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J. Biol. Chem., Vol. 278, Issue 35, 33284-33289, August 29, 2003
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From the Unitat de Senyalització Cel·lular, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, C/ Dr. Aiguader 80, Barcelona 08003, Spain
Received for publication, March 19, 2003 , and in revised form, May 16, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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subunit) but not caveolin-1. Transfection of C1300 cells with an antisense
oligonucleotide directed against the specific membrane leader sequence of VDAC
markedly reduced both VDAC immunostaining and antiestrogen-activated Maxi
Cl– currents, suggesting that VDAC forms the plasma membrane
Maxi Cl– channel or a part thereof. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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The molecular identity of Maxi Cl– channels1 has been even more difficult to prove. The fact that Maxi Cl– channel electrophysiological properties resemble those of the mitochondrial voltage-dependent anion channel (VDAC) (11) encouraged some investigators to assume that the two channels were one and the same protein (12, 13). This hypothesis was based on early observations suggesting the presence of VDAC protein on the plasma membrane (12, 14) but was questioned by others (15). The proposal suggesting the presence of VDAC in extramitochondrial locations has received recent strong support from two independent reports: 1) the identification of a VDAC isoform (pl-VDAC) that contains a leader sequence for its trafficking to the plasma membrane (16) and 2) the presence of VDAC in caveolae (17). In the present study, we have evaluated the presence of VDAC in the plasma membrane of C1300 neuroblastoma cells and, more interestingly, its contribution to the Maxi Cl– currents activated by antiestrogens in these cells.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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Preparation of Cell Lysates and Plasma Membrane Fractions—T-75 flasks containing confluent cells were washed three times with 5 ml of PBS without calcium, magnesium, and sodium bicarbonate (Invitrogen). After the final wash, 600–1,000 µl of ice-cold cell lysis buffer (50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 0.5% Brij-35, 10 µg/ml phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml leupeptin, and 10 µg/ml aprotinin) was added per T-75 flask. The cell lysate was transferred to a 2-ml Eppendorf tube, rocked for 30 min at 4 °C, and subsequently centrifuged at 10,000 x g for 10 min at 4 °C. A 50-µl aliquot was removed from the supernatant and stored at –20 °C for subsequent determination of protein concentration (Method DC protein assay, BioRad). The remaining supernatant was aliquoted into cryotubes and frozen rapidly in liquid nitrogen before being stored at –70 °C. Plasma membrane proteins were isolated using a previously described protocol (18, 19).
Immunoblotting—Proteins were resolved by SDS-PAGE (12%) and blotted onto nitrocellulose. The primary antibodies used were: monoclonal mouse anti-porin 31 HL (Calbiochem) raised against the N terminus of human VDAC1 (1:2,000 dilution), monoclonal mouse anti-cytochrome oxidase subunit IV (1:1,000; Molecular Probes) to the corresponding complex of the mitochondrial oxidative phosphorylation system, and goat polyclonal anti-ribophorin I (1:100; Santa Cruz Biotechnology) that recognizes integral membrane glycoproteins that localize exclusively to the rough endoplasmic reticulum. Non-specific binding was avoided by incubating the nitrocellulose membranes in a blocking solution consisting of Tween 20-Tris buffer solution (100 mM Tris-HCl, pH 7.5, 150 mM NaCl) supplemented with 5% non-fat milk for 1 h at room temperature or overnight at 4 °C. Mouse and rabbit antibodies were detected either with alkaline-phosphatase-conjugated antibodies (goat anti-mouse IgG at a 1:500 dilution (Calbiochem)) or peroxidase-conjugated antibodies (sheep anti-mouse IgG or donkey anti-rabbit IgG, both at a 1:2,000 dilution (Amersham Biosciences)). The membranes were then washed, and the bands were visualized by using the enhanced chemiluminescence substrate Super Signal (Pierce) and autoradiographed on either Amersham Biosciences Hyperfilm ECL or nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate toluidine salt solution for detection of alkaline phosphatase.
Confocal Microscopy—Double-label analysis was carried out on
C1300 cells adhered to glass coverslips coated with poly-D-lysine.
Cells were fixed in 2% paraformaldehyde, 0.15 M sucrose, and 0.1%
glutaraldehyde for 10 min and permeabilized with 0.1% Triton for 10 min. Prior
to antibody incubation, cells were treated with NH4Cl for 30 min to
minimize the number of reactive aldehyde groups and blocked for 30–60
min (room temperature) with 5% fetal bovine serum and 1% bovine serum albumin
in washing buffer. Cells were then incubated with anti-porin 31 HL
(20)(1:80) or rabbit
anti-caveolin-1 (N20, Santa Cruz Biotechnology) for 2 h at room temperature.
Unbound antibody was removed by washing the cells three times with 1 ml of
blocking solution for 10 min each time. Staining with FITC-labeled cholera
toxin subunit (CTX) was performed under non-permeabilized
conditions by incubating the cells with blocking solution for 30 min and then
staining them with FITC-CTX for 30 min at room temperature.
Subsequently, cells were washed, fixed, and permeabilized as described above.
Following several washes in blocking solution, cells were incubated with
secondary Alexa Fluor 488 goat anti-rabbit (1:500) and/or goat anti-mouse IgG
coupled to the fluorochrome Cy3 (1:2,000) for1hat room temperature. Prior to
incubation, the secondary antibody was centrifuged at 13,000 x
g for 15 min at 4 °C to pellet any precipitated constituents.
Negative controls were performed in which the cells were solely incubated with
the secondary antibody. Digital images were taken with a Leica TCS SP confocal
microscope and analyzed with Leica confocal software (Heidelberg,
Germany).
Antisense Oligonucleotides—Cells were seeded in 24-well
dishes (NUNC) at a concentration of 104 cells/well 2 days before
transfection. On reaching 60% confluency, the cells were exposed to an
antisense oligonucleotide hybridizing to the pl-VDAC specific leader sequence
(5'-CAC GAG AAA GAA TGA ACA C-3') and then plated onto
poly-D-lysine-coated coverslips. -Globin antisense
oligonucleotide (5'-CCT CTT ACC TCA GTT ACA ATT TAT A-3') was used
as the negative control. Immediately before transfection, cells were washed
and placed in 200 µl of fresh serum-free Dulbecco's modified Eagle's
medium. Each well received 200 ng of antisense pl-VDAC plus 100 ng of pEGFP
plasmid (Clontech) diluted into 25 µl of serum-free DMEM. Cells were
transfected by a LipofectAMINE Plus (Invitrogen) procedure following the
manufacturer's instructions. Each transfection experiment was carried out in
triplicate, and the cells were used for both path-clamp and immunofluorescence
experiments.
RNA Extraction and RT-PCR—RNA extraction and RT-PCR were performed as described previously (19). In brief, RNA was isolated from C1300 cells using the Nucleospin RNA II kit (Macherey-Nagel, Düven, Germany) according to the manufacturer's instructions. Total RNA (1 µg) was reverse-transcribed to cDNA. We used the following primer pair for PCR amplification of pl-VDAC (16): forward, 5'-TGT GTT CAT TCT TTC TCG TGC-3', and reverse, 5'-CCA GTG TTC GGC GAG AAT GAC-3'. PCR products were analyzed on a 2% agarose gel containing a final concentration of 0.5 µg/ml ethidium bromide.
Electrophysiology and Statistics—C1300-transfected cells were plated on 13-mm poly-D-lysine-coated glass coverslips and mounted on the stage of an inverted Olympus IX70 microscope. Ion currents were recorded 48–72 h after transfection using the inside-out or whole-cell patch clamp modes as described previously (8). The pClamp8 software (Axon Instruments, Foster City, CA) was used for pulse generation, data acquisition through an Axon Digidata A/D interface, and subsequent analysis. Borosilicate glass patch pipettes had 3–5 megaohms resistance and were filled with a solution containing 140 mM N-methyl-D-glucamine chloride, 1.2 mM MgCl2, 1 mM EGTA, and 10 mM HEPES (295 mosmol/liter, pH 7.3). ATP (4 mM) and GTP (0.1 mM) were added to the pipette solution for whole-cell experiments. Cells were bathed in a solution containing 140 mM N-methyl-D-glucamine chloride, 1.3 mM CaCl2, 0.5 mM MgCl2, 10 mM HEPES (305 mosmol/liter, pH 7.4), which was also the solution bathing the cytoplasmic face of the patch membrane in inside-out experiments. Data were collected using a List Medical D-6100 Darmstadt or Axopatch 200A amplifier. Inside-out currents were acquired at 1 kHz and low pass-filtered at 1 kHz. Membrane patches were clamped at 0 mV and pulsed for 5 s to both –80 mV and +80 mV. Whole-cell currents were acquired at 5 kHz and low pass-filtered at 1 kHz. Cells were clamped at 0 mV and pulsed for 500 ms from –80 to +80 mV in 40-mV steps before and after addition of toremifene (10 µM) to the bath solution. All experiments were performed at room temperature (22–26 °C). Data are expressed as means ±S.E. Differences between the three different groups were compared by one-way analysis of variance followed by the Bonferroni post-test for multiple comparisons. Data are taken as significant at a probability of 0.05.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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32 kDa, which is the expected size for VDAC, was
obtained from both preparations, indicating the absence of cross-reactivity
with other cellular proteins. Contamination of the membrane fraction with
organelle membranes was tested with antibodies against the cytochrome oxidase
IV (a mitochondrial marker) or ribophorin I (endoplasmic reticulum marker).
Fig. 1B shows that our
membrane preparation is negative for the mitochondrial marker but positive for
the marker of the endoplasmic reticulum, an organelle where the presence of
VDAC has already been reported
(22).
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The experiments shown in Fig. 1, A and B, are consistent with the targeting of VDAC through two different pathways: the plasma membrane (via the endoplasmic reticulum/golgi pathway) and the mitochondria. The lack of a mechanism to explain how this protein is selected for its targeting to the plasma membrane, instead of the outer mitochondrial membrane, has fuelled the reticence to accept its extramitochondrial localization (15). However, the recent identification of an alternative exon in the murine vdac-1 gene evidenced a plasmalemmal form of VDAC (pl-VDAC) with an N-terminal leader sequence for its targeting to the plasma membrane (16). The presence of a pl-VDAC form in C1300 cells was confirmed by RT-PCR using primers specific to the pl-VDAC, including the leader sequence (Fig. 1C). A band of the expected size (350 bp) was identified and confirmed to be pl-VDAC by sequencing.
Another piece of evidence suggesting the presence of VDAC in the plasma
membrane has been its isolation from liquid-ordered membrane microdomains
called lipid rafts and functional reconstitution in artificial bilayers
(17). To test whether VDAC was
also present in such specialized membrane microdomains in C1300 cells, we
carried out colocalization experiments using confocal microscopy. Two markers
for lipid raft microdomains were used: the 1 subunit of
cholera toxin, which binds to GM1 ganglioside, with which lipid
rafts are enriched, and caveolin-1, a protein present in more structured lipid
rafts with flask-shaped membrane invaginations termed caveolae
(23,
24).
Fig. 2 shows confocal images of
C1300 cells probed with anti-VDAC antibody
(Fig. 2, A and
D, in red) and shows anti-caveolin 1 antibody
(Fig. 2B, in
green) or cholera toxin 1 subunit
(CTX-1) conjugated to FITC
(Fig. 2E, in
green). Merged images (Fig. 2,
C and F) show an almost complete overlap of
CTX-1 and VDAC at the plasma membrane level, whereas no clear
VDAC overlapping was observed with caveolin-1. These results reinforced
previous observations suggesting the presence of VDAC at the plasma membrane,
and similar to other cell types
(17), VDAC in C1300 cells
segregates to GM1-containing lipid rafts. Caveolin-1 has been
detected in cells from neuronal origin such as pheochromocytoma PC12 cells
(25), although its
localization varies with the degree of cell differentiation, mainly appearing
in the soma of undifferentiated PC12 cells, similar to the pattern observed in
our study with C1300 neuroblastoma cells. The apparent lack of colocalization
of VDAC with caveolin-1 in C1300 cells might be related to the
undifferentiated state of the cells and, therefore, the low presence of the
caveolin-1 at the plasma membrane. However, experiments using differentiated
C1300 cells, confirmed by the appearance of well developed neurites, did not
modify the localization pattern of either caveolin-1 or
VDAC.2 Further
immunolocalization experiments using antibodies against other caveolin
proteins expressed in neuronal cells will be necessary to elucidate the
presence of VDAC in caveolae of C1300 cells.
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The large conductance chloride channel, Maxi Cl– channel,
has been recorded under both cell-attached and whole-cell patch clamp
conditions from C1300 cells exposed to triphenylethylene antiestrogens such as
tamoxifene and toremifene (8).
The activation of Maxi Cl– channels appears to involve the
interaction of antiestrogens with an external plasma membrane binding site and
the generation of intracellular signals
(8). The plasma membrane Maxi
Cl– channel shares many of the electrophysiological and
pharmacological characteristics of the mitochondrial VDAC
(11), implying that VDAC might
be the molecular correlate of Maxi Cl– channels
(13). This hypothesis received
additional experimental support from excisedpatch recording studies of Maxi
Cl– channels from cells expressing heterologous VDAC protein
(16,
26) and their functional
inhibition with VDAC antibodies
(26). However, these
approaches can be criticized based on the fact that control cells used for
transfection already express the channel of interest and, secondly, the
absence of a correlation between immunodetection of VDAC protein and Maxi
Cl– channel activity in VDAC-transfected cells. Therefore, we
set out to investigate whether endogenous pl-VDAC might underlie the
antiestrogen-activated Maxi Cl– current in C1300 cells. For
that purpose, we transfected C1300 cells with antisense oligonucleotides
against the 5' end of the murine vdac-1 gene. The
oligonucleotide hybridized with the leader sequence required for targeting
VDAC to the plasma membrane
(16). The control antisense
oligonucleotide was directed against an unrelated protein, -globin. The
use of an EGFP-expressing plasmid served as a reporter gene for the
identification of transfected cells. Fig.
3 shows images of cells transfected with pl-VDAC antisense/EGFP
(Fig. 3, A–C) or
-globin antisense/EGFP (Fig. 3,
D–F). VDAC levels in transfected cells were
determined by immunofluorescence confocal microscopy with the anti-VDAC
antibody (Fig. 3, A and
D) and the efficiency of the transfection by the
production of EGFP protein (shown as the fluorescence signal in
Fig. 3, B and
E). From the images shown in
Fig. 3 and the graph presented
in Fig. 3G, a clear
inverse correlation can be seen between the EGFP and VDAC signals in those
cells transfected with pl-VDAC antisense and EGFP but not in those cells
transfected with -globin antisense/EGFP. Once we demonstrated that the
pl-VDAC antisense oligonucleotide reduced VDAC levels, we proceeded to analyze
the activity of Maxi Cl– channels.
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Whole-cell Maxi Cl– currents were recorded under control
conditions and following the addition of 10 µM toremifene to the
bathing solution in cells transfected with either -globin antisense
(Fig. 4A) or pl-VDAC
antisense oligonucleotides (Fig.
4B). Transfection with the latter greatly reduced the
magnitude of the toremifene-activated Maxi Cl– currents,
whereas transfection with -globin antisense oligonucleotides did not
modify the channel response. The mean Maxi Cl– channel
activity recorded from untransfected, -globin antisense-, and pl-VDAC
antisense-transfected cells exposed to toremifene is shown in
Fig. 4C. We also
tested by Western blot and confocal microscopy that VDAC expression was not
affected by the treatment of C1300 cells with antiestrogens (results not
shown).
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Maxi Cl– channels can also be activated following the excision of the membrane patch containing the channel (1, 8). This stimulus was also used to evaluate the Maxi Cl– channel activity in cells exposed to pl-VDAC antisense. Fig. 5 shows representative excised inside-out single channel recordings obtained from control cells (Fig. 4A) or pl-VDAC antisense-treated cells (Fig. 5B). In the control cells, typical Maxi Cl– channel transitions of about 300 picosiemens were present. The voltage dependence was identical to the whole-cell currents described above with rapid inactivation at negative potentials. On the traces obtained from pl-VDAC antisense-treated cells, no such channel activity was observed. The probability of finding Maxi Cl– channels in the excised patches should decrease with reduction in the amount of VDAC protein. Accordingly, Maxi Cl– channel activity was detected in 100% (8/8) of patches obtained from control cells but also in 60% (10/16) of patches obtained from pl-VDAC-treated cells, without changes in the single channel conductance (280 ± 15 picosiemens in control cells, n = 8, and 270 ± 10 picosiemens in pl-VDAC antisense-treated cells, n = 10; p = 0.5) or the open probability (results not shown). However, we acknowledge that these experiments are less representative than the whole-cell experiments described above, where the activity of all channels present in the plasma membrane was studied. Nevertheless, the Maxi Cl– channel activity was reduced in pl-VDAC antisense-treated cells, regardless of the stimuli used (activation by toremifene or membrane excision). Mitochondrial VDAC is inhibited by 4,4'-diisothiocyanatostilbene-2,2'-sisulfonic acid (DIDS). Therefore, we carried out experiments to check whether the plasma membrane Maxi Cl– channel was also inhibited by DIDS. Fig. 5C shows current-voltage relationships obtained from control cells treated with toremifene in the presence or absence of 100 µM DIDS. Addition of DIDS completely abolished the toremifene-activated Maxi Cl– currents of C1300 cells.
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Voltage-dependent anion channels (VDAC), also known as porins, are integral membrane proteins that form a pore slightly more permeable to anions than cations and also permeable to small solutes. They have been identified in the mitochondrial outer membrane, where they provide a major pathway for the transport of metabolites, e.g. ATP (27) and cholesterol (28), among others. They have also been involved in the mitochondrial events leading to apoptosis (4, 5). Besides their mitochondrial location, several reports claim the presence of VDAC in the plasma membrane of different cell types (13, 14, 16, 17). In the present study, in addition to demonstrating the association of VDAC with membrane lipid rafts of C1300 neuroblastoma cells, we have provided the first molecular evidence relating VDAC with plasma membrane Maxi Cl– channels activated by antiestrogens. The identification of the molecular nature of the Maxi Cl– channel will allow us to study whether its regulation by estrogen and antiestrogens presents a double pathway, involving the generation of intracellular signals and a direct interaction of the hormones with the channel protein, similar to the modulation of Maxi K+ channels by estrogens (29, 30).
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 34-93-542-2832; Fax:
34-93-542-2802; E-mail:
miguel.valverde{at}cexs.upf.es.
1 The abbreviations used are: Maxi Cl– channel, large
conductance voltage-dependent chloride channel; Maxi K+ channels,
large conductance Ca2+-activated K+ channels;
CTX, cholera toxin subunit; DIDS,
4,4'-diisothiocyanatostilbene-2,2'-sisulfonic acid; EGFP, enhanced
green fluorescent protein; VDAC, voltage-dependent anion channel; pl-VDAC,
plasma membrane VDAC; FITC, fluorescein isothiocyanate; RT, reverse
transcription.
2 M. I. Bahamonde, unpublished observations.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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), in the presence of 10
µM toremifene (tore, •), and following the
superfusion of toremifene plus 100 µM DIDS (
).