Patch-clamp experiments on mitoplasts (mitochondria with the
inner membrane exposed) have detected a multiple conductance channel,
MCC (
)(also referred to as mitochondrial megachannel, MMC),
which is activated by
calcium(1, 2, 3, 4, 5, 6) .
This multisubstate channel activity is voltage-dependent and has a peak
conductance of 1-1.5 nanosiemens (see (2) and (4) for recent reviews). Szabó and
Zoratti (5) have proposed that MCC may be responsible for a
permeability transition in mitochondria. Halestrap and Davidson (7) have postulated that the adenine nucleotide translocator
(ANT) is intrinsic to a permeability transition. Recently, Zoratti and
Szabó(4) have speculated on the
possibility that MCC may be associated with a dimer of ANT plus other
proteins.
The protein(s) responsible for MCC activity have not been
identified. However, it is possible that MCC may be associated with a
membrane transporter like ANT since many transporters display channel
activity when reconstituted in bilayers and proteoliposomes. These
transporters include the
(Na
/K
)-ATPase, the chloroplast triose
phosphate/phosphate translocator, and members of the ATP binding
cassette superfamily of transporters, e.g. cystic fibrosis
transmembrane conductance regulator and multidrug-resistant
transporters(8, 9, 10, 11, 12, 13, 14) .
In fact, channel activity similar to that of MCC has been reported in
black lipid membranes fused with proteoliposomes containing protein
fractions of ANT(15) . Possible relationships between MCC and
transporters can now be examined by using deletion mutants of
mitochondrial transporters in yeast. Unfortunately, the other dominant
inner mitochondrial membrane channel (mitochondrial centum picosiemen
channel) has not yet been detected in yeast(16) .
In the
present work, we have used patch-clamp techniques to compare the MCC
activity in proteoliposomes reconstituted with mitochondrial inner
membranes from ANT-deficient and wild-type strains of Saccharomyces
cerevisiae. In addition, we have applied similar techniques to
mouse kidney mitoplasts to see whether MCC activity is affected by
carboxyatractyloside, which is a specific inhibitor of
ANT(17) .
MATERIALS AND METHODS
Mitochondria, Mitoplast, and Proteoliposome
Preparations
Mouse kidney mitochondria were isolated as
described previously(18) , and mitoplasts were prepared using
the French press method (at 2000 p.s.i.) of Decker and
Greenawalt(19) . Mitochondria from S. cerevisiae strain M3 (wild type, graciously provided by M. Forte, Vollum
Institute) and strain JL-1-3 (a triple mutant in which all three
genes (AAC1, AAC2, AAC3) encoding
mitochondrial ANT have been disrupted) (20) were isolated by a
modification of the method of Daum et al.(21) . In
general, yeast channel activities were studied after reconstitution
into proteoliposomes. Isolated yeast mitoplasts (prepared by French
press) were osmotically lysed and spun on a sucrose gradient according
to the method of Mannella(22) . Pellets from the gradient spin
were used as inner membranes, which were reconstituted into giant
proteoliposomes using the dehydration-rehydration method of Criado and
Keller (23) in 0.15 M KCl, 5 mM HEPES, pH
7.4, as described previously(6, 16) . Inner membrane
yields were
10 and 2.5 mg of protein from overnight cultures of 20
g (2 liters) and 17 g (1.5 liters) of strains M3 and JL-1-3 cell
pellets, respectively. Approximately 10% of each inner membrane
preparation (0.25-1 mg of protein) was added to liposomes (
5
mg of Sigma type IV-S soybean L-
-phosphatidylcholine) for
a typical reconstitution.
Patch Clamping
The procedures and conditions used
for patch clamping mitoplasts and proteoliposomes are essentially the
same as previously reported(18, 24) . The reported
voltage is that of the mitochondrial matrix in inside-out excised
patches from mitoplasts, where V = V
- V
. Voltages are reported relative to
the bath for proteoliposome patches since the orientation of MCC (as
indicated by voltage dependence and kinetics) is the same as in
mitoplasts. Open probability is calculated as the fraction of the total
time spent in the fully open state from amplitude histograms using the
PAT program (Strathclyde Electrophysiological Software, courtesy of J.
Dempster, University of Strathclyde, UK). All yeast experiments were in
done in 0.15 M KCl, 5 mM HEPES, 1 mM EGTA,
1.05 mM CaCl
(10
M free Ca
), pH 7.4. Selectivity was estimated from
the reversal potential with a 5:1 KCl gradient as described previously (16) . The level of MCC activity in mammalian mitoplasts was
varied by exposure to calcium as in (3) and the patching media
as indicated in Table 1.
Effectors
Peptides were prepared by the Wadsworth
Center's peptide synthesis core facility on an Applied Biosystems
431A automated peptide synthesizer as described previously(6) .
The targeting peptides used were the amino termini of cytochrome
oxidase subunit VI (
MLSRAIFRNPVINRTLLRAR
) and
subunit IV (
MLSLRQSIRFFKY
) of S.
cerevisiae and subunit IV (
RAPALRRSIATTVVRCNAET
) of Neurospora
crassa. The control peptide was the amino terminus from N.
crassa voltage-dependent anion-selective channel (
MAVPAFSDIAKSANDLLNKD
). Carboxyatractyloside
(Sigma) was included in the medium in the patch pipette (and usually
the bath) at concentrations of 5 or 10 µM. The presence or
absence of carboxyatractyloside was alternated every 3-5 patches
within each preparation of mammalian mitoplasts.
Verification of Phenotypes by Western Blot
Analysis
Western blot analysis was performed on both wild-type
and ANT-deficient purified mitochondrial inner membranes. Samples (10
µg of protein) were boiled in SDS-polyacrylamide gel
electrophoresis sample buffer, electrophoresed in 14% polyacrylamide
gel, and blotted onto a polyvinylidene difluoride membrane (Novex, San
Diego, CA). Samples were then probed with anti-ANT antibodies
recognizing all three isoforms of ANT (graciously provided by J.
Kolarov and I. Hapala) and visualized with a horseradish
peroxidase-coupled second antibody (Pierce) with diaminobenzidine as a
substrate(25) .
RESULTS
MCC Is Present in ANT-deficient
Yeast
Patch-clamp techniques were used to characterize the MCC
activity in proteoliposomes containing mitochondrial inner membranes
from a wild-type strain of yeast and a strain in which the three ANT
genes were disrupted. Western blot analysis confirmed the absence of
ANT in the inner membranes prepared from the later strain as shown in Fig. 1. Fig. 2illustrates typical channel activity
detected in proteoliposomes derived from the ANT-deficient strain (n = 8 patches). The peak conductance state (
1.1
nanosiemens in Fig. 2) and half-open substate are typical of MCC
transitions observed in preparations from wild-type
yeast(6, 16) . MCC activity from the ANT-deficient
strain is voltage-dependent, occupying near-peak conductance levels in
the potential range between -40 mV and +20 mV. At larger
amplitude transmembrane potentials of either polarity, the channel
tends to occupy lower conductance levels including a low noise closed
state (occurring at +40 mV in Fig. 2). This
voltage-dependent behavior is the same as that of reconstituted MCC
from wild-type strains of yeast(2, 6, 16) .
Figure 1:
ANT is
absent in AAC1, AAC2, AAC3 deletion mutant. Western blot
analysis using an antibody recognizing all three isoforms of ANT was
performed on mitochondrial inner membranes. The blot shows the presence
of ANT in membranes from wild-type yeast (lane 1) and the
absence of ANT in membranes from ANT-deficient (lane 2) yeast
mitochondria.
Figure 2:
MCC activity is present in ANT-deficient
yeast. A, current traces of MCC were recorded at various
voltages from an excised patch from a proteoliposome reconstituted with
mitochondrial inner membranes isolated from ANT-deficient yeast. Data
were bandwidth-limited to 2 kHz and sampled at 5 kHz. Total amplitude
histograms of current traces at -40 mV (B) and 30 mV (C) (bin width, 0.4 pA; duration, 15-20 s) show the
occupancy of open (O), substate (S), and closed (C) conductance levels.
No significant differences between the MCC activity of wild-type and
ANT-deficient yeast were resolved. MCC activity of the ANT-deficient
strain is slightly cation-selective (P
/P
6, n = 2, data not shown) like that of the wild-type
strain (P
/P
6, (16) ). Peptides whose sequences target precursor
proteins to the mitochondrial inner membrane specifically induce a
transient blockade of MCC from the ANT-deficient strain (n = 7 patches) as previously reported for MCC from the
wild-type strain (6) (Fig. 3). Furthermore, the
frequency of recording MCC is about the same from proteoliposomes from
the wild-type (75%, n = 12 patches) and ANT-deficient
(88%, n = 8 patches) yeast strains. We also noted that
the 50 picosiemens channel activity often detected in wild-type yeast
proteoliposomes (16) was also present in the ANT-deficient
yeast proteoliposomes (data not shown).
Figure 3:
MCC activity from ANT-deficient yeast is
peptide sensitive. A, current traces show the transient
blockade of MCC in the presence of 50 µM targeting peptide
(amino terminus of yeast cytochrome oxidase subunit VI). B,
amplitude histograms show a shift in the occupation of conductance
levels in the presence (
) and absence (
) of the peptide.
Other conditions are the same as in Fig. 2.
Mammalian MCC Is Not Affected by Carboxyatractyloside, an
ANT Inhibitor
Carboxyatractyloside stabilizes the nucleotide
binding site of ANT on the cytoplasmic side of the inner membrane,
thereby blocking the exchange of matrix ATP and cytoplasmic ADP (K
10
M)(17) . The effect of this ANT inhibitor on MCC
activity was determined in mouse kidney mitoplasts. As shown in Table 1, the frequency of recording MCC was not changed by the
presence of carboxyatractyloside under a variety of conditions. That
is, carboxyatractyloside does not appear to activate MCC under low
calcium conditions that normally favor quiescent MCC(3) .
Likewise, this ANT inhibitor does not appear to inhibit MCC activated
by calcium. As shown in Table 1, the seal resistance was
unchanged by the presence of carboxyatractyloside, indicating that
there was no masking of MCC in a stabilized open state. (If MCC were
immobilized in an open state, resistance would be below 0.5-1
gigaohm). The studies were restricted to the potential range of
±30 mV to avoid voltage activation of MCC(26) . (In
general, the presence of MCC in patches scored inactive was verified by
activation of MCC by higher voltages (±60-100 mV) in the
presence and absence of carboxyatractyloside.)
DISCUSSION
ANT Is Not Responsible for MCC Activity
The
protein(s) responsible for MCC have not yet been identified but Zoratti
and Szabó(4) have speculated that this
channel activity might be associated with a dimer of the transport
protein ANT (plus other proteins). Tikhonova et al.(15) detected channel activity similar to that of MCC in
black lipid membranes fused with proteoliposomes containing purified
fractions of ANT after mersalyl addition. In addition, mersalyl induced
a high conductance pathway in the proteoliposomes that was not
sensitive to carboxyatractyloside. Tikhonova et al.(15) suggest that mersalyl may modify ANT so that it can
operate as a channel and suggest that ANT may function as a
permeability transition pore-forming protein. Similarly, Dierks et
al.(27) report mersalyl induces an ATP efflux from
proteoliposomes co-reconstituted with ANT and the aspartate/glutamate
carrier. This efflux was inhibited 50% by concentrations of
carboxyatractyloside that also eliminated ANT-mediated nucleotide
exchange (27) but had no effect on aspartate efflux.We used
a molecular genetics approach to test the hypothesis that ANT was
responsible for MCC activity. This has advantages over biochemical
approaches, which may be compromised or confounded by trace
contamination by channel proteins that are detectable with patch-clamp
techniques. Channel activity with the same conductance, ion
selectivity, voltage dependence, and peptide sensitivity as MCC was
reconstituted from inner membrane fractions derived from mitochondria
of ANT-deficient and wild-type yeast strains. These results strongly
indicate that ANT is not responsible for MCC. This conclusion was
supported by experiments which showed carboxyatractyloside, a known
inhibitor of ANT and an activator of a permeability transition, had no
effect on mammalian MCC. However, this work does not eliminate the
possibility that ANT may have channel activity unrelated to MCC.
MCC and the Mitochondrial Permeability
Transition
A permeability transition can be induced in
mitochondria by calcium and a trigger, e.g. phosphate.
Similarly, carboxyatractyloside is known to induce a permeability
transition in mitochondria(7, 28) . Furthermore, a
model has been proposed in which the c conformation of ANT (favored by
carboxyatractyloside) is integral to a permeability
transition(7) . Szabó and Zoratti have
proposed that MCC activity is responsible for a permeability transition (5) since they share many characteristics including estimated
pore size and pharmacology (see (2) and (4) for
recent reviews). However, it is not likely MCC is associated with the
permeability transition induced by carboxyatractyloside since, in this
study, MCC activity was insensitive to this transition inducer. This
apparent conflict may, however, be resolved since studies indicate that
the induction of the permeability transition by carboxyatractyloside
may be indirect by modifying adenine nucleotide
levels(4, 29) .The diversity of permeability
transition effectors leads one to speculate that more than one high
permeability pathway may be involved in these transitions. For example,
cyclosporine inhibits the calcium-induced transition but not the
butylated hydroxytoluene-induced transition(30) . If multiple
pathways exist, our findings suggest MCC is not responsible for the
permeability pathway induced by carboxyatractyloside.
Conclusions
Reconstituted MCC activity is the same
in yeast lacking all three ANT genes as it is in the wild type. In
addition, mammalian MCC is not affected by carboxyatractyloside, a
known inhibitor of ANT. These findings indicate that the inner membrane
translocator ANT is not responsible for the multiple conductance
channel activity.