescence (binned in 0.25 arbitrary units).
Open squares and open diamonds, cells
co-transfected with NCS-1. Closed circles and closed
triangles, cells co-transfected with NCS-1(G2A).
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Because expression levels in transfection experiments are highly
variable, we also plotted PH-EGFP fluorescence intensities binned from
the whole population of analyzed cells (Fig. 6C). NCS-1
expression led to a decrease in the number of cells with low amounts of
plasma membrane PH-EGFP staining and a concomitant increase in the
number of cells with larger amounts of plasma membrane PH-EGFP staining
(Fig. 6C). These data suggest that NCS-1 overexpression
leads to a specific increase of PtdIns(4,5)P2 levels at the
plasma membrane, in agreement with its proposed role in activating
PI4K
.
PAO Treatment Leads to a Decrease in Plasma Membrane
PtdIns(4,5)P2 Levels--
To confirm that PAO indeed
inhibits the activity of PI4K, leading to a decrease of plasma membrane
PtdIns(4,5)P2 levels, cells were transfected with the
PH-EGFP construct before treatment with PAO. In live cells, PH-EGFP
labeling of the plasma membrane could still be observed, but cytosol
staining was increased (data not shown). The intensity of cytosolic
PH-EGFP staining in cells transfected with PH-EGFP and treated for 15 min with PAO prior to fixation was counted from 300 individual cells
and compared with the intensity of cytosolic PH-EGFP staining in
transfected cells treated for 15 min with Me2SO (Fig.
7). PAO treatment led to a decrease in the number of cells with small amounts of cytosolic PH-EGFP staining and a concomitant increase in the number of cells with larger amounts
of cytosolic PH-EGFP staining (Fig. 7A). However, the total
amount of PH-EGFP staining in both cytosol and plasma membrane was not
changed upon PAO treatment, indicating that the increase in PH-EGFP
staining in the cytosol was accompanied by a decrease in PH-EGFP
staining at the plasma membrane (Fig. 7, B and
C). Overall, treatment of cells with PAO resulted in a
45 ± 6% increase in cytosolic PH-EGFP staining as compared with
control Me2SO treatment, indicating that PAO treatment
results in a partial knock-off of PH-EGFP staining from the plasma
membrane because of a resultant decrease in plasma membrane
PtdIns(4,5)P2 levels (Fig. 7C).

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Fig. 7.
PAO treatment leads to decrease in plasma
membrane PtdIns(4,5)P2 levels. Cells were transfected
with PH-EGFP, and 2 days after transfection, cells were treated with 5 µM PAO or Me2SO vehicle alone for 15 min
prior to fixation. Fluorescence intensities were quantified using
rectangles of identical pixel size centered within (for cytosol) or
around (for total) 100 EGFP-positive cells for each condition.
Distribution of cells expressing identical cytosolic (A) or
total (B) PH-EGFP fluorescence (binned in 0.25 arbitrary
units) is shown. Open squares, cells treated with
Me2SO only. Closed circles, cells treated with 5 µM PAO in Me2SO. C, averaged
arbitrary fluorescence intensities (means ± S.E.,
n = 3) from a total of 300 cells expressing PH-EGFP in
either the absence or presence of PAO, measuring cytosolic
(left) and total (right) GFP fluorescence,
respectively. *, p < 0.001.
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Overexpression of NCS-1 Leads to Increased PtdIns(4)P
Production--
The effect of overexpressing NCS-1 on the
phosphorylation of endogenous phosphatidylinositol (61, 62) was
also examined in permeabilized PC12 cells. Expression of NCS-1 caused a
significant increase in 32P labeling of PtdIns(4)P and
PtdIns(4,5)P2 in cell lysates and membrane fractions, as
compared with cells transfected with empty control vector (Fig.
8). The NCS-1-dependent
increase in the labeling of PtdIns(4)P was abolished upon incubation
with 10 µM wortmannin (data not shown), indicating that
it was because of enhanced type III PI4K
activity (Fig. 8). The
substantial amount of radiolabeled PtdIns(4)P produced in the absence
of NCS-1 overexpression was likely because of the activity of a type II
enzyme, which is responsible for a large amount of PI4K activity in
membranes of mammalian cells, as it was not sensitive to wortmannin and
significantly stimulated with Triton X-100 (data not shown). These data
are in agreement with a recent report indicating that endogenous
PtdIns(4)P and PtdIns(4,5)P2 levels were increased in a
stable NCS-1-expressing cell line as compared with control cells
(63), which further validifies the present approach in
permeabilized cells to measure PI4K
-dependent changes in
PtdIns(4)P and PtdIns(4,5)P2 levels upon transient
overexpression of NCS-1.

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Fig. 8.
Effect of NCS-1 overexpression on
phosphorylation of endogenous lipid substrate in permeabilized PC12
cells. Cells were electroporated with either pcDNA3.1
(C) or NCS-1 (N) constructs. Two days later, cell
lysates or membrane fractions were analyzed for
[32P]phosphate incorporation into phospholipids from
[ -32P]ATP. A, representative TLC
analysis of radiolabeled PtdIns(4)P and PtdIns(4,5)P2
production of entire cell lysates or membrane fractions. B,
summary of quantitative data from several experiments
(n = 6 for cell lysate, n = 3 for
membrane fractions). *, p < 0.04. Phosphorylation of
lipids was quantified by phosphorimaging analysis (PhosphorImager;
Molecular Dynamics).
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Novel NCS-1 Mutations Defective in Enhancing
Ca2+-dependent Secretion--
NCS-1 is
structurally similar to recoverin (53, 54) and can functionally replace
the activating effect of recoverin on rhodopsin kinase in
vitro (55). The residues within the N-terminal region of recoverin
(Phe-22, Glu-26, Phe-55, Thr-92) implicated in making contacts with
rhodopsin kinase (56) are all conserved in NCS-1. Moreover, the
recently solved crystal structure of human NCS-1, which is identical in
sequence to the rat homologue, revealed that three of these residues
(Phe-22, Phe-55, Thr-92) are solvent-exposed and located in the wide
hydrophobic crevice at the surface of NCS-1, which interacts with its
protein ligand (54). The fourth, Glu-26, makes important side-chain
contacts with helix J, the C-terminal region of NCS-1 that lies
adjacent to the hydrophobic crevice and is thought to be important for
ligand recognition (54). To determine whether these residues are
essential for the function of NCS-1, we generated a series of novel
NCS-1 mutations and tested their effects on hGH secretion (Fig.
9). Mutation E26A, which may interfere
with the proper positioning of helix J, and mutations F22A and F55A,
which may alter the ability of the hydrophobic crevice of NCS-1 to
interact with its downstream target, abolished the ability of NCS-1 to
enhance regulated secretion (Fig. 9, A and B).
Mutation T92A was still functional in enhancing release, suggesting
that the effects of F22A and F55A are specific and that not all
solvent-exposed residues in the hydrophobic crevice interact with a
protein ligand. In addition, the localization, overexpression levels,
and co-transfection efficiencies of F22A, F55A, and E26A were
equivalent to those of wild-type NCS-1 (Fig. 9C) (data not
shown), and overexpressed mutant constructs were not degraded (Fig.
9C), indicating that their impaired ability to enhance
secretion is not because of mislocalization or lowered expression
levels. The in vivo effect of these mutants, together with
the structural information mentioned above, suggests that residues
Phe-22, Phe-55, and Glu-26 may function to allow protein ligand
binding, which may directly or indirectly lead to activation of
PI4K
.

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Fig. 9.
Three novel NCS-1 mutants defective in
enhancing Ca2+-dependent secretion.
A, representative experiment comparing effects of control
vector, NCS-1, and NCS-1 mutants on hGH secretion in cells
co-transfected with pCMV5-hGH. B, to standardize results
from the same plasmids from repeated experiments, secretion observed in
the control vector transfections was set to 100% for all experiments,
and relative enhancement of secretion of test plasmids was normalized
to the control (normalized enhancement of release). Values shown
represent means ± S.E. from multiple (n = 3)
experiments. Statistically significant differences are marked by
asterisks (*, p < 0.15; **, p < 0.05). C, extracts (60 µg) of PC12 cells transfected with
various NCS-1 constructs were analyzed by Western blotting.
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DISCUSSION |
We examined the function of NCS-1, a neuronal Ca2+
sensor protein, in stimulus-secretion coupling in PC12 cells. Studies
in Drosophila and Xenopus indicate that NCS-1
enhances synaptic efficacy (6, 7), and recent data demonstrate that
NCS-1 regulates associative learning and memory in vivo in
Caenorhabditis elegans (57). Although the effect of
NCS-1 on enhancing transmitter release and/or synaptic plasticity is
well established, the mechanism(s) of its action are poorly understood.
In the present study, several independent lines of evidence indicate
that NCS-1 enhances release by enhancing the activity of PI4K. 1)
Secretagogues that activate PLC result in a pronounced
NCS-1-dependent enhancement of secretion, whereas
PLC-independent secretagogues have either none or only a slight effect
on release. 2) Overexpression of NCS-1 leads to a shift in the
dose-response curve of inhibition of release using PAO, a specific
inhibitor of PI4K. 3) Overexpression of NCS-1 leads to an increase in
plasma membrane-localized PtdIns(4,5)P2, as imaged with a
PH-domain GFP construct. 4) Extracts from NCS-1-overexpressing cells
display enhanced production of PtdIns(4)P in vitro,
indicating increased PI4K activity. 4) Mutants of NCS-1 that are likely
to be incapable of binding ligand or that are incapable of binding to
membranes, have lost their ability to enhance secretion.
Overexpression of NCS-1 resulted in enhanced hGH secretion when cells
were stimulated by ATP, a purinergic receptor agonist that activates
PLC. Enhanced secretion using ATP as a secretagogue may thus reflect a
PI4K
-mediated increase in the amounts of PtdIns(4,5)P2 present at the plasma membrane, which act as substrate for PLC. However, the observation that NCS-1 also moderately enhanced
ionomycin-triggered release suggests that increased levels of
PtdIns(4,5)P2 have a more direct role in mediating
exocytosis independent of PLC. Overexpression of NCS-1 has been
reported to have no effect on Ca2+-dependent
release from permeabilized PC12 cells (8), which is in contrast to the
enhanced ionomycin-triggered release observed in this study. However,
it seems possible that permeabilization would lead to the partial loss
of NCS-1 and/or PI4K
, which, at least in part, are both soluble in
the cytoplasm.
The present study indicates that NCS-1 enhances large dense-core
vesicle-mediated secretion from PC12 cells by activating PI4K
.
Because large dense-core vesicles contain high concentrations of
intravesicular ATP, NCS-1 may elicit facilitation of neuropeptide and
amine release by a purinergic receptor-mediated process. However, NCS-1
has also been reported to enhance secretion from small synaptic vesicles. Although the evidence for a requirement for
PtdIns(4,5)P2 synthesis in synaptic vesicle secretion is
conflicting (39, 50), NCS-1 may enhance exocytosis of synaptic vesicles
by a similar mechanism. Alternatively, biochemical studies suggest that
NCS-1 may have a diverse array of target proteins, some of which may be
channel proteins (13-16). Thus, in addition to regulating PI4K
activity, and dependent on the cell system, NCS-1 overexpression may
lead to a combined effect on PtdIns(4,5)P2 levels and
channel activity. Modulation of channel activity may be either direct or indirect through regulating the amount of channels expressed on the
cell surface. For example, overexpression of NCS-1 leads to its
localization in the cell periphery, in addition to a diffuse distribution throughout the cytoplasm, and to a concomitant increase in
Kv4 channel localization at the plasma membrane (16), suggesting that
NCS-1 may increase membrane trafficking of channel proteins, which by
itself may be a PtdIns(4,5)P2-dependent process.
Activation of PI4K
by NCS-1 will lead to concomitant de
novo synthesis of PtdIns(4,5)P2, which has been shown
to be essential for vesicle priming reactions in neuroendocrine cells
(25, 26, 29, 58). During priming, PI4K and PtdIns(4)P-5-kinases may act
in concert to modify the two newly juxtaposed membranes and contribute
to the acquisition of fusion competence. Lipid polymorphism as
exemplified by a localized increase in PtdIns(4,5)P2 may
have a direct physiological role in the generation of membrane
curvature as required during membrane fusion (59). Alternatively, the localized increase in PtdIns(4,5)P2 may allow recruitment
of proteins involved in mediating secretion, such as CAPS (31,
60). In summary, our present study indicates that in neuroendocrine
cells, the NCS-1-mediated enhancement of secretion is because of
enhanced PI4K
activity and concomitant increases in plasma membrane
PtdIns(4,5)P2 levels.