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Volume 16, Number 23,
Issue of December 1, 1996
pp. 7557-7565
Copyright ©1996 Society for Neuroscience
Expression and Subunit Interaction of Voltage-Dependent
Ca2+ Channels in PC12 Cells
Hongyan Liu2,
Ricardo Felix1,
Christina A. Gurnett1,
Michel De Waard1,
Derrick R. Witcher1, and
Kevin P. Campbell1
1 Department of Physiology and Biophysics, The Howard
Hughes Medical Institute, and 2 Program in Neuroscience,
University of Iowa College of Medicine, Iowa City, Iowa 52242
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES
ABSTRACT
Nerve growth factor (NGF)-induced differentiation in PC12 cells is
accompanied by changes in the expression of voltage-dependent Ca2+ channels. Ca2+ channels are multimeric
complexes composed of at least three subunits ( 1, ,
and 2 ) and are involved in neuronal migration, gene
expression, and neurotransmitter release. Although attempts have been
undertaken to elucidate NGF regulation of Ca2+ channel
expression, the changes in subunit composition of these channels during
differentiation still remain uncertain. In the present study,
patch-clamp recordings show that in addition to the previously
documented L-type and N-type Ca2+ currents,
undifferentiated PC12 cells also express an -agatoxin-IVA-sensitive (P/Q-type) component. In addition, the corresponding mRNA encoding the
pore-forming 1 subunits for these channels (C, B, and A, respectively) was detected. Likewise, mRNA for three distinct auxiliary
subunits (1, 2, 3) were also found, 3 protein being dominantly expressed. Immunoprecipitation experiments show that the
N-type Ca2+ channel is associated with either a
2 or 3 subunit and that NGF increases the
channel expression without affecting its subunit association. These
results (1) indicate that the diversity of Ca2+ currents in
PC12 cells arise from the expression of three distinct 1
and three different subunit genes; (2) support a model for heterogenous subunit association of the N-type Ca2+
channel in a single cell type; and (3) suggest that the regulation of
the N-type Ca2+ channel during NGF-mediated differentiation
involves an increase in the number of functional channels with no
apparent changes in subunit composition.
Key words:
calcium channels;
1B subunit;
subunit;
PC12;
nerve growth factor;
P/Q-type;
N-type;
-agatoxin-IVA;
-conotoxin GVIA
INTRODUCTION
Voltage-dependent Ca2+ channels play
an important role in regulating cellular Ca2+ concentration
in excitable cells (Tsien et al., 1995 ). These Ca2+
channels not only are central in controlling neurotransmitter release,
excitation-contraction coupling, and excitation-secretion coupling,
they are also involved in gene expression and neuronal migration (Beam
et al., 1992 ; Komuro and Rakic, 1992 ; Ghosh et al., 1994 ; Dunlap et
al., 1995 ). Several voltage-dependent Ca2+ channels have
been identified and carry out diverse functions: T-, L-, N-, P-, Q-,
and R-type (Llinas et al., 1992 ; Zhang et al., 1993 ; Basarsky et al.,
1994 ). The structure of the Ca2+ channels was elucidated
with the purification of skeletal muscle L-type and brain N-type
channel, which are composed of at least three protein subunits
( 1, , and 2 ) (Leung et al., 1987 ;
Takahashi et al., 1987 ; McEnery et al., 1991 ; Witcher et al., 1993 ).
The 1 subunit forms the Ca2+ channel pore
and binds Ca2+ channel blockers (Tanabe et al., 1987 ;
Hockerman et al., 1995 ). Six different 1 genes have been
identified thus far (S, A, B, C, D, and E), and with the exception of
S, all are expressed in the nervous system (Birnbaumer et al., 1994 ).
The class B 1 gene encodes for the N-type
Ca2+ channel (Dubel et al., 1992 ; Williams et al., 1992 ;
Witcher et al., 1993 ), whereas the product of class A 1
gene is a component of the P/Q-type Ca2+ channel (Mori et
al., 1991 ; Liu et al., 1996 ).
The subunit directly associates with the 1 subunit
(De Waard et al., 1994 ; Pragnell et al., 1994 ) and is essential for normal function and localization of the 1 subunit
(Castellano et al., 1993a ,b; Olcese et al., 1994 ; Chien et al., 1995 ).
Similar to the multitude of the 1 subunits, at least
four different genes (1, 2, 3, and 4) have been identified
(Birnbaumer et al., 1994 ).
Rat PC12 pheochromocytoma cell line has been a model system to study
neuronal differentiation (Greene and Tischler, 1976 ; Shafer and
Atchison, 1991 ; Chao, 1992 ). PC12 cells are chromaffin-like cells that
begin to resemble sympathetic neurons when exposed to nerve growth
factor (NGF). Previously, functional N- and L-type Ca2+
channels were detected in PC12 cells (Usowicz et al., 1990 ; Avidor et
al., 1994 ). In addition, it has been shown that NGF increases mRNA
expression of several Ca2+ channel 1
subunits (Lievano et al., 1994). Although in the highly heterogeneous
brain, it has been reported recently that 3,
4, and 1b subunits all are capable of
associating with the 1B subunit to form distinct brain
N-type channels (Scott et al., 1996 ), it is not clear whether different
subunits can associate with the N-type channels in a single cell
type. Furthermore, it is uncertain whether there is any change of the
subunit composition of the Ca2+ channels during NGF-induced
differentiation. To address these questions, we have investigated, at
the molecular level, the expression of Ca2+ channel
1 and subunits in PC12 cells. In addition, we have characterized the subunit composition of the PC12 N-type
Ca2+ channel during NGF-induced differentiation.
MATERIALS AND METHODS
Cell culture. PC12 cells (American Type Culture
Collection, Rockville, MD) were grown on rat collagen type I
(Collaborative Biomedical Products, Bedford, MA) coated dishes in RPMI
1640 medium supplemented with 5% horse serum and 10% fetal calf serum
in a 5% CO2 air-humidified atmosphere. The medium was
changed every 3 d, and the cells were subcultured approximately
every 10 d in a ratio of 1:6. Cells were plated 1 d before
the NGF (Promega, Madison, WI) addition. NGF (50 ng/ml) was added to
the culture medium for a 3 d period to induce PC12
differentiation.
Patch-clamp experiments. For electrophysiological recording,
undifferentiated PC12 cells were plated on
poly-L-lysine-coated glass coverslips (10 × 25 mm)
and grown in RPMI 1640 medium supplemented with 5% fetal bovine serum,
1% L-glutamine, 100 U/ml penicillin, and 100 µg/ml
streptomycin. After 3-5 d in culture, the cells were subjected to the
standard whole-cell patch-clamp technique (Hamill et al., 1981 ) using
an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). Current
signals were filtered at 1 kHz (internal four-pole Bessel filter),
digitized at 50 kHz, and analyzed with pClamp software (Axon
Instruments). Patch pipettes were pulled from borosilicate glass
capillaries (KIMAX-51; Kimble Division, Owens-Illinois, Toledo, OH) on
a horizontal puller (Sutter Instrument, Novato, CA), and pipette tips
were fire-polished with a microforge (Narishige, Tokyo, Japan). Typical
electrode resistances were 2-5 M . The bath solution contained (in
mM): 20 BaCl2, 125 tetraethylammonium chloride
(TEA-Cl), 10 HEPES, and 10 glucose, pH 7.3. The internal (patch-pipette) solution consisted of (in mM): 130 CsCl, 2 MgCl2, 11 EGTA, 20 HEPES, 2 Na2ATP, 0.1 GTP,
and 10 glucose, pH 7.3. After establishing the whole-cell mode,
capacitative transients were canceled with the amplifier. Currents were
obtained by applying 150 msec test pulses every 30 sec from a holding
potential of 90 mV. Leakage currents were usually less than 20 pA
at holding potential. Data were leak-subtracted on line by a standard
P/4 procedure. -agatoxin-IVA ( -Aga-IVA, Peptides International, Louisville, KY) was prepared as a 10 µM stock in
distilled water, and aliquots were stored at 20°C. A fresh aliquot
was diluted in the bath solution for each experiment to give a final
concentration of 200 nM. All experiments were performed at
20-22°C.
Membrane preparations and immunoblot analysis. Crude rat
brain membranes and cardiac microsome membranes were prepared as detailed elsewhere (Sakamoto and Campbell, 1991 ). PC12 cell membranes were prepared as follows. Preconfluent PC12 cells were washed with PBS
and harvested with a cell scraper. Cells were collected by
centrifugation at 500 × g and homogenized in buffer A
(5 mM HEPES, pH 7.4, 0.3 M sucrose, 0.1 mM phenylmethylsulfonyl fluoride, 0.75 mM
benzamidine, 0.6 µg/ml pepstatin A, 0.5 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.01 mg/ml lysozyme). Cell membranes were collected
by centrifugation at 135,000 × g for 30 min,
resuspended in buffer A, and stored at 80°C. PC12 and rat tissue
membranes (100-250 µg of protein/lane) were resolved on 3-12%
gradient SDS-polyacrylamide gels and transferred to nitrocellulose.
ECL detection system (Amersham, Arlington Heights, IL) was used
according to the manufacturer's instructions.
Total RNA isolation, RT-PCR, and sequencing. Total RNA was
isolated by homogenization in RNAzol according to the protocol of
Cinna/Biotecx (Friendswood, TX) followed by chloroform extraction. The
RNA was stored at 80°C as precipitates in 70% ethanol. To eliminate any possible contamination by genomic DNA, the total RNA was
treated with DNase RQ 1 (Promega, Madison, WI) for 30 min at 37°C
before reverse transcription PCR (RT-PCR).
cDNA was synthesized from 2 µg total RNA by 400 U Moloney murine
leukemia virus reverse transcriptase in a total volume of 30 µl.
Typically, 4 µl of cDNA was used in 50-100 µl PCR. The PCR
reactions were usually carried out for 32-36 cycles of 94°C 1 min,
56°C 1 min, and 72°C 1 min. The primers used are as follows: rat
1b forward 5 -TCCAGGGACCCTACCTTGTTTCC-3 (1391-1413,
rat 1b GenBank no. X61394[GenBank]); rat 1b
reverse 5 -CCTCCAGCTCATTCTTATTGCGC-3 (1806-1828); rat
2 forward 5 -TCGGATCCGAAGAAGAA- CCTTGTCTGG-3 (1759-1777, GenBank no. 80545); rat 2 reverse
5 -TCGAATTCAGTAGCGATCCTTAGATTTATGC-3 (2087-2110); rat
3 forward 5 -GTGGTGTTGGATGCTGAC-3 (892-909, GenBank
no. M88751[GenBank]); rat 3 reverse 5 -ATTGTGGTCATGCTCCGA-3 (1483-1500); rat 4 forward
5 -TTGGATCCACAGCTCTCTCACCGTA- TCC-3 (1457-1479, GenBank no. L02315[GenBank]),
rat 4 reverse 5 -TAGGAT- CCAGGGTAGTGATCTCGGCTG-3
(1639-1664); rat 1A forward
5 -CCAGTCTGTGGAGATGAGAGAAATGGG-3 (6042-6068, GenBank no.
M64373[GenBank]); rat 1A reverse 5 -TTTGGAGGGCAGGTCACCCG- ATTG-3
(6412-6435); rat 1B forward 5 -GCCGTCTCAGCCGCG-
GCCTTTCT-3 (6668-6690, GenBank no. M92905[GenBank]); 1B reverse
5 -CAAAGGTGAGTGTATCCTCAGGC-3 (6810-6832); 1C
forward 5 -GGAAGGATCCGAGCGAAAGAAGCTGGC-3 (3064-3090 in rat
1C rbc-I, GenBank no. M67516[GenBank]); 1C reverse
5 -AGGTGAATTCGG- CCCACAGGCATCTCG-3 (3307-3333 rat 1C
rbc-I); 1D-1 forward 5 -GGAGAGGAGGGCAAACGAAACACTAGC-3 (1892-1918, GenBank no. M57682[GenBank]); 1D-1 reverse
5 -CGTACACACCGGAACACAGAGA- CGC-3 (2364-2388); 1D-2
forward 5 -GTGCCCTGCACACAGTAGGC- GC-3 (485-506);
1D-2 reverse 5 -GGCACGGGCAGGTCGGCT- GTTAG-3
(816-838); 1E forward 5 -TCGGATCCAAATGTGAAG-
AGGAGCGTATC-3 (2569-2597, GenBank no. L15453[GenBank]),
1E reverse 5 -AGTCAAGCTTCACGTCAGGGATGGCGACTG-3 (3262-3291).
The 1 and 3 RT-PCR products from PC12
total RNA were directly sequenced by the dideoxy chain termination
method using total PCR products as sequencing templates (Barnard et
al., 1994 ). The 1 PCR product was also subcloned into
pBluescript, and its sequence was confirmed by cycle sequencing from
both directions. All others were subcloned into pGEX 2T or pBluescript
and sequenced by the dideoxy chain termination method according to
United States Biochemicals (Cleveland, Ohio).
Enrichment of 2 subunit using 2
antibody affinity column. Affinity-purified rabbit 143 ( 2) antibody was coupled to Hydrazide Avidgel AX
according to the manufacturer's instructions (Unisyn Technologies, San
Diego, CA). PC12 membranes (25 mg) were solubilized for 1 hr at 4°C
in solubilization buffer (1% digitonin, 1 M NaCl, 10 mM HEPES, pH 7.4, 0.75 mM benzamidine, 0.6 µg/ml pepstatin A, 0.5 µg/ml aprotinin, 0.5 µg/ml leupeptin). The
solubilized materials were sedimented by centrifugation in a Beckman TL
100 ultracentrifuge for 10 min at 400,000 × g. Then
the supernatant was diluted eightfold with 10 mM HEPES
containing the above-mentioned protease inhibitors. The diluted
material (50 ml) was applied to the antibody affinity column. After
washing the column extensively with 50 ml of buffer B (10 mM HEPES, pH 7.4, 0.1% digitonin, 100 mM
NaCl), the column was eluted with glycine buffer (50 mM
glycine, pH 2.5, 100 mM NaCl, 0.1% digitonin) containing
the protease inhibitors. A 125 µl volume of 2 M Tris, pH
8.0, was added to every milliliter of eluate immediately after the
elution. The eluted material was concentrated with Centricon-100
(Amicon, Beverly, WA) to 1-2 ml, and 0.1 ml per lane was loaded onto
SDS-polyacrylamide gel for analysis.
Immunoprecipitation of N-type Ca2+ channels.
PC12 cell membranes were solubilized and diluted as described
above. The diluted extract was labeled with 50 pM
[125I] -conotoxin GVIA ( -CgTX) (Amersham, Arlington
Heights, IL) for 1 hr at room temperature. Subsequently, aliquots (1 ml) of the labeled extract were incubated overnight at 4°C with 50 µl antibody-protein G Sepharose beads (Pharmacia, Piscataway, NJ). The beads were extensively washed, and total binding was quantified by
counting. Nonspecific binding was determined by the addition of
1000-fold excess unlabeled -CgTX (Bachem, Torrance, CA) before the
addition of the labeled toxin. The nonspecific counts were subtracted
from total counts to give specific counts. Each sample point represents
the average of triplicates.
RESULTS
Detection of P/Q-type Ca2+ channels
Ca2+ currents in undifferentiated PC12 cells consist
of dihydropyridine (L-type) and -CgTX (N-type)-sensitive components
(Usowicz et al., 1990 ), as well as a component that is resistant to
both pharmacological agents (Rane and Pollock, 1994 ). Likewise, it is
known that undifferentiated PC12 cells resemble normal chromaffin cells
(Greene and Tischler, 1976 ), which in addition to N- and L-type
Ca2+ currents, possess a component sensitive to the
P/Q-type Ca2+ channel blocker -Aga-IVA (Artalejo et al.,
1994 ; Gandia et al., 1995 ). To investigate the presence of P/Q-type
functional Ca2+ channels, -Aga-IVA was applied to PC12
cells, and whole-cell Ba2+ inward currents were recorded.
As described previously (Randall and Tsien, 1995 ), although some of the
-Aga-IVA sensitive current is inhibited quickly, blockage increases
progressively until a stable plateau is reached ~5 min after toxin
application. Therefore, to ensure a saturating degree of inhibition and
to minimize the impact of the rundown on Ca2+ current,
cells were preincubated for 30 min with 200 nM -Aga-IVA before initiating whole-cell recording. This concentration of the toxin
produces maximal block of P/Q-type Ca2+ currents (Pearson
et al., 1995 ).
The average amplitude of Ba2+ currents
(IBa) in the control condition and after the
preincubation with -Aga-IVA is plotted as a function of membrane
potential (Vm) in Figure
1A. In both cases, the peak current
increases steeply over the range between 20 and 10 mV, reaching a
maximum value at +20 mV. The shape of these current-voltage
(I-V) relationships suggests that PC12 cells exhibit
a large component of high-voltage-activated Ca2+ channels,
whereas low-voltage-activated Ca2+ channels are virtually
absent. As illustrated in the I-V curve indicated by filled
circles, the preincubation with -Aga-IVA induced a decrease in peak
current amplitude at all potentials examined.
Fig. 1.
Voltage dependence of Ba2+ current in
PC12 cells and its inhibition by -Aga-IVA. A, Peak
current-voltage relationships obtained from two groups of cells after
30 min incubation in the absence (open circles) or
presence of 200 nM -Aga-IVA (filled
circles). The currents were recorded in response to 150 msec
depolarization from a holding potential of 90 mV with 10 mV increase
in the pulse amplitude per step. Symbols represent mean
peak current values of sham preincubated (n = 9)
and toxin-treated cells (n = 11). B,
Illustrative traces of peak inward current from a control (upper
trace) and an -Aga-IVA-treated (lower trace)
cell. The voltage protocol is shown above the traces,
and the dotted line represents baseline current.
C, Comparison of peak current amplitudes at +20 mV in
both control and -Aga-IVA-treated cells. Data are given as mean ± SE, and the number of recorded cells is indicated in
parentheses. Asterisks denote significant
differences (p < 0.05).
[View Larger Version of this Image (11K GIF file)]
Typical Ba2+ currents from two distinct PC12 cells are
shown in Figure 1B, one cell subjected to a sham
preincubation (upper trace) and the other preincubated with
-Aga-IVA (lower trace), as described above. Under the
conditions of these experiments, test pulses elicited a rapidly
activating inward sustained current, the average amplitude of which was
significantly reduced in the toxin-preincubated cells. On average,
IBa was inhibited ~33% from a mean control
value of 109 ± 9 pA to 73 ± 17 pA on treatment with
-Aga-IVA (Fig. 1C).
To gain insight into the -Aga-IVA sensitive component of the
whole-cell Ca2+ channel current in PC12 cells, we recorded
Ba2+ currents immediately before and after 1-6 min of
exposure to 200 nM -Aga-IVA in individual cells. The
results of these experiments indicated that PC12 cells differ markedly
in their response to the toxin. We found that -Aga-IVA was capable
of blocking a fraction of Ca2+ channel currents in about
one-third of the investigated cells (n = 16) over a 6 min period. The curve indicated by filled circles in Figure
2A shows the inhibition of
-Aga-IVA-sensitive current, as evidenced by a decrease in normalized
total current from 1.0 to 0.62 ± 0.07 after 6 min toxin exposure.
Note that under our recording conditions, IBa
amplitude in control cells (open circles) decreased from 1.0 to 0.91 ± 0.02 with no detectable changes in waveform. A straight
line provided a close fit to these rundown data, which were given to
demonstrate the specific inhibitory effect of -Aga-IVA.
Fig. 2.
Properties of the -Aga-IVA-sensitive
Ca2+ channel current in PC12 cells. A, Plot
of peak IBa versus time in which recordings were sequentially taken before and after -Aga-IVA application. Filled circles represent average current blocked by the
toxin (n = 5). Currents were obtained by applying
150 msec steps from 90 to +20 mV, and peak currents were normalized
by the values observed before toxin application. Arrow
indicates -Aga-IVA addition. Open circles denote
time-dependent changes in the amplitude of the current (rundown) in
control cells, and the straight line represents the best
fit to the data. B, Representative superimposed traces
of Ba2+ currents taken from one of the cells indicated in
A, before (b) and 6 min after exposure to
200 nM -Aga-IVA (a). Lower
trace exemplifies the currents (b-a) obtained by
subtracting the current in the absence and in the presence of the toxin
and represents the -Aga-IVA-sensitive component. The inactivation
phase of these currents was fitted with a single exponential
(solid line).
[View Larger Version of this Image (11K GIF file)]
Upper traces in Figure 2B show representative
currents from one -Aga-IVA responsive cell immediately before
(b) and 6 min after (a) the application of the
toxin. Subtraction of the current remained after exposure to the toxin
from the current before treatment enabled us to dissect out the blocked
component, as illustrated in lower trace (b-a). According to
this analysis, -Aga-IVA-sensitive current averaged 51 ± 10 pA
in amplitude and accounted for 32 ± 2% of the total
IBa. In addition, the inactivation phase of these subtracted currents were fitted with a single exponential equation of the form: Aexp( t/ ) + C, where A is the initial amplitude (pA),
t is time (msec), is the time constant for inactivation, and C is a constant. The application of the toxin resulted
in the inhibition of a current component that exhibited a degree of
decay of 17.5 ± 3.1% over the 150 msec voltage step and an average time constant ( ) of 88.7 ± 9.4 msec.
mRNA expression of multiple 1 and subunits in
PC12 cells
Because several different types of Ca2+ channels are
present in PC12 cells, these cells should express mRNA of various
1 subunits. RT-PCR was performed on PC12 total RNA to
investigate the 1 and subunit genes expressed in
these cells. PCR of total RNA, without reverse transcription, was
performed as a negative control in parallel with RT-PCR (data not
shown). RT-PCR with three pairs of 1 subunit primers
amplified PC12 cDNA of the predicted sizes (Fig.
3A). Sequencing of these PCR products
confirmed the expression of three 1 subunit genes
(A, B, C) in PC12 cells (Fig.
3B). The sequences of the RT-PCR products are identical to
nucleotide 6259-6386 of rat 1A (GenBank accession no.
M64373[GenBank]), nucleotide 6685-6835 of rat 1B (GenBank
accession no. M92905[GenBank]), and nucleotide 3162-3316 of a splice variant
rbc-I of rat 1C (GenBank accession no. M67516[GenBank]) (Snutch
et al., 1991 ), respectively. These data are in agreement with the
expression of L-type ( 1C), N-type ( 1B),
and P/Q-type ( 1A) Ca2+ channels.
1D and 1E subunits were not detected
using RT-PCR either because of their absence or very low level of
expression.
Fig. 3.
mRNA expression of three Ca2+ channel
1 subunits in PC12 cells. A, RT-PCR from
PC12 cell total RNA with 1A, 1B, and
1C primers. The PCR products were separated on a 1%
agarose gel. The molecular weight standards are on the
left. B, Schematic representation of the
regions of published rat 1A, 1B, and
1C rbc-I sequences, which are identical to the sequences
of PC12 1A, 1B, and 1C RT-PCR products, respectively. The numbers stand for the
nucleotide positions in the published rat sequences.
[View Larger Version of this Image (17K GIF file)]
Because Ca2+ channel subunits have two highly
homologous domains, two pairs of primers that specifically amplified a
portion of 2 or 3 control cDNA in PCR
reactions were used to amplify PC12 cDNA (Fig.
4A). PCR of PC12 cDNA with these
primers amplified DNA fragments of identical molecular weights as the
products of control cDNA. The sequence of 2 RT-PCR
product from PC12 cells is 100% identical to nucleotide 1759-1990 of
rat 2 (GenBank accession no. M80545[GenBank]) in a 232-nucleotide
stretch. The 3 RT-PCR product is 100% identical to
nucleotide 984-1122 of rat 3 (GenBank accession no.
M88751[GenBank]) in the 143 bases sequenced. The 1 primers
efficiently amplify control 1 cDNA, although they also
weakly amplify control 4 cDNA. However, no
4 sequence was identified in sequencing 14 subcloned
1 PCR products. The sequence of the 1
RT-PCR product is 99% identical to rat 1b (GenBank accession no. X61394[GenBank]) (Pragnell et al., 1991 ) with only three
nucleotide differences between the PCR product and the rat 1b sequence in a stretch of 435 nucleotides (Fig.
4B). Two of the three single nucleotide changes
(T1537 C and T1595 C) result in W492R and V511A amino acid changes.
The last two different nucleotides (C188, C202) in PC12
1 are the same as the corresponding nucleotides in human
1 sequence (Collin et al., 1993 ). Therefore, PC12 cells
express a 1 isoform that is very similar to the
1b isoform expressed in brain. 4 was not
detected using either the 1 primers or a pair of
4 primers, suggesting an absence or extremely low level
of expression. These results demonstrate that PC12 cells express mRNA
that encode for 1, 2, and
3 subunits.
Fig. 4.
Expression of mRNA of three Ca2+
channel subunits in PC12 cells. A, RT-PCR from PC12
cell total RNA with 1, 2, and
3 primers. Left panel, PCR products
obtained from PC12 cDNA, control 1, 2,
3, and 4 cDNA with 1
primers. Middle panel, PCR products amplified with
2 primers from the same DNA samples as left panel. Right panel, PCR products amplified with
3 primers from the same DNA samples as left panel.
B, Alignment of 1 sequence of PC12 RT-PCR
product with rat 1b sequence. The different nucleotides are in italics, and dots represent
identical nucleotides.
[View Larger Version of this Image (44K GIF file)]
Association of different subunits with the
1B subunit
The N-type Ca2+ channel represents the most abundant
Ca2+ channel in PC12 cells (Usowicz et al., 1990 ) and has
been shown to be heterogeneous in its subunit composition in brain
(Scott et al., 1996 ). Because several subunits are expressed in
PC12 cells, it is important to identify the subunit(s) associated
with the N-type channel. We first examined the protein expression of
these subunits in PC12 cells. Four subtype-specific antibodies
were produced against 1b, 2,
3, and 4 subunits, respectively (Liu et
al., 1996 ; Scott et al., 1996 ). These polyclonal subunit-specific antibodies recognize a 1 subunit of 78 and 80 kDa, a
3 subunit of 58 kDa, and a 4 subunit of
59 and 55 kDa in crude rat brain membranes, and 2
subunits of 87, 74, and 70 kDa in rat cardiac microsome membranes (Fig.
5A). The 2 antibody only
detected the 74 kDa 2 in rat brain (data not shown).
These results demonstrate that the four subunit-specific antibodies
recognize native rat subunit proteins. There is also evidence that
at least four subunits are present in rat whole brain
( 1, 2, 3, and
4) and that 2 subunit is present in rat
cardiac tissue. The multiple bands seen in immunoblot of
1, 2, and 4 subunit may be
attributable to post-translational modification or multiple subunit
isoforms (Collin et al., 1993 ; Chien et al., 1995 ). These same four
polyclonal antibodies were used to detect the subunits in PC12
cells. 3 subunit was easily detected from crude
membranes of both undifferentiated and differentiated PC12 cells,
whereas no other subunits were ever detected (Fig. 5B).
This suggests a dominant expression of 3 subunit in PC12
cells. An ~10-fold enrichment by a 2 antibody affinity
column was required to visualize the 2 subunit by
immunoblot analysis. As shown in Figure 5B, a 70 kDa
2 subunit appeared in the eluate of the 2
antibody affinity column. The size of this 2 subunit is
comparable to the post-translational modified form of this subunit
expressed in human embryonic kidney cells (Chien et al., 1995 ).
Fig. 5.
3 subunit is dominantly expressed
and is the major form of subunit of the N-type Ca2+
channels in PC12 cells. A, Four subunit-specific
antibodies recognize native subunits from rat tissues. Shown are
ECL immunoblots of crude rat tissue membranes stained by four
affinity-purified subunit-specific antibodies. Crude rat brain
membranes were probed with polyclonal antibodies to 1,
3, and 4 subunits, and rat cardiac
microsome membranes were probed with polyclonal antibodies to
2 subunit. B, Expression of
3 and 2 subunit protein in PC12 cells.
Shown are an ECL immunoblot of crude PC12 membranes stained with
affinity-purified 3 subunit antibody and an ECL immunoblot of the eluate from a 2 antibody affinity
column stained with 2 antibody. C,
Association of different subunits with the 1B
subunit of the PC12 N-type Ca2+ channel. Shown is the
immunoprecipitation of [125I] -CgTX GVIA receptors by
protein G (PG), four subunit antibodies, and
polyclonal antibodies against 1B subunit from
undifferentiated and differentiated PC12 cells. The error bar
represents the SE of three replicates.
[View Larger Version of this Image (30K GIF file)]
Because expression of the 1B subunit of the N-type
Ca2+ channel is upregulated by NGF (Lievano et al., 1994),
we investigated whether the subunit composition of this channel is
modified by NGF treatment. To examine the interaction between
1B and subunits in PC12 cells, the above-mentioned
four subunit-specific antibodies and polyclonal antibodies against
1B GST fusion protein (Y006) were used for
immunoprecipitation of [125I] -CgTX-labeled N-type
Ca2+ channels (Fig. 5C). Polyclonal antibodies
against 3 immunoprecipitated 74 ± 4%
(n = 3) of [125I] -CgTX labeled
receptors from undifferentiated PC12 membrane extracts, followed by
2 antibody 22 ± 3% (n = 3). No
immunoprecipitation was detected with protein G beads alone used as a
negative control. In addition, polyclonal antibodies against
1b and 4 subunits were unable to
immunoprecipitate significant amounts of labeled receptors from
undifferentiated PC12 membrane extracts (<10%), which may result from
either the low level of expression or the sensitivity of the methods.
Therefore, the 1B subunits are associated with either
3 or 2 subunits in the N-type
Ca2+ channels of undifferentiated PC12 cells.
NGF treatment induced striking morphological changes in PC12 cells.
After 3 d of NGF treatment, >70% of PC12 cells in culture had
grown neurites longer than the cell diameter (data not shown). Lievano
et al. (1994) has reported that the upregulation of the 1B mRNA expression reached the maximum plateau as early
as day 1 of NGF application. Therefore, the association of the
different subunits with the 1B subunit was examined
in the 3 d NGF-treated PC12 cells (Fig. 5C). The total
expression of 1B subunits per milligram of membrane
protein was increased ~59%. Notably, the polyclonal antibody against
3 immunoprecipitated 74 ± 2% (n = 3) of [125I] -CgTX-labeled receptors, followed by
2 antibody 21 ± 3% (n = 3).
Again, polyclonal antibodies against 1b and
4 subunits immunoprecipitated negligible amounts of
[125I] -CgTX-labeled receptors. Therefore, the same
proportion of 1B subunits in both undifferentiated and
differentiated PC12 cells is associated with either the
3 or 2 subunits, although the expression
of N-type Ca2+ channels increased significantly after NGF
treatment.
DISCUSSION
In this paper, we provide evidence for the presence of
-Aga-IVA-sensitive Ca2+ channels in undifferentiated
PC12 cells. This component of the whole-cell Ca2+ current
has not been reported previously in this cell line. Our results confirm
previous reports (Usowicz et al., 1990 ; Rane and Pollock, 1994 ) that
describe global Ca2+ channel current in undifferentiated
PC12 cells as consisting of a large component of high-voltage-activated
Ca2+ current and no low-voltage-activated Ca2+
current. In addition, preincubation experiments indicated that exposure
to nanomolar concentrations of -Aga-IVA resulted in a significant
decrease in average peak IBa amplitude (Fig. 1). This inhibitory effect was confirmed when IBa
was sequentially recorded from patch-clamped cells before and after
toxin application, particularly in cells with large Ca2+
channel activity.
Sequential recording experiments also reveal that PC12 cell cultures
were heterogeneous in their response to the spider toxin. This
functional heterogeneity was characterized by the presence of a subset
of cells apparently insensitive to -Aga-IVA during 6 min exposure.
It is known that -Aga-IVA blockage of Ca2+ channel
current in neurons is greatly time-dependent (Pearson et al., 1995 ;
Randall and Tsien, 1995 ), thus differences in sensitivity to the toxin
among PC12 cells could account for the lack of response observed in
some cases: less susceptible cells could not be inhibited after short
periods of toxin exposure. If this were the case, long periods of time
would be necessary to recruit the entire fraction of
-Aga-IVA-sensitive cells; however, as illustrated in Figure
2A, blockage in PC12 cells is gradual, and
Ca2+ channel activity runs down during continuous
whole-cell recording, hampering the analysis of slow-developing effects
of the toxin in individual cells. A second possibility could be that
PC12 cultures were indeed composed of -Aga-IVA-sensitive and
-insensitive cell subpopulations. The presence of subpopulations with
differential expression of distinct Ca2+ channel types has
been documented previously in neurons (Christenson et al., 1993 ) and
endocrine cells (Felix et al., 1993 ) in culture. Likewise, a
combination of both possibilities cannot be ruled out.
Taken together, these data indicate that -Aga-IVA partially inhibits
a high-threshold Ca2+ current in undifferentiated PC12
cells and strongly suggest that a significant proportion of the current
in these cells can be attributed to the activity of P/Q-type
Ca2+ channels in their plasma membrane. Interestingly, when
analyzed by subtraction (lower trace in Fig.
2B), the waveform of the current blocked by
-Aga-IVA showed attributes described previously for the current
induced by 1A Ca2+ channel subunit
expression in Xenopus oocytes (Sather et al., 1993 ; Stea et
al., 1994 ; De Waard and Campbell, 1995 ), and it is comparable in
relative magnitude and the degree of decay to the Q-type
Ca2+ channel current in cerebellar neurons (Randall and
Tsien, 1995 ). Although we have no information so far regarding the
possible functional significance of the -Aga-IVA-sensitive current
component in PC12 cells, it may be associated with neurotransmitter
release as has been documented in chromaffin cells (Artalejo et al.,
1994 ).
The presence of functional -Aga-IVA-sensitive Ca2+
current is in agreement with the expression of the 1A
subunit mRNA in undifferentiated PC12 cells. In addition, mRNA
expression of 1B and 1C (isoform rbc-I)
is consistent with the presence of N-type and L-type Ca2+
currents in PC12 cells, and the detection of specific receptors for
125I- -CgTX (N-type) and [3H]PN200-110
(L-type) in the PC12 membranes (data not shown). Therefore, the
expression of these different 1 subunits provides the
molecular basis for the heterogeneity of Ca2+ currents in
PC12 cells. Furthermore, our data demonstrate that not only several
1 subunits ( 1A, 1B, and
1C) but also multiple subunits are expressed in the
undifferentiated PC12 cells. Using a sensitive RT-PCR assay, mRNA
expression of three subunits ( 1, 2,
and 3) was detected from PC12 cell. In contrast, control experiments from RNA without reverse transcription failed to amplify any signal, indicating that RT-PCR products were amplified from PC12
cDNA. The inability to detect 1 in immunoblot analysis
is probably attributable to technical difficulty because of a low expression level of Ca2+ channel subunits. The novel
finding that three 1 and three subunits are
expressed in PC12 cells suggests that multiple 1 and subunits are likely to be expressed in other neuronal cell types,
because several different types of Ca2+ currents can be
recorded from many neurons such as sympathetic neurons and cerebellum
granule cells (Mintz and Bean, 1993 ; Pearson et al., 1995 ; Randall and
Tsien, 1995 ). Considering the relative uniformity of PC12 cells, it is
possible that a single PC12 cell may express multiple 1
and subunits, which raises the question how these 1
and subunits are assembled in a single cell.
Protein level of the Ca2+ channel subunits in PC12 cells is
very low for immunoblot analysis. However, 3 and an
2 subunit (data not shown) can be detected on ECL
immunoblot. There did not appear to be a significant change of
expression level per milligram of total protein for 3 or
the 2 subunit after NGF treatment. This may be
attributable to the experimental error associated with ECL immunoblot
analysis, which makes it difficult to detect less than a twofold change
in protein level. On the other hand, immunoprecipitation experiments
showed ~59% increase of the 1B subunit after NGF
treatment. Similarly, the level of the subunits associated with the
N-type channel increased in approximately the same proportion after NGF
induction.
In our study, NGF treatment did not significantly alter the subunit
composition of the N-type Ca2+ channels despite an increase
in total number of N-type channels. 3 subunit remains
the major subunit of the N-type Ca2+ channel after NGF
treatment. This indicates that NGF regulates the expression of
different subunits in a similar manner. Furthermore, this suggests
that NGF upregulates the expression of functional N-type channels
without changing the properties of the channels.
Our results indicate that either 3 or 2
subunits are associated with the 1B subunits of the
N-type Ca2+ channel in PC12 cells, which is in agreement
with the subunit heterogeneity of N-type Ca2+ channels
observed in rabbit brain (Scott et al., 1996 ). Although we detected the
same order of association
( 3> 4> 1b) for different subunits with the 1B subunit in rat brain tissue as
in rabbit brain (data not shown), the order of association for subunits in PC12 cells ( 3> 2) is clearly
different from that of mammalian brain. Whereas numerous neuronal cell
types are present in brain, PC12 cells are rather homogeneous, although
individual cells may differ to some extent. It is therefore important
to demonstrate that in a single cell type, the 1B
subunit is associated with either of the two subunits. In addition,
this is the first evidence that 2 subunit can associate
with the N-type channel. Noticeably, the association of
3 subunit with the 1B subunit correlates with the dominant expression of this subunit in PC12 cells suggesting a
role for the expression level of subunits in determining the subunit association with the calcium channels.
The diversity of Ca2+ channel currents in PC12 cells arise
from the expression of at least three 1 subunits and
three subunits. Whereas 1 subunits determine the
major properties of the current type (N, L, P/Q), different subunits may further modify the properties of the Ca2+
channels. In support of a previous finding that three of brain subunits are associated with the N-type channel (Scott et al., 1996 ),
at least two populations of N-type channels ( 1B
3 and 1B 2) are present in
a single PC12 cell line that may differ in their function and/or
localization. However, NGF does not appear to modify the fraction of
each population over the 3 d period we examined. Therefore, the
properties of the N-type channels resulting from the subunit
association remain unchanged before and after NGF induced
differentiation. In the future, PC12 cells can serve as a neuronal cell
model for additional investigation of the regulation and function of
N-, L-, and P/Q-type Ca2+ channels during development.
FOOTNOTES
Received July 8, 1996; revised Sept. 18, 1996; accepted Sept. 24, 1996.
K.P.C. is an Investigator of The Howard Hughes Medical Institute. H.L.
is supported by a predoctoral fellowship from the American Heart
Association, Iowa Affiliate. R.F. is supported by a postdoctoral fellowship from the Human Frontier Science Program. We thank the University of Iowa DNA Core Facility for DNA sequencing.
Correspondence should be addressed to Dr. Kevin P. Campbell, The Howard
Hughes Medical Institute, University of Iowa College of Medicine, 400 EMRB, Iowa City, IA 52242.
Dr. De Waard's present address: INSERM U374, Institute Jean Roche,
Marseille Cedex 20, France 13916.
Dr. De Witcher's present address: Protein Products, Protein
Biochemistry, Pioneer Hi-Bred International Inc., 7300 NW 62nd Avenue,
Johnston, IA 50131-1004.
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J. M. Schjott, S.-C. Hsu, and M. R. Plummer
The Neuronal {beta}4 Subunit Increases the Unitary Conductance of L-type Voltage-gated Calcium Channels in PC12 Cells
J. Biol. Chem.,
September 5, 2003;
278(36):
33936 - 33942.
[Abstract]
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R. Del Toro, K. L. Levitsky, J. Lopez-Barneo, and M. D. Chiara
Induction of T-type Calcium Channel Gene Expression by Chronic Hypoxia
J. Biol. Chem.,
June 13, 2003;
278(25):
22316 - 22324.
[Abstract]
[Full Text]
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M. B. Kulick and I. von Kugelgen
P2Y-Receptors Mediating an Inhibition of the Evoked Entry of Calcium through N-Type Calcium Channels at Neuronal Processes
J. Pharmacol. Exp. Ther.,
November 1, 2002;
303(2):
520 - 526.
[Abstract]
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K. N Green, J. P Boyle, and C. Peers
Hypoxia potentiates exocytosis and Ca2+ channels in PC12 cells via increased amyloid {beta} peptide formation and reactive oxygen species generation
J. Physiol.,
June 15, 2002;
541(3):
1013 - 1023.
[Abstract]
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S. C. Taylor, K. N. Green, I. F. Smith, and C. Peers
Prion protein fragment 106-126 potentiates catecholamine secretion from PC-12 cells
Am J Physiol Cell Physiol,
December 1, 2001;
281(6):
C1850 - C1857.
[Abstract]
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L. G Weigl, M. Hohenegger, and H. G Kress
Dihydropyridine-induced Ca2+ release from ryanodine-sensitive Ca2+ pools in human skeletal muscle cells
J. Physiol.,
June 1, 2000;
525(2):
461 - 469.
[Abstract]
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A. Elhamdani, M. E. Brown, C. R. Artalejo, and H. C. Palfrey
Enhancement of the Dense-Core Vesicle Secretory Cycle by Glucocorticoid Differentiation of PC12 Cells: Characteristics of Rapid Exocytosis and Endocytosis
J. Neurosci.,
April 1, 2000;
20(7):
2495 - 2503.
[Abstract]
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M. Jia, M. Li, X.-W. Liu, H. Jiang, P. G. Nelson, and G. Guroff
Voltage-Sensitive Calcium Currents Are Acutely Increased by Nerve Growth Factor in PC12 Cells
J Neurophysiol,
December 1, 1999;
82(6):
2847 - 2852.
[Abstract]
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L. Taupenot, M. Mahata, S. K. Mahata, and D. T. O’Connor
Time-Dependent Effects of the Neuropeptide PACAP on Catecholamine Secretion : Stimulation and Desensitization
Hypertension,
November 1, 1999;
34(5):
1152 - 1162.
[Abstract]
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S. C. Taylor, T. F. C. Batten, and C. Peers
Hypoxic Enhancement of Quantal Catecholamine Secretion. EVIDENCE FOR THE INVOLVEMENT OF AMYLOID beta -PEPTIDES
J. Biol. Chem.,
October 29, 1999;
274(44):
31217 - 31222.
[Abstract]
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S C Taylor, M L Roberts, and C Peers
Acid-evoked quantal catecholamine secretion from rat phaeochromocytoma cells and its interaction with hypoxia-evoked secretion
J. Physiol.,
September 15, 1999;
519(3):
765 - 774.
[Abstract]
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H. Jiang, K. Takeda, P. Lazarovici, Y. Katagiri, Z.-X. Yu, G. Dickens, A. Chabuk, X.-W. Liu, V. Ferrans, and G. Guroff
Nerve Growth Factor (NGF)-induced Calcium Influx and Intracellular Calcium Mobilization in 3T3 Cells Expressing NGF Receptors
J. Biol. Chem.,
September 10, 1999;
274(37):
26209 - 26216.
[Abstract]
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S. C. Taylor, E. Carpenter, M. L. Roberts, and C. Peers
Potentiation of Quantal Catecholamine Secretion by Glibenclamide: Evidence for a Novel Role of Sulphonylurea Receptors in Regulating the Ca2+ Sensitivity of Exocytosis
J. Neurosci.,
July 15, 1999;
19(14):
5741 - 5749.
[Abstract]
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E. H. Gerstin Jr., T. McMahon, J. Dadgar, and R. O. Messing
Protein Kinase Cdelta Mediates Ethanol-induced Up-regulation of L-type Calcium Channels
J. Biol. Chem.,
June 26, 1998;
273(26):
16409 - 16414.
[Abstract]
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C. L. Vance, C. M. Begg, W.-L. Lee, H. Haase, T. D. Copeland, and M. W. McEnery
Differential Expression and Association of Calcium Channel alpha 1B and beta Subunits during Rat Brain Ontogeny
J. Biol. Chem.,
June 5, 1998;
273(23):
14495 - 14502.
[Abstract]
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B. Ligon, A. E. Boyd III, and K. Dunlap
Class A Calcium Channel Variants in Pancreatic Islets and Their Role in Insulin Secretion
J. Biol. Chem.,
May 29, 1998;
273(22):
13905 - 13911.
[Abstract]
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M. Solem, T. McMahon, and R. O. Messing
Protein Kinase A Regulates Inhibition of N- and P/Q-type Calcium Channels by Ethanol in PC12 Cells
J. Pharmacol. Exp. Ther.,
September 1, 1997;
282(3):
1487 - 1495.
[Abstract]
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O. T. Jones, G. M. Bernstein, E. J. Jones, D. G. M. Jugloff, M. Law, W. Wong, and L. R. Mills
N-Type Calcium Channels in the Developing Rat Hippocampus: Subunit, Complex, and Regional Expression
J. Neurosci.,
August 15, 1997;
17(16):
6152 - 6164.
[Abstract]
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S. C. Taylor, K. N. Green, E. Carpenter, and C. Peers
Protein Kinase C Evokes Quantal Catecholamine Release from PC12 Cells via Activation of L-type Ca2+ Channels
J. Biol. Chem.,
August 25, 2000;
275(35):
26786 - 26791.
[Abstract]
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S. Berjukow, R. Marksteiner, F. Gapp, M. J. Sinnegger, and S. Hering
Molecular Mechanism of Calcium Channel Block by Isradipine. ROLE OF A DRUG-INDUCED INACTIVATED CHANNEL CONFORMATION
J. Biol. Chem.,
July 14, 2000;
275(29):
22114 - 22120.
[Abstract]
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H. J. Walter, T. McMahon, J. Dadgar, D. Wang, and R. O. Messing
Ethanol Regulates Calcium Channel Subunits by Protein Kinase C delta -dependent and -independent Mechanisms
J. Biol. Chem.,
August 11, 2000;
275(33):
25717 - 25722.
[Abstract]
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