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* This work was supported in part by Conte Center Grant MH48108.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. § Contributed equally to the results of this work. ¶ Howard Hughes Medical Institute predoctoral fellow. ** Supported by Grant NIHGM 08327 from the Jointly Sponsored National Institutes of Health Predoctoral Training Program in the Neurosciences.
The molecular mechanisms underlying the Ca2+ regulation of hormone and neurotransmitter release are largely unknown. Using a reconstituted [3H]norepinephrine release assay in permeabilized PC12 cells, we found that essential proteins that support the triggering stage of Ca2+-stimulated exocytosis are enriched in an EGTA extract of brain membranes. Fractionation of this extract allowed purification of two factors that stimulate secretion in the absence of any other cytosolic proteins. These are calmodulin and protein kinase Cα (PKCα). Their effects on secretion were confirmed using commercial and recombinant proteins. Calmodulin enhances secretion in the absence of ATP, whereas PKC requires ATP to increase secretion, suggesting that phosphorylation is involved in PKC- but not calmodulin-mediated stimulation. Both proteins modulate release events that occur in the triggering stage of exocytosis. The half-maximal increase was elicited by 3 nm PKC and 75 nmcalmodulin. These results suggest that calmodulin and PKC increase Ca2+-activated exocytosis by directly modulating the membrane- or cytoskeleton-attached exocytic machinery downstream of Ca2+ elevation.
The molecular mechanisms of presynaptic vesicle release have been extensively examined by a combination of biochemical, genetic, and electrophysiological techniques. A series of protein-protein interaction cascades have been proposed to lead to vesicle docking and fusion (
). The SNARE protein family, including syntaxin, SNAP-25, and vesicle-associated membrane protein (VAMP, also called synaptobrevin), plays an essential role in promoting membrane fusion, and is thought to comprise the basic fusion machinery (
). In Ca2+-stimulated exocytosis, many additional proteins are important in the Ca2+ regulation of the basic membrane trafficking apparatus. Calcium not only triggers rapid fusion of release-competent vesicles, but is also involved in earlier processes which replenish the pool of readily releasable vesicles (
). In this study, we used an established cracked cell assay, in which [3H]norepinephrine (NE)1 labeled PC12 cells are permeabilized by mechanical “cracking” and then reconstituted for secretion of NE in the presence of test proteins (
). These cracked cells readily release NE upon addition of ATP, brain cytosol, and 1 μm free Ca2+ at an elevated temperature. We term this a “composite assay,” as all essential components are added into one reaction mixture. Alternatively, cracked cells can be first primed with cytosol and ATP, washed, then reconstituted for NE release with cytosol and Ca2+ (
). This sequential priming-triggering protocol is useful for determining whether a protein acts early or late in the exocytic pathway and whether its effect is dependent on Ca2+ or ATP.
This semi-intact cell system serves as a bridge between an in vitro system comprised of purified components and electrophysiological systems that monitor release in vivo. It provides information on protein functions in a cell with an intact membrane infrastructure while being easily manipulatable. Ca2+ regulation by membrane depolarization is no longer a concern, as intracellular Ca2+ concentration can be controlled by a buffered solution. Indirect readout of neurotransmitter release using a postsynaptic cell is replaced by direct readout of [3H]NE released into the buffer. Complications associated with interpreting overlapping exo- and endocytotic signals are also eliminated as only one round of exocytosis is measured. Finally, concentration estimates are likely to be accurate, since added compounds do not need to diffuse long distances along axons and dendrites to their sites of action.
Using this assay, several proteins required for NE release have been purified from rat brain cytosol, including phosphatidylinositol transfer protein (
Calmodulin is the most ubiquitous calcium mediator in eukaryotic cells, yet its involvement in membrane trafficking has not been well established. Some early studies showed that calmodulin inhibitors (
) inhibited Ca2+-activated exocytosis. However, in other studies, calmodulin-binding peptides and an anti-calmodulin antibody led to the conclusion that calmodulin is only involved in endocytosis, not exocytosis (
). More recently, it was reported that Ca2+/calmodulin signals the completion of docking and triggers a late step of homotypic vacuole fusion in yeast, thus suggesting an essential role for Ca2+/calmodulin in constitutive intracellular membrane fusion (
). If calmodulin indeed plays an important role in exocytosis, a likely target of calmodulin is Ca2+/calmodulin-dependent protein kinase II (CaMKII), a multifunctional kinase that is found on synaptic vesicles (
). It is usually assumed that phorbol esters effect on exocytosis is through activation of PKC, but Munc13-1 was recently shown to be a presynaptic phorbol ester receptor that enhances neurotransmitter release (
), which complicates the interpretation of some earlier reports. The mode of action of PKC remains controversial. There is evidence that PKC increases the intracellular Ca2+ levels by modulating plasma membrane Ca2+ channels (
). It is believed that upon phosphorylation, these PKC substrates might interact differently with their binding partners, which, in turn, leads to the enhancement of exocytosis. In addition, evidence is accumulating that PKC and calmodulin interfere with each others actions, as PKC phosphorylation sites are embedded in the calmodulin-binding domains of substrates such as neuromodulin and neurogranin (
). It is therefore possible that PKC could modulate exocytosis via a calmodulin-dependent pathway by synchronously releasing calmodulin from storage proteins.
In this study, we fractionated an EGTA extract of brain membranes in order to identify active components that could reconstitute release in the cracked cell assay system. We identified calmodulin and PKC as two active factors. Thus, we demonstrate that calmodulin and PKC play a role in the Ca2+ regulation of exocytosis, and provide further insight into the mechanisms of their action.
Bisindolylmaleimide I, PKC inhibitor peptide(19–36), human recombinant PKCα, rat brain PKC, and anti-PKC monoclonal antibody (Ab2) were purchased from Calbiochem (San Diego, CA). Calmodulin from bovine brain was obtained from Sigma, annexin VI (annexin 67 kDa) from bovine liver was obtained from Sigma and Biodesign (Kennebunk, ME), and anti-calmodulin monoclonal antibody was purchased from Upstate Biotechnology (Lake Placid, NY). Calmodulin-binding peptide (CaMKII aa 291–312) was synthesized at Research Genetics (Huntsville, AL) and high pressure liquid chromatography purified.
Rat Brain Cytosol Preparation
Frozen rat brains (Harlan, IN) were homogenized in 3 ml/g of ice-cold homogenization buffer (20 mm Hepes, pH 7.5, 0.25 mm sucrose, 2 mm EGTA, 2 mm EDTA, 2 μg/ml leupeptin, 4 μg/ml aprotinin, 1 mmdithiothreitol, and 1 mm phenylmethylsulfonyl fluoride) using a VirTishear homogenizer (Virtis, NY). The homogenate was centrifuged at 30,000 × g for 20 min, and the supernatant was centrifuged again at 100,000 × g for 1 h. The resulting supernatant was rapidly frozen in aliquots and stored at −80 °C.
Membrane EGTA Extract Preparation
Frozen rat or bovine brains (RJO Biologicals, Kansas City, MO) were homogenized as above, except that the buffer lacks 2 mm EGTA and 2 mm EDTA (1 liter/bovine brain). The homogenate was centrifuged twice at 3,000 × g for 15 min, and the supernatant centrifuged at 100,000 × g for 1 h. The 100,000 × g supernatant, if kept, was rapidly frozen and stored as cytosol. It is referred to in the text as “cytosol prepared in the absence of EGTA.” The pellet of the 100,000 × g spin was washed once by rehomogenization into homogenization buffer without protease inhibitors, using a Teflon/glass homogenizer, and centrifuged at 100,000 ×g for 1 h. The washed membrane pellet was then rehomogenized into homogenization buffer without protease inhibitors at about 0.5 ml/g of starting material. EGTA was added to reach 2 mm final concentration, and the extraction was carried out at 4 °C for a few hours or overnight. The membrane homogenate was then centrifuged at 100,000 × g for 1 h, and the supernatant collected as “membrane EGTA extract.”
The protein concentration of cytosol and membrane EGTA extract was estimated using a Bradford protein assay (Bio-Rad), using bovine serum albumin as a standard. In a typical preparation, the protein concentration of rat brain cytosol ranges from 7 to 9 mg/ml, and the membrane EGTA extract 0.3 to 0.6 mg/ml. About 3% of total membrane proteins can be extracted into the EGTA extract.
Cracked Cell Assay
PC12 cells were maintained and [3H]NE labeled as described previously (
). Labeled cells were harvested by pipetting with ice-cold potassium glutamate buffer (50 mm Hepes, pH 7.2, 105 mm potassium glutamate, 20 mmpotassium acetate, 2 mm EGTA) containing 0.1% bovine serum albumin. All subsequent manipulations were carried out at 0–4 °C unless otherwise stated. Labeled cells (1–1.5 ml/dish) were mechanically permeabilized by a single passage through a chilled stainless steel ball homogenizer. The cracked cells were adjusted to 11 mm EGTA and incubated on ice for 0.5–3 h, followed by three washes in which the cells were centrifuged at 800 ×g for 5 min and resuspended in potassium glutamate buffer containing 0.1% bovine serum albumin.
Each release reaction contains 0.5–1 million cracked cells, 1.5 μm free Ca2+, 2 mm MgATP, and the protein solution to be tested in a total volume of 200 μl of potassium glutamate buffer. Release reactions were initiated by incubation at 30 °C and terminated by returning to ice after a 15-min incubation. The supernatant of each reaction was isolated by centrifugation at 2,500 × g for 30 min at 4 °C, and the released [3H]NE was quantified by scintillation counting (Beckman LS6000IC). Cell pellets were dissolved in 1% Triton X-100, 0.02% azide and similarly counted. NE release was calculated as a percentage of total [3H] in the supernatant.
A priming reaction contains about 1–2 million cracked cells, 2 mm MgATP, and the protein solution to be tested in a total volume of 200 μl of potassium glutamate buffer. Note that Ca2+ is omitted. The reaction was carried out at 30 °C for 30 min. The primed cells were then spun down, washed once with fresh potassium glutamate buffer, and distributed into two triggering reactions, each containing 10 μl of rat brain cytosol and 1.5 μm free Ca2+ in a total volume of 200 μl of potassium glutamate buffer. The triggering reaction was performed at 30 °C for 3 min, and the NE release was measured as in a composite assay.
Cracked cells were primed in potassium glutamate buffer containing 0.7–1 mg/ml rat brain cytosol and 2 mm MgATP at 30 °C for 30 min. The primed cells were centrifuged, washed once with potassium glutamate buffer, and distributed into triggering reactions containing 1.5 μmfree Ca2+ and the protein solution to be tested. To inhibit any ATP dependent activity in the triggering reaction, where indicated, an ATP depletion system of 5 units/ml hexokinase (Sigma), 2 mm MgCl2, 10 mm glucose or a non-hydrolyzable ATP analogue AMP-PNP (5 mm; Sigma) was added into the triggering reaction. NE release was measured as above.
Free Ca2+ Concentration Determination
The range of [Ca2+]free in the release reaction (Fig. 2B) was achieved by adding Ca2+into potassium glutamate buffer to reach final [Ca2+]total values of 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 1.9, and 2.0 mm. The pH of the reaction was 7.24 when no Ca2+ was added and 7.04 when 2.0 mmCa2+ was added, in the absence of protein extracts or cracked cells. Free Ca2+ concentrations were determined using video microscopic measurements of fura-2 fluorescence. Imaging was performed as described previously (
). The values of Rmin, Rmax, and Kd* were determined in the following solutions: 1) Rmin: potassium glutamate buffer containing 8 × 106 cracked cells/ml, 2 mm MgATP, and 10 mm additional EGTA; 2)Rmax: potassium glutamate buffer containing 8 × 106 cracked cells/ml, 2 mm MgATP, and 10 mm total Ca2+; 3) Kd*: potassium glutamate buffer containing 8 × 106 cracked cells/ml, 2 mm MgATP, 28 mm additional EGTA, and 18 mm total Ca2+, pH 7.2 ([Ca2+]free = 169 nm, determined in the absence of cells and MgATP based on fura-2 calibration in cell-free solutions). These solutions were incubated at 37 °C for 3 min, mixed with fura-2 pentapotassium salt (100 μm; Molecular Probes, Eugene, OR), and imaged. This procedure allowed us to take into account changes in fura-2 properties caused by the presence of permeabilized cells. Duplicate measurements of the above range of [Ca2+]total gave the following average [Ca2+]free values: 106, 146, 277, 462, 971, 1468, 1847, and 2484 nm.
Purification of Active Proteins
All procedures were carried out at 4 °C or on ice unless noted otherwise. Membrane EGTA extract of one or two bovine brain(s) (usually 150 ml/brain) was filtered through cheesecloth and loaded overnight onto a 10- or 20-ml DEAE column packed with DEAE-Sepharose CL-6B beads (Amersham Pharmacia Biotech). The column was washed with wash buffer (20 mm Hepes, pH 7.5, 0.25 mmsucrose, 2 mm EGTA, 1 mm dithiothreitol) and step eluted with 10 column volumes of elution buffer (20 mmHepes, pH 7.5, 2 mm EGTA, 400 mm KCl, 1 mm dithiothreitol). 100 μl of every other fraction was dialyzed overnight into potassium glutamate buffer, and 20 μl tested in a composite release assay for activity. The active fractions were pooled and dialyzed into zero salt buffer (20 mm Hepes, pH 7.5, 2 mm EGTA) and batch bound to 10 ml of Affi-Gel Blue beads (Bio-Rad) or DyeMatrex-Green A beads (Amicon) for a few hours. Blue beads were used in earlier experiments, and Green beads were used later to specifically deplete CAPS, which was known to bind to Green beads (
). The unbound material was collected, concentrated to about 2 ml using a Centriprep-10 (Amicon), and loaded onto a 120-ml HiPrep Sephacryl S-200 gel filtration column (Amersham Pharmacia Biotech). Samples were run on the S-200 column in potassium glutamate buffer at a flow rate of 7 ml/h. 10–50 μl of every other fraction was tested for activity in the cracked cell composite assay, and two peaks of activity were observed (Fig. 3).
The first peak of activity had a predicted molecular mass of 85 kDa. The corresponding material was adjusted to 10 mm potassium phosphate concentration (pH 7.2) and loaded onto a 1-ml hydroxyapatite column packed with hydroxyapatite Bio-Gel HT (Bio-Rad). The bound material was eluted with a linear potassium phosphate gradient from 10 to 500 mm (pH 7.2) in a 30-ml total volume at a flow rate of about 0.1 ml/min, and 0.4–0.5-ml fractions were collected. 200 μl of each fraction was dialyzed into potassium glutamate buffer and 20 μl was tested for activity. Meanwhile, the fractions (10 μl each) were also analyzed by SDS-PAGE and silver staining (Sigma silver stain kit). The active material was concentrated and resolved on an 8% polyacrylamide gel. Two Coomassie-stained protein bands that matched the activity profile (Fig. 6) were excised from the gel, minced into small pieces, dried, and sequenced by the Stanford PAN facility. The two polypeptide sequences obtained from the upper band were: LLNQEEGEYYNVPIXEGD and IRSTLNPRWDESFT. The only bovine protein that contains both polypeptides is PKCα. The four polypeptide sequences obtained from the lower band were: YELTGKFERLIVGLMRPPAY, LIEILASRTNEQIHQLVAA, MLVVLLQGTREEDDVVSEDL, and EMSGDVRDVFVAIVQSVK. Based on these sequences, the protein band was unambiguously identified to be bovine annexin VI.
The second S-200 peak has a predicted molecular mass of 25 kDa. The corresponding material was dialyzed into zero salt buffer (20 mm Tris, pH 7.5, 1 mm EGTA) and injected onto a Mono-Q HR 5/5 FPLC column (Pharmacia). The FPLC run was performed at 18 °C at 1 ml/min and 1-ml fractions were collected with a linear salt gradient from 0 to 1 m KCl over 71 ml. The fractions containing proteins (determined by A280) were dialyzed into potassium glutamate buffer and tested in the cracked cell assay.
Anti-calmodulin antibody and anti-PKC antibody were used at 1:2000 and 1:100 dilution at room temperature for 1 h. ECL (Amersham) was used for detection.
In this study, we first identified an EGTA extract of brain membranes as a protein source capable of reconstituting Ca2+-activated exocytosis in cracked PC12 cells. EGTA only extracts a small pool of Ca2+-dependent membrane-associating proteins, and thus it served as an efficient initial purification step. Further protein chromatography led to the identification of two active factors in the starting extract, calmodulin and PKC, which together accounted for about half of the starting activity. Upon confirmation with commercially obtained proteins, this result unambiguously demonstrated that calmodulin and PKC mediate aspects of Ca2+-dependent processes in exocytosis.
The finding that brain membrane EGTA extract alone is able to replace cytosol in supporting Ca2+-triggered NE secretion in PC12 cells is somewhat surprising. We suggest that the likely explanation is 2-fold. First, some cytosolic proteins essential for exocytosis have a membrane-bound pool within permeabilized cells, whose activity might be sufficient for a normal level of exocytosis. Second, although the 100,000 × g membrane pellet was washed to remove as many cytosolic proteins as possible, some cytosolic proteins that associate with membranes in a Ca2+-independent manner are probably present in the membrane EGTA extract. However, these proteins likely constitute only a small percentage of the proteins in the extract, as the characteristics of the activity triggered by the membrane extract are quite different to that of cytosol (Fig. 2).
Using an unbiased biochemical purification method, we demonstrated that calmodulin and PKC directly modulate the exocytotic machinery downstream of Ca2+ entry, and furthermore, that they signal through membrane-attached molecules to increase exocytosis. These targets include integral and peripheral membrane proteins, and cytosolic proteins that have a significant membrane-bound pool. The modest stimulation by calmodulin and PKC on secretion might suggest a regulatory role. However, it is also possible that some intermediates in their signaling pathways are in limiting amounts in the cell ghosts, so that their full effects were not observed. Half-maximal stimulation was obtained at about 3 nm for PKC and at about 75 nm for calmodulin. This is consistent with an enzymatic role for PKC, and predicts a high-affinity interaction between calmodulin and its substrate protein.
Ca2+ regulates exocytosis at many different levels. Prior studies indicated that Ca2+ signaling occurs in the priming steps as well as in triggering steps (
). Our priming triggering protocol does not allow Ca2+-dependent priming events to be assayed, as EGTA is present in the priming reaction. However, a different approach revealed the existence of both high and low Ca2+-dependent processes (Fig. 2). Moreover, this analysis indicated that late triggering events require high [Ca2+], whereas early priming events require low [Ca2+]. If, as proposed, there is indeed a pronounced intracellular spatial and temporal [Ca2+] gradient from the point of Ca2+ entry during depolarization (
), then, perhaps triggered events occur closer to the point of Ca2+entry, while Ca2+-dependent priming events occur further away from the point of Ca2+ entry. Distinct Ca2+ sensors at these stages might be appropriately tuned to different [Ca2+] to handle different tasks. By analyzing the Ca2+ sensitivity of calmodulin- and PKC-stimulated release, we addressed the question of whether calmodulin and PKC plays an early or a late role in vesicle release. We showed that they both require relatively high [Ca2+] (Fig.8B), implying that calmodulin and PKC both mediate late triggering events, consistent with some earlier reports (
), we are able to limit the possible modes of PKC action in our system to an increase in the readily releasable vesicle pool or release sites, or an enhancement of the probability of release of individual vesicles upon Ca2+ influx.
The experiments assaying the calcium sensitivity of release (Figs. 2,5, and 8) demonstrated a drop in release at very high [Ca2+]. This decline in release at high [Ca2+] has been previously reported (
), and may represent the true Ca2+ sensitivity of the Ca2+-sensing mechanism inside cells. However, in our system, it could also be due to the activation of a variety of Ca2+-activated proteases, as experiments are usually performed in the presence of crude extracts, which include unsequestered proteases.
What might the molecular targets of PKC and calmodulin be? An obvious calmodulin target molecule is CaMKII, as noted earlier. However, we showed that calmodulin's effect on exocytosis is ATP-independent, rendering the involvement of a kinase extremely unlikely. Calmodulin has also been shown to associate with synaptic vesicles in a Ca2+-dependent fashion through synaptotagmin (
). However, we found that there was little calcium-dependent binding of calmodulin to synaptotagmin either on synaptic vesicles, in a bead binding assay with recombinant proteins, or in a calmodulin overlay (data not shown). In addition, using immobilized calmodulin, we did not see significant Ca2+-dependent pull-down of synaptotagmin or Rab3A from rat brain extract (data not shown). Recent work has suggested three other candidate targets for calmodulin, Munc13, Pollux, and CRAG (
). Pollux has similarity to a portion of a yeast Rab GTPase-activating protein, while CRAG is related to Rab3 GTPase exchange proteins. Further work is required to investigate the role of their interactions with calmodulin in vivo.
The recent report that calmodulin mediates yeast vacuole fusion (
) is intriguing, as it raises the possibility that calmodulin, a highly conserved ubiquitous molecule, may mediate many membrane trafficking events. It is not yet known if the effector molecule of calmodulin is conserved or variable across species and different trafficking steps. It is enticing to propose a model for Ca2+ sensing whereby calmodulin is a high affinity Ca2+ sensor for both constitutive and regulated membrane fusion. In the case of constitutive fusion, calmodulin may be the predominant Ca2+ sensor. In the case of slow, non-local exocytosis of large dense core granules, an additional requirement for the concerted actions of other molecule(s) that are better tuned to intermediate rises in [Ca2+] might exist. At the highly localized sites of fast exocytosis of small clear vesicles where high [Ca2+] is reached, specialized low affinity sensor(s) are likely required in addition to calmodulin to achieve membrane fusion. Therefore, although calmodulin participates in multiple types of vesicle fusion, the impact of Ca2+sensing by calmodulin on vesicle release likely varies.
Due to the fact that calmodulin binding to some proteins can be modulated by PKC phosphorylation, one might suspect that PKC action on exocytosis proceeds through a calmodulin-dependent pathway. However, we found that the effects of calmodulin and PKC are additive within our system, suggesting that PKC does not act by releasing calmodulin from a substrate that functions as a calmodulin storage protein.
How Ca2+ regulates presynaptic vesicle release has been an open question for many years. By identifying calmodulin and PKC as modulators of Ca2+-regulated exocytosis and clarifying their functions, we have extended our knowledge of the release process. While the basic machinery of membrane fusion is becoming better understood, the multiple effects of Ca2+ on exocytosis remain to be elucidated at the molecular level. In addition, the ways that Ca2+ regulation may be important to the mechanisms of synaptic plasticity in the central nervous system will be a major focus in the future.
We thank Diana Bautista and Dr. Richard S. Lewis for generous help with [Ca2+]freedetermination; Dr. Ching Kung for providing the Paramecium calmodulin mutants, and Dr. Anthony R. Means for providing the chicken calmodulin mutants. We also thank Dr. Jesse C. Hay for the initial setup of the cracked cell assay, and Dr. Suzie J. Scales for helpful comments on the manuscript.