| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Neurobiology, Pharmacology, and Physiology, The University of Chicago, Chicago, Illinois 60637 2 Tufts University School of Medicine, New England Medical Center, Boston, Massachusetts 02111
Submitted 5 May 2003; accepted in final form 22 June 2003
 |
ABSTRACT |
|---|
|
1B,
2a,2
(Cav2.2), the cells expressed robust Ca2+ currents that were blocked by 
-conotoxin GVIA. Activation of N-type Ca2+ currents caused rapid increases in membrane capacitance of the MPC 9/3L cells, indicating that the Ca2+ influx was linked to exocytosis of vesicles similar to that reported in chromaffin or PC12 cells. Synaptic protein interaction (synprint) sites, like those found on N-type Ca2+ channels, are thought to link voltage-dependent Ca2+ channels to SNARE proteins involved in synaptic transmission. Interestingly, MPC 9/3L cells transfected with either LC-type (1C, 2a, 2, Cav1.2) or T-type (1G, 2a, 2, Cav3.1) Ca2+ channel subunits, which do not express synprint sites, expressed appropriate Ca2+ currents that supported rapid exocytosis. Thus MPC 9/3L cells provide a unique model for the study of exocytosis in cells expressing specific Ca2+ channels of defined subunit composition without complicating contributions from endogenous channels. This model may help to distinguish the roles that different Ca2+ channels play in Ca2+-dependent secretion. |
|
INTRODUCTION |
|---|
|
). They are rare in the human population at large (Beard et al. 1983) but occur with increased frequency in patients with the hereditary disease, neurofibromatosis type 1 (Riccardi 1981). Jacks et al. developed a knockout mouse with a germline mutation in the murine homologue of the NF1 gene (Jacks et al. 1994). Although the homozygous mutation is lethal, heterozygous animals show an increased frequency of pheochromocytomas (Jacks et al. 1994) compared with wild-type mice (Tischler et al. 1996). Recently, Powers et al. established immortal mouse pheochromocytoma (MPC) cell lines from those tumors (Powers et al. 2000).
The rat pheochromocytoma (PC12) cell line is widely utilized as a model to study synthesis, storage, and secretion of catecholamines and regulation of nervous system development. PC12 cells are chromaffin-like cells that differentiate toward a neuronal phenotype in response to nerve growth factor (NGF), which causes these cells to extend long processes and become electrically excitable (Greene and Rein 1977; Greene and Tischler 1976). In contrast, dexamethasone (DEX) treatment induces a more endocrine-like phenotype and upregulates catecholamine synthesis and storage (Tischler et al. 1983). Untreated PC12 cells express high-voltage-activated Ca2+ channels that include L (Usowicz et al. 1990), N (Usowicz et al. 1990), P/Q (Liu et al. 1996), and R types (Rane and Pollock 1994), and neuronally differentiated PC12 cells typically exhibit enhanced expression of those channels (Garber et al. 1989; Powers et al. 2000) in addition to low-voltage-activated (T-type) Ca2+ channels (Garber et al. 1989). PC12 cells are a useful model for studies of secretory mechanisms as they rapidly release vesicles in a Ca2+-dependent manner (Elhamdani et al. 2000; Harkins and Fox 1998; Kasai et al. 1996). However, because they express multiple Ca2+ channel subtypes, the contribution of individual channels to secretion cannot be unambiguously discerned. To overcome this problem, we have developed a novel model that employs recombinant ion channels and MPC cells.
We report here on one of the MPC cell lines established by Powers et al. (Powers et al. 2000). We show that this cell line expresses proteins associated with secretion. The MPC cell line does not express endogenous Ca2+ channels and so exhibited no exocytosis when stimulated by trains of depolarizations. N-type Ca2+ channels, that contain the synprint site, initiate neurotransmitter release from neurons and from secretory cells on activation (Artalejo et al. 1994; Takahashi and Momiyama 1993; Wheeler et al. 1994). When MPC cells were transfected with N-type Ca2+ channel subunits, appropriate Ca2+ currents were observed, which when activated, rapidly released vesicles in a Ca2+-dependent manner. Robust secretion was also observed when cells transfected with either LC- or T-type Ca2+ channels, which have no synprint site, were stimulated. MPC cells represent a new mammalian expression system that secretes in a manner similar to PC12 cells and adrenal chromaffin cells, yet is deficient in endogenous voltage-dependent Ca2+-channels. Our study shows that secretion can be elicited by recombinant Ca2+ channels. We expect that MPC cells in conjunction with recombinant ion channels will be useful for studying molecular mechanisms of secretion at the single-cell level.
|
|
METHODS |
|---|
|
MPC 9/3L cell line was grown in culture medium that consisted of RPMI-1640, 10% heat-inactivated horse serum, 5% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin in a humidified 5% CO2 incubator at 37°C. To test the effects of various differentiation agents, the cells were treated for 8–12 days with medium that contained nerve growth factor (NGF, 100 ng/ml), epidermal growth factor (EGF, 50 ng/ml), basic fibroblast growth factor (bFGF, 25 µg/ml), DEX (10 µM), retinoic acid (RA, 1 µM), or dibutyryl adenosine cAMP (cAMP, 1 mM), which was used with 2 µM 5-fluorodeoxyuridine. All of the stock solutions were stored at –20°C and mixed with medium just prior to use.
Immunoblot analysis
MPC 9/3L cells were maintained for 4 days in control medium or medium supplemented with glial cell line-derived neurotrophic factor (GDNF; 50 ng/ml) or dexamethasone (10 µM). Cell proteins were extracted in RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.5% wt/vol sodium deoxycholate, 0.1% wt/vol sodium dodecyl sulfate, and 1% vol/vol NP-40) plus protease and phosphatase inhibitors (100 mM NaF, 2 mM sodium pyrophosphate, 1 mM sodium vanadate, 4 µg/ml leupeptin, 20 µg/ml aprotinin, and 2 mM phenylmethylsulfonyl fluoride) and quantitated using BCA protein assay (Pierce Chemicals). Proteins (40 µg/lane) were separated on a 10% polyacrylamide gel and transferred to a nylon membrane (Nytran, Schleicher, and Schuell) (Towbin et al. 1979). Blots were incubated in 1% (wt/vol) casein in Tris-buffered saline to block nonspecific staining and then probed with the following antibodies: anti-tyrosine hydroxylase (1: 5000, DiaSorin), anti-chromogranin A and anti-chromogranin B [each 1:1000, gifts from R. Fischer-Colbrie, (Fischer-Colbrie and Schober 1987)], anti-syntaxin (1:1000, Calbiochem), and anti-SNAP25 (1: 1000, Alomone laboratories). Bound antibodies were detected by chemiluminescence using goat anti-rabbit or anti-mouse IgG conjugated to alkaline phosphatase (1:5000, Tropix) and CDP-Star (Tropix) as a reporter. Luminescence was digitally recorded using a Kodak 440CF Image Station.
Transfection with Ca2+ channel cDNAs
Cloning of the bovine chromaffin cell 1B (GenBank AF173882
[GenBank]
) and 2a (GenBank AF174417
[GenBank]
) subunits of the N-type Ca2+ channel has been described previously (Cahill et al. 2000). The rat 2 Ca2+ channel subunit cDNA (GenBank M86621
[GenBank]
) was a kind gift from Terry Snutch (Univ. of British Columbia). Rabbit heart 1C (GenBank X15339
[GenBank]
) was a kind gift from Dr. Gerald W. Zamponi (University of Calgary). Rat brain clone 1G (GenBank AF027984
[GenBank]
) was a kind gift from Dr. Edward Perez-Reyes (University of Virginia). The day before transfection, the cells were replated to collagen-coated coverslips. Cells were transfected with Qiagen-purified plasmids in a 5:5: 5:1 µg ratio of the 1B:2a:2:EGFP DNAs, by using lipofectamine plus. Recordings took place 48–72 h after transfection.
[Ca2+]i measurements
Intracellular Ca2+ concentrations ([Ca2+]i) were measured by ratiometric fluorescence imaging with fura-2. Cells were plated onto glass coverslips the day prior to an experiment. A coverslip was placed in Hank's balanced salt solution (HBSS), which contained (in mM) 138 NaCl, 5 KCl, 1.3 CaCl2, 0.3 KH2PO4, 0.8 MgSO4, 0.3 Na2HPO4, 5.6 D-glucose, and 20 HEPES (pH = 7.3) with 0.1% bovine serum albumin to load 3 µM fura-2 for 15 min. The cells were washed in a fura-2-free solution for 30 min. The coverslip was transferred to an experimental chamber for recording. Backgrounds at 340 and 380 nm were obtained using an area of the coverslip devoid of cells. For each experiment, 10–20 small, round MPC cells were selected and individually imaged. Image pairs (1 at 340 and 380 nm) were obtained every 10 s by averaging 16 frames at each wavelength. Backgrounds were subtracted from each wavelength, and the 340-nm image was divided by the 380-nm image to provide a ratiometric image. Ratios were converted to free [Ca2+]i by comparing data to fura-2 calibration curves made in vitro by adding fura-2 (50 µM free acid) to solutions that contained known [Ca2+] (0–2,000 nM). Cells were exposed to high-K+ solutions that contained (in mM) 50 KCl, 87 NaCl, 1 MgCl2, 5 CaCl2, 10 D-glucose, and 12 HEPES, and after wash in HBSS for a few minutes, were exposed to ionomycin (20 µM).
Electrophysiology
A coverslip of cells was set into a recording chamber and perfused with an external Na+ solution that contained (in mM) 135 NaCl, 2 KCl, 1 MgCl2, 5 CaCl2, 12 HEPES, and 10 glucose (pH = 7.3, osmolality 
295 mOsm). Single cells were visualized and voltage-clamped in the whole cell configuration (Hamill et al. 1981) with an Axopatch-1C amplifier modified for capacitance measurements. Transfected cells were identified by their fluorescence due to EGFP expression. Gigaohm seals were obtained with electrodes filled with an internal solution that contained (in mM) 120 CsAsp, 5 MgCl2, 0.1 EGTA, 40 HEPES, 2 ATP, and 0.3 GTP (pH = 7.3, osmolality 310 mOsm). Ca2+ currents (ICa) were isolated with a Na+-free external solution that contained (in mM) 140 TEA-Cl, 0.0001 tetrodotoxin (TTX), 5 CaCl2, 10 HEPES, and 10 glucose (pH = 7.3, osmolality 300 mOsm). Changes in membrane capacitance (
Cap) were measured with the phase-tracking technique (Joshi and Fernandez 1988; Neher and Marty 1982) and have been described previously in detail (Harkins and Fox 1998). All current records were compensated for series resistance and whole cell capacitance, and leak subtracted by averaging 16 hyperpolarizing sweeps. To block N-type Ca2+ channels, -conotoxin GVIA (-CgTx GVIA) was mixed as a 1 µM solution with the Na+-free external solution.
Data analysis
Statistical analysis of the data are expressed as means ± SE, and the Student's t-test was used to compare significance.
|
|
RESULTS |
|---|
|
MPC 9/3L cells appear as either groups of cells or single cells 20 µm in diameter that occasionally have long processes but typically do not (see, for example, Fig. 1A, left). Because differentiating agents can profoundly affect morphology, survival, neurite outgrowth, and receptor and channel expression in cultured neurons and PC12 cells, we treated the MPC 9/3L cells with a variety of agents and assessed changes in morphology and in expression of a number of proteins known to be contained in secretory vesicles or involved in exocytosis. After 3, 5, 7, and 9 days of treatment with NGF, EGF, GDNF, or RA (see METHODS), there were no visual gross morphological changes of the treated cells compared with untreated control cells. In contrast, both bFGF and DEX caused many of the cells to become elongated and extend short processes (Fig. 1A, right). The majority of cells died within the first 4 days of treatment with cAMP plus 2-fluorodeoxyuridine, and the few cells that remained had large cell bodies and extremely long processes (not shown). There were no effects of NGF, EGF, GDNF, bFGF, DEX, or RA on cell proliferation or survival (not shown).
|
To determine whether MPC 9/3L cells have proteins that are typically associated with synaptic vesicles, immunoblotting experiments were carried out with cells that were either untreated or treated with GDNF (50 ng/ml) or DEX (10 µM) for 4 days. Both chromogranin A (CGA) and chromogranin B (CGB), the major proteins contained in large dense-core vesicles, were expressed in the MPC 9/3L cells; the expression of CGA was increased by treatment with DEX (Fig. 1B). The increase in CGA but not CGB in response to DEX is consistent with the finding that maintenance of CGA levels in rat adrenal medulla depends on corticosteroids, whereas levels of CGB do not (Sietzen et al. 1987). Syntaxin and SNAP-25, the plasma membrane localized components of the SNARE complex, were also present in MPC 9/3L cells, and the levels of these proteins were not changed by GDNF or DEX. Syntaxin appears as a doublet in Fig. 1B because both the 1A and the 1B isoforms of syntaxin are present and recognized by the antibody (Foletti et al. 2000). Finally tyrosine hydroxylase, the rate limiting enzyme in the biosynthesis of the catecholamine neurotransmitters was clearly expressed in the MPC 9/3L cells, and levels of this enzyme were increased in response to DEX (Fig. 1B).
Despite the presence of tyrosine hydroxylase, HPLC analysis showed that MPC 9/3L cells stored only very small quantities of dopamine (0.02 nmol/106 cells) or 2% of the dopamine levels found in PC12 cells (2.9 nmol/106 cells) (Greene and Tischler 1976). Norepinephrine was not detectable in these cells. DEX-treatment of the cells (10 µM DEX for 6 days) increased dopamine levels about fivefold and increased norepinephrine to detectable levels. MPC 9/3L cells did not secrete detectable quantities of dopamine after treatment with 56 mM K+, presumably because of the lack of endogenous Ca2+ channels (see following text). However, in response to 20 µM ionomycin (a Ca2+ ionophore), 10% of the cellular content was secreted into the medium within 15 min. The low levels of catecholamines in MPC 9/3L cells may mean that each secretory vesicle has little catecholamine or that only a fraction of the vesicles are catecholaminergic.
MPC 9/3L cells have endogenous Na+ current but little or no endogenous Ca2+ current
Endogenous, voltage-dependent currents were characterized using cells patch-clamped in the whole cell configuration. We recorded from phase-bright cells without processes that were 20 µm in diameter (10 ± 0.21 pF, n = 589 cells). Currents were elicited by step depolarizations to different potentials from a holding potential of –80 mV. Of 126 cells, 119 had an endogenous Na+ current (INa) as shown in Fig. 2A. Endogenous INa was blocked by bath application of TTX [either 100 nM (n = 3) or 2 µM (n = 3)] as shown in the superimposed top current trace in Fig. 2A. Only 23 of the 126 cells tested had a small endogenous ICa that averaged 70 ± 8 pA and ranged from 30–164 pA. Of the 126 cells tested, 3 cells had a small outward current and 7 cells had no detectable current whatsoever.
|
When vesicles fuse with the plasma membrane, an increase in membrane surface area can be detected as a change in membrane capacitance (Cap) (Neher and Marty 1982). Ca2+-dependent exocytosis can be elicited by interrupting the capacitance measurement for brief stimulations with voltage steps to activate Ca2+ channels, thereby allowing an influx of extracellular Ca2+ into the cells. Cells were stimulated with trains of 10 depolarizations to 10 mV (50-ms step duration, 100-ms interpulse duration). As expected, the endogenous INa did not support secretion (n = 27) as no change in Cap was observed on stimulation (Fig. 2B). No measurable secretion was observed in MPC 9/3L cells with the small endogenous ICa when cells were depolarized with a train of either 5 or 10 depolarizations to 10 or 20 mV (n = 10).
After treatment with NGF, PC12 cells exhibit increased Ca2+ channel expression (Garber et al. 1989; Plummer et al. 1989). In contrast, endogenous currents in the MPC 9/3L cells were not significantly changed after treatment of the cells with differentiating agents. We treated MPC 9/3L cells with several differentiating agents: 100 ng/ml NGF, 50 ng/ml EGF, 25 ng/ml bFGF, 10 µM DEX, 1 µM RA, and 1 mM cAMP. Whole cell currents were recorded after 8–12 days of treatment and compared with control cells in culture for the same duration. There was no easily discernable difference in either the types of currents expressed or the endogenous Na+ or Ca2+ current density in cells treated with any of the differentiating agents compared with control cells, although one cell treated with cAMP exhibited an endogenous Ca2+ current that was slightly larger than any seen in control cells (Table 1).
|
All subsequent results in this manuscript are from cells that were not treated with differentiating agents. [Ca2+]i levels were monitored by loading MPC 9/3L cells with fura-2 (Fig. 3). For each cell, data were averaged for the first 120 s to determine resting [Ca2+]i, and was, on average, 54 ± 4 nM (n = 55). A high-K+ solution (50 mM) was applied to the cells and resulted in a small increase in [Ca2+]i to 67 ± 5 nM (P > 0.05, n = 52). After washout, as a control the cells were exposed to 20 µM ionomycin, a Ca2+ ionophore, which increased [Ca2+]i to 490 ± 23 nM (P < 0.001 compared with resting levels, n = 52). Typically, excitable cells that contain Ca2+ channels respond to high-K+ depolarizations with a large elevation in [Ca2+]i. The results in MPC 9/3L cells suggest that these cells have few Ca2+ channels.
|
Transient expression of exogenous Ca2+ channels in MPC 9/3L cells supports secretion
N-TYPE CA2+ CHANNELS. To determine whether MPC 9/3L cells could support Ca2+-dependent release, the cells were transiently transfected with the N-type Ca2+ channel subunit 1B, along with 2a, 2, and co-transfected with GFP as a marker. Whole cell currents were recorded from GFP-positive cells. The external Na+ solution was exchanged with a Na+-free solution (5 mM Ca2+) designed to isolate the Ca2+ currents. Of the GFP-expressing cells recorded, 75% of the cells expressed N-type Ca2+ current. Figure 4A plots current versus voltage data, averaged from 7 cells, showing that inward Ca2+ currents were observed in these transfected cells. The peak current for each voltage step was normalized to the whole cell capacitance (pA/pF). Figure 4B, a plot of current as a function of time, shows that ICa was completely blocked by the N-type Ca2+ channel blocker -CgTx GVIA (1 µM). The * in Fig. 4B depict the times where the two examples of ICa, shown in Fig. 4C, were recorded. The first current trace (Fig. 4C, top) was obtained prior to blockade by -CgTx GVIA, whereas the second trace (Fig. 4C, bottom) was obtained in the presence of the toxin.
|
Cells that expressed ICa were stimulated with a train of five depolarizations to 20 mV (peak current) for 200 ms with a 50-ms interpulse duration while capacitance was measured. Figure 5A plots capacitance (Cap) as a function of time. The capacitance trace was interrupted by the depolarizations (gaps in the trace). In this cell, membrane capacitance increased by 900 fF (as indicated by - - -). ICa recorded in response to the first of the five depolarizations is shown in Fig. 5C. Integrating the area under the currents supplied the number of Ca2+ ions that entered the cell during each depolarization; 1,300 x 106 Ca2+ ions entered the cell during the first stimulation, while 1,630 x 106 ions entered for all five depolarizations. The maximal rate of release was 1,289 fF/s for this cell (calculated as the maximal secretion that occurred for a single depolarization divided by the duration of the depolarization). After 5 min, the cell was washed with 1 µM -CgTx GVIA to block the N-type Ca2+ channels and then stimulated with a second train of depolarizations. Figure 5, B and D, shows that both ICa and changes in membrane capacitance were blocked by -CgTx GVIA.
|
On average, peak exocytosis was 584 ± 108 fF (n = 44) for MPC 9/3L cells transfected with N-type Ca2+ channel subunits. The number of Ca2+ ions that entered the cell during all five of the stimulations was 859 x 106 ions (±100 x 106, n = 44). The averaged maximal rate of release was 657 ± 129 fF/s (n = 34). Although the MPC 9/3L cells did not have endogenous ICa, when they were transfected with exogenous N-type Ca2+ channels, they exhibited robust currents and rapid exocytosis. A rough estimate of "secretion efficiency" can be defined as the ratio of secretion (fF) divided by the number of Ca2+ ions (x106). The ratio in MPC 9/3L cells was similar to the ratio in PC12 cells but about half that in chromaffin cells (unpublished observation).
The secretory responses of MPC 9/3L cells showed some run-down when cells were stimulated every 5 min even though ICa was relatively unchanged. In eight cells that were stimulated with multiple trains of depolarizations, the average peak secretion was 309 ± 62 fF for the first stimulation and 230 ± 54 fF (P = 0.83) for the second stimulation. The mean total number of Ca2+ ions that entered the cell during the first stimulation was 646 x 106 ± 116 x 106 and 614 x 106 ± 98 x 106 ions (P = 0.36) for the second stimulation. Rundown of secretion has been observed in other secretory cells (Harkins and Fox 1998). A number of MPC 9/3L cells were stimulated more than twice, up to as many as seven times, with decreasing but detectable secretion (data not shown).
LC- AND T-TYPE CA2+ CHANNELS. When either the 1C or the 1G subunits were substituted for the 1B subunit, the MPC 9/3L cells expressed Ca2+ currents with typical biophysical properties of the LC- and T-type Ca2+ channel, respectively. MPC 9/3L cells with exogenously expressed 1C subunits produced an ICa that averaged 391 ± 50 pA and ranged from 141 to 724 pA (n = 13), values almost 10-fold less than that observed for N-type channels. A representative inward ICa with a peak of 533 pA is shown in Fig. 6A (left) for the LC-type Ca2+ channel (1C). MPC 9/3L cells that were transiently transfected with the T-type Ca2+ channel (1G) expressed a rapidly inactivating ICa that averaged 576 ± 142 pA and ranged from 85 to 1,421 pA (n = 10). A representative cell expressing T-type Ca2+ channels is shown in Fig. 6B (left) with a peak ICa of 1,289 pA. Note the rapid inactivation exhibited during the depolarization is characteristic of this family of Ca2+ channels. Current versus voltage plots are shown for cells expressing either LC (Fig. 6A, middle)- or T-type Ca2+ channels (Fig. 6B, middle). Each plot shows the average from nine cells in which the peak current for each voltage step was normalized to the whole cell capacitance (pA/pF). The peak ICa for the LC-type Ca2+ channels was observed at 20 mV, whereas the peak ICa for the T-type Ca2+ channels was between –20 and –10 mV. The threshold for LC Ca2+ channel activation was 10 mV more negative than that for N-type Ca2+ channels, while the T-type current activation was 40 mV more negative than that of the N-type channels.
|
MPC 9/3L cells were stimulated with a train of five step depolarizations to +20 mV, while capacitance changes were monitored, to determine whether LC-type Ca2+ channels supported secretion. A representative capacitance trace (Fig. 6A, right), shows that membrane capacitance increased by 265 fF. The maximal rate of release was 188 fF/s; 258 x 106 Ca2+ ions entered the cell during the first depolarization while 580 x 106 Ca2+ ions entered for all five depolarizations. To study secretion in cells expressing the T-type Ca2+ channel, cells were stimulated with a train of either 5 or 10 depolarizations to –20 mV. A representative capacitance trace (Fig. 6B, right) shows that membrane increased by 133 fF. The maximal rate of release was 50 fF/s; 50 x 106 Ca2+ ions entered the cell during the first depolarization, whereas 233 x 106 Ca2+ ions entered the cell for all five depolarizations. Thus both LC- and T-type Ca2+ channels support secretion in MPC 9/3L cells.
On average, the number of Ca2+ ions that entered during all five of the stimulations was 238 x 106 ± 51 x 106 ions (n = 12) for the LC-type Ca2+ channel (1C) and 204 x 106 ± 46 x 106 ions (n = 7) for the T-type Ca2+ channel (1G). Both of these averages were much less than the Ca2+ entry for the N-type Ca2+ channel (1B), which averaged 859 x 106 ± 100 x 106 ions (n = 44) during five depolarizations. Average peak exocytosis was 106 ± 18 fF (n = 12) for the LC-type channel, 113 ± 27 fF (n = 7) for the T-type channel, and 584 ± 108 fF (n = 44) for the N-type channel.
|
|
DISCUSSION |
|---|
|
1B, 1C or 1G Ca2+ channel subunits (along with the 2a and 2 accessory subunits), the MPC 9/3L cells express Ca2+currents with the appropriate electrophysiological characteristics and exhibit rapid, Ca2+-dependent exocytosis. MPC 9/3L cells therefore represent an excellent single-cell system in which to study the roles of specific Ca2+ channel subunits in secretion. Subtypes of known subunit composition can be expressed in isolation or in assorted combinations. We have used the MPC 9/3L cells to show that N (1B)-, LC (1C)-, and T (1G)-type Ca2+ channels couple to secretion.
Voltage-gated Ca2+-channels are composed of a pore forming 1 subunit and accessory subunits (, 2, 
). Ten different 1 genes have been found (Catterall 2000). Members of the Cav2 Ca2+-channel family (P/Q-, N-, and R-type) are thought to initiate synaptic transmission (Hirning et al. 1988; Takahashi and Momiyama 1993; Turner et al. 1992; Wheeler et al. 1994; Wu et al. 1998). Binding of N- and P/Q-type Ca2+ channels to SNARE proteins is thought to couple the influx of Ca2+ ions to rapid exocytosis (Catterall 1998). Co-immunoprecipitation studies have shown that Ca2+ channels associate with syntaxin and synaptotagmin in situ (Bennett et al. 1992; Leveque et al. 1992; Yoshida et al. 1992), and in vitro binding assays have identified a region of the 1B and 1A Ca2+ channel subunits called the synprint site that is necessary for binding of syntaxin (Rettig et al. 1996; Sheng et al. 1994). This synprint site not only targets N- and P/Q-type Ca2+ channels to presynaptic nerve terminals (Mochida et al. 2003b) and anchors them there (Bezprozvanny et al. 2000) but also binds SNARE proteins, resulting in changes in channel function (Bezprozvanny et al. 1995; Mochida et al. 2003b; Wiser et al. 1997). The 1 subunits of L- and T-type Ca2+ channels do not have recognizable synprint sites (Mochida et al. 2003b). Functional coupling between SNARE proteins and the 1C Ca2+ channel is somewhat controversial. One study suggested that syntaxin 1A did not alter L-type Ca2+ channel gating (Bezprozvanny et al. 1995), whereas other studies suggested that syntaxin could alter gating in this channel (Wiser et al. 1996, 1999). Injecting peptides containing the synprint site inhibited synaptic transmission in neurons, whereas corresponding peptides made from the II-III linker of L-type channels had no effect (Mochida et al. 1996). In contrast, insulin release from pancreatic beta cells is initiated by activation of L-type Ca2+ channel. Insulin secretion was inhibited by a peptide made to the II-III loop of the L-type Ca2+ channel (Wiser et al. 1999). Although synaptic transmission appears, in general, to be mediated by members of the CaV2 family (N, P/Q and R type) (Mochida et al. 2003a; Takahashi and Momiyama 1993; Wheeler et al. 1994), there are exceptions. For instance, release from goldfish retinal bipolar neurons is mediated by L-type channels (Heidelberger and Matthews 1992; Kobayashi and Tachibana 1995) and L-type Ca2+ channels contribute to neurotransmitter release in Xenopus neuromuscular synapses (Sand et al. 2001; Thaler et al. 2001). Interestingly, the Ca2+ channels that mediate neurotransmitter release in Drosophila have no recognizable synprint site (Kawasaki et al. 2000; Littleton and Ganetzky 2000). Comparisons of secretion produced by different classes of Ca2+ channels was complicated by different levels of channel expression in MPC 9/3L cells. Because release is typically a steep function of Ca2+ current, keeping Ca2+-influx constant is important for quantitative comparisons. Unfortunately, N-type channels expressed in MPC cells at significantly higher levels than did LC and T-type Ca2+ channels. To get a rough estimate of secretory efficiency, we normalized total secretion by Ca2+ influx; we observed that LC- and T-type Ca2+ channels supported secretion with an efficiency that was similar to that of N-type Ca2+ channel (data not shown). These results are not due to nonspecific overexpression of Ca2+ channels as expression levels of LC was low. Our results in MPC 9/3L cells suggest that all types of Ca2+ channels can functionally couple to release sites. The fact that synaptic transmission is mediated primarily by CaV2 family members may be due to differential targeting of these channels to presynaptic nerve terminals (Mochida et al. 2003b).
Other model cells that have been used to study secretory mechanisms include PC12 cells, adrenal chromaffin cells, and pituitary cells. However, a major drawback of each of these cells is multiplicity of endogenous Ca2+ channel subtypes (Garber et al. 1989; Kongsamut and Miller 1986; Liu et al. 1996; Usowicz et al. 1990), which contributes to difficulties in correlation of secretion with a specific Ca2+ channel. Many laboratories have tried to identify specific channel subtypes that contribute to rapid release (Artalejo et al. 1994; Liu and Tsien 1995; Lukyanetz and Neher 1999; Santana et al. 1999; Takahashi and Momiyama 1993; Turner et al. 1995), and in each case, expression of multiple types of Ca2+ channels complicated the analysis of data. In PC12 cells, 30–40% of the whole cell, high-voltage-activated Ca2+ current remained even after inhibition with specific pharmacological agents that block N-, L-, and P/Q-type Ca2+ channels (unpublished results; Rane and Pollock 1994). Because of their complicated endogenous backgrounds, cells commonly used for secretion studies do not allow the subunit composition of Ca2+ channels to be altered in a controlled manner. In contrast to these other models, MPC 9/3L cells that lack endogenous Ca2+ channels allow the subunit composition of the Ca2+ channels to be altered in a controlled manner. Moreover, the efficiency of transmitter release in MPC 9/3L cells with transfected and defined Ca2+ channels is similar to that seen with both adrenal chromaffin cells and PC12 cells.
MPC cell lines differ from PC12 cells in that they show little or no response to NGF (Powers et al. 2000). Although some MPC lines respond to GDNF in a manner comparable to the NGF response of PC12 cells (Powers et al. 2002), such a response does not occur with MPC 9/3L. In the present study, we detected limited morphological responses of MPC 9/3L to several agents, but those changes were not accompanied by altered expression or function of Ca2+ channels. It remains to be determined whether other MPC lines will offer additional experimental applications.
Other expression systems may potentially be used to study Ca2+ channels. However, MPC 9/3L cells offer an advantage over other commonly used systems in that they provide a pseudo-neuronal environment. For instance, HEK-293 cells, derived from human embryonic kidney cells, do not express the molecular machinery for regulated vesicular release. Another example, the Xenopus oocyte, has a number of drawbacks that include voltage-clamp difficulties and endogenous Ca2+-dependent Cl– currents (Wagner et al. 2000). Thus MPC 9/3L cells represent a unique expression system for investigating Ca2+-dependent signaling.
|
|
DISCLOSURES |
|---|
|
|
|
ACKNOWLEDGMENTS |
|---|
|
|
|
FOOTNOTES |
|---|
Present address and address for reprint requests and other correspondence: A. B. Harkins, Dept. of Pharmacological and Physiological Science, Saint Louis University, 1402 S. Grand Blvd., St. Louis, MO 63104 (E-mail: harkinsa{at}slu.edu).
|
|
REFERENCES |
|---|
|
Beard CM, Sheps SG, Kurland LT, Carney JA, and Lie JT. Occurrence of pheochromocytoma in Rochester, Minnesota, 1950 through 1979. Mayo Clin Proc 58: 802–804, 1983.[Web of Science][Medline]
Bennett MK, Calakos N, and Scheller RH. Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Science 257: 255–259, 1992.
Bezprozvanny I, Scheller RH, and Tsien RW. Functional impact of syntaxin on gating of N-type and Q-type calcium channels. Nature 378: 623–626, 1995.[Medline]
Bezprozvanny I, Zhong P, Scheller RH, and Tsien RW. Molecular determinants of the functional interaction between syntaxin and N-type Ca2+ channel gating. Proc Natl Acad Sci USA 97: 13943–13948, 2000.
Cahill AL, Hurley JH, and Fox AP. Coexpression of cloned alpha(1B), beta(2a), and alpha(2)/delta subunits produces non-inactivating calcium currents similar to those found in bovine chromaffin cells. J Neurosci 20: 1685–1693, 2000.
Catterall WA. Structure and function of neuronal Ca2+ channels and their role in neurotransmitter release. Cell Calcium 24: 307–323, 1998.[Web of Science][Medline]
Catterall WA. Structure and regulation of voltage-gated Ca2+ channels. Annu Rev Cell Dev Biol 16: 521–555, 2000.[Web of Science][Medline]
Elhamdani A, Brown ME, Artalejo CR, and Palfrey HC. Enhancement of the dense-core vesicle secretory cycle by glucocorticoid differentiation of PC12 cells: characteristics of rapid exocytosis and endocytosis. J Neurosci 20: 2495–2503, 2000.
Fischer-Colbrie R and Schober M. Isolation and characterization of chromogranins A, B, and C from bovine chromaffin granules and a rat pheochromocytoma. J Neurochem 48: 262–270, 1987.[Web of Science][Medline]
Foletti DL, Lin R, Finley MA, and Scheller RH. Phosphorylated syntaxin 1 is localized to discrete domains along a subset of axons. J Neurosci 20: 4535–4544, 2000.
Garber SS, Hoshi T, and Aldrich RW. Regulation of ionic currents in pheochromocytoma cells by nerve growth factor and dexamethasone. J Neurosci 9: 3976–3987, 1989.[Abstract]
Greene LA and Rein G. Release, storage and uptake of catecholamines by a clonal cell line of nerve growth factor (NGF) responsive pheochromocytoma cells. Brain Res 129: 247–263, 1977.[Web of Science][Medline]
Greene LA and Tischler AS. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci USA 73: 2424–2428, 1976.
Hamill OP, Marty A, Neher E, Sakmann B, and Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch Eur J Physiol 391: 85–100, 1981.[Web of Science][Medline]
Harkins AB and Fox AP. Activation of nicotinic acetylcholine receptors augments calcium channel-mediated exocytosis in rat pheochromocytoma (PC12) cells. J Gen Physiol 111: 257–269, 1998.
Heidelberger R and Matthews G. Calcium influx and calcium current in single synaptic terminals of goldfish retinal bipolar neurons. J Physiol 447: 235–256, 1992.
Hirning LD, Fox AP, McCleskey EW, Olivera BM, Thayer SA, Miller RJ, and Tsien RW. Dominant role of N-type Ca2+ channels in evoked release of norepinephrine from sympathetic neurons. Science 239: 57–61, 1988.
Jacks T, Shih TS, Schmitt EM, Bronson RT, Bernards A, and Weinberg RA. Tumour predisposition in mice heterozygous for a targeted mutation in Nf1. Nat Genet 7: 353–361, 1994.[Web of Science][Medline]
Joshi C and Fernandez JM. Capacitance measurements. An analysis of the phase detector technique used to study exocytosis and endocytosis. Biophys J 53: 885–892, 1988.[Web of Science][Medline]
Kasai H, Takagi H, Ninomiya Y, Kishimoto T, Ito K, Yoshida A, Yoshioka T, and Miyashita Y. Two components of exocytosis and endocytosis in phaeochromocytoma cells studied using caged Ca2+ compounds. J Physiol 494: 53–65, 1996.
Kawasaki F, Felling R, and Ordway RW. A temperature-sensitive paralytic mutant defines a primary synaptic calcium channel in Drosophila. J Neurosci 20: 4885–4889, 2000.
Kobayashi K and Tachibana M. Ca2+ regulation in the presynaptic terminals of goldfish retinal bipolar cells. J Physiol 483: 79–94, 1995.
Kongsamut S and Miller RJ. Nerve growth factor modulates the drug sensitivity of neurotransmitter release from PC-12 cells. Proc Natl Acad Sci USA 83: 2243–2247, 1986.
Leveque C, Hoshino T, David P, Shoji-Kasai Y, Leys K, Omori A, Lang B, el Far O, Sato K, Martin-Moutot N, Newsome-Davis J, Takahashi M, and Seagar MJ. The synaptic vesicle protein synaptotagmin associates with calcium channels and is a putative Lambert-Eaton myasthenic syndrome antigen. Proc Natl Acad Sci USA 89: 3625–3629, 1992.
Littleton JT and Ganetzky B. Ion channels and synaptic organization: analysis of the Drosophila genome. Neuron 26: 35–43, 2000.[Web of Science][Medline]
Liu G and Tsien RW. Properties of synaptic transmission at single hippocampal synaptic boutons. Nature 375: 404–408, 1995.[Medline]
Liu H, Felix R, Gurnett CA, De Waard M, Witcher DR, and Campbell KP. Expression and subunit interaction of voltage-dependent Ca2+ channels in PC12 cells. J Neurosci 16: 7557–7565, 1996.
Lukyanetz EA and Neher E. Different types of calcium channels and secretion from bovine chromaffin cells. Eur J Neurosci 11: 2865–2873, 1999.[Web of Science][Medline]
Mochida S, Sheng ZH, Baker C, Kobayashi H, and Catterall WA. Inhibition of neurotransmission by peptides containing the synaptic protein interaction site of N-type Ca2+ channels. Neuron 17: 781–788, 1996.[Web of Science][Medline]
Mochida S, Westenbroek RE, Yokoyama CT, Itoh K, and Catterall WA. Subtype-selective reconstitution of synaptic transmission in sympathetic ganglion neurons by expression of exogenous calcium channels. Proc Natl Acad Sci USA 100: 2813–2818, 2003a.
Mochida S, Westenbroek RE, Yokoyama CT, Zhong H, Myers SJ, Scheuer T, Itoh K, and Catterall WA. Requirement for the synaptic protein interaction site for reconstitution of synaptic transmission by P/Q-type calcium channels. Proc Natl Acad Sci USA 100: 2819–2824, 2003b.
Neher E and Marty A. Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin cells. Proc Natl Acad Sci USA 79: 6712–6716, 1982.
Plummer MR, Logothetis DE, and Hess P. Elementary properties and pharmacological sensitivities of calcium channels in mammalian peripheral neurons. Neuron 2: 1453–1463, 1989.[Web of Science][Medline]
Powers JF, Evinger MJ, Tsokas P, Bedri S, Alroy J, Shahsavari M, and Tischler AS. Pheochromocytoma cell lines from heterozygous neurofibromatosis knockout mice. Cell Tissue Res 302: 309–320, 2000.[Web of Science][Medline]
Powers JF, Schelling KH, Brachold JM, and Tischler AS. Plasticity of pheochromocytoma cell lines from neurofibromatosis knockout mice. Ann NY Acad Sci 971: 371–378, 2002.[Web of Science][Medline]
Rane SG and Pollock JD. Fibroblast growth factor-induced increases in calcium currents in the PC12 pheochromocytoma cell line are tyrosine phosphorylation dependent. J Neurosci Res 38: 590–598, 1994.[Web of Science][Medline]
Rettig J, Sheng ZH, Kim DK, Hodson CD, Snutch TP, and Catterall WA. Isoform-specific interaction of the alpha1A subunits of brain Ca2+ channels with the presynaptic proteins syntaxin and SNAP-25. Proc Natl Acad Sci USA 93: 7363–7368, 1996.
Riccardi VM. Von Recklinghausen neurofibromatosis. N Eng J Med 305: 1617–1627, 1981.[Web of Science][Medline]
Sand O, Chen BM, and Grinnell AD. Contribution of L-type Ca(2+) channels to evoked transmitter release in cultured Xenopus nerve-muscle synapses. J Physiol 536: 21–33, 2001.
Santana F, Michelena P, Jaen R, Garcia AG, and Borges R. Calcium channel subtypes and exocytosis in chromaffin cells: a different view from the intact rat adrenal. Naunyn Schmiedebergs Arch Pharmacol 360: 33–37, 1999.[Web of Science][Medline]
Sheng ZH, Rettig J, Takahashi M, and Catterall WA. Identification of a syntaxin-binding site on N-type calcium channels. Neuron 13: 1303–1313, 1994.[Web of Science][Medline]
Sietzen M, Schober M, Fischer-Colbrie R, Scherman D, Sperk G, and Winkler H. Rat adrenal medulla: levels of chromogranins, enkephalins, dopamine beta-hydroxylase and of the amine transporter are changed by nervous activity and hypophysectomy. Neuroscience 22: 131–139, 1987.[Web of Science][Medline]
Takahashi T and Momiyama A. Different types of calcium channels mediate central synaptic transmission. Nature 366: 156–158, 1993.[Medline]
Thaler C, Li W, and Brehm P. Calcium channel isoforms underlying synaptic transmission at embryonic Xenopus neuromuscular junctions. J Neurosci 21: 412–422, 2001.
Tischler AS, Perlman RL, Morse GM, and Sheard BE. Glucocorticoids increase catecholamine synthesis and storage in PC12 pheochromocytoma cell cultures. J Neurochem 40: 364–370, 1983.[Web of Science][Medline]
Tischler AS, Sheldon W, and Gray R. Immunohistochemical and morphological characterization of spontaneously occurring pheochromocytomas in the aging mouse. Vet Pathol 33: 512–520, 1996.[Abstract]
Towbin H, Staehelin T, and Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354, 1979.
Turner TJ, Adams ME, and Dunlap K. Calcium channels coupled to glutamate release identified by omega-Aga-IVA. Science 258: 310–313, 1992.
Turner TJ, Lampe RA and Dunlap K. Characterization of presynaptic calcium channels with omega-conotoxin MVIIC and omega-grammotoxin SIA: role for a resistant calcium channel type in neurosecretion. Mol Pharmacol 47: 348–353, 1995.[Abstract]
Usowicz MM, Porzig H, Becker C, and Reuter H. Differential expression by nerve growth factor of two types of Ca2+ channels in rat phaeochromocytoma cell lines. J Physiol 426: 95–116, 1990.
Wagner CA, Friedrich B, Setiawan I, Lang F, and Broer S. The use of Xenopus laevis oocytes for the functional characterization of heterologously expressed membrane proteins. Cell Physiol Biochem 10: 1–12, 2000.[Web of Science][Medline]
Wheeler DB, Randall A, and Tsien RW. Roles of N-type and Q-type Ca2+ channels in supporting hippocampal synaptic transmission. Science 264: 107–111, 1994.
Wiser O, Bennett MK, and Atlas D. Functional interaction of syntaxin and SNAP-25 with voltage-sensitive L- and N-type Ca2+ channels. Embo J 15: 4100–4110, 1996.[Web of Science][Medline]
Wiser O, Tobi D, Trus M, and Atlas D. Synaptotagmin restores kinetic properties of a syntaxin-associated N-type voltage sensitive calcium channel. FEBS Lett 404: 203–207, 1997.[Web of Science][Medline]
Wiser O, Trus M, Hernandez A, Renstrom E, Barg S, Rorsman P, and Atlas D. The voltage sensitive Lc-type Ca2+ channel is functionally coupled to the exocytotic machinery. Proc Natl Acad Sci USA 96: 248–253, 1999.
Wu LG, Borst JG, and Sakmann B. R-type Ca2+ currents evoke transmitter release at a rat central synapse. Proc Natl Acad Sci USA 95: 4720–4725, 1998.
Yoshida A, Oho C, Omori A, Kuwahara R, Ito T, and Takahashi M. HPC-1 is associated with synaptotagmin and omega-conotoxin receptor. J Biol Chem 267: 24925–24928, 1992.
This article has been cited by other articles:
|
|
 |
|
  M. R. Kasten, B. Rudy, and M. P. Anderson Differential regulation of action potential firing in adult murine thalamocortical neurons by Kv3.2, Kv1, and SK potassium and N-type calcium channels J. Physiol., October 15, 2007; 584(2): 565 - 582. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Moore, J. B. Papke, A. L. Cahill, and A. B. Harkins Stable gene silencing of synaptotagmin I in rat PC12 cells inhibits Ca2+-evoked release of catecholamine Am J Physiol Cell Physiol, August 1, 2006; 291(2): C270 - C281. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. B. Harkins, A. L. Cahill, J. F. Powers, A. S. Tischler, and A. P. Fox Deletion of the synaptic protein interaction site of the N-type (CaV2.2) calcium channel inhibits secretion in mouse pheochromocytoma cells PNAS, October 19, 2004; 101(42): 15219 - 15224. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
TOP
ABSTRACT
INTRODUCTION
