(Received for publication, February 24, 1995; and in revised form, April 19, 1995)
From the
The inner membrane of mitochondria from various strains of Saccharomyces cerevisiae has been analyzed with the patch
clamp technique for comparison with the better known homologous
membrane in mammals (Sorgato, M. C., and Moran, O.(1993) CRC Crit.
Rev. Biochem. Mol. Biol. 18, 127-171). Differently than in
mammals, the yeast inner membrane was found to harbor essentially two
channels with similar anionic selectivity but otherwise different
functional behavior. One had a conductance of around 45 picosiemens (in
symmetrical 150 mM KCl) and an activity only marginally
sensitive to voltage. The other channel was prominent for the higher
outwardly rectifying current and for the dependence upon voltage of the
open probability that induced rapid closure at physiological (negative)
membrane potentials. Particularly interesting was the effect of ATP
(Mg free) added on the matrix side of the membrane.
In the case of the lower conducting channel, the nucleotide caused an
immediate block of activity (IC
, 0.240 mM),
whereas it locked the larger conductance in the open state at both
positive and negative potentials. In proteoliposomes containing both
mitochondrial membranes, the small conductance was clearly evident,
whereas a larger channel, cationic and without the voltage dependence
typical of that in the native inner membrane, was found.
A novel aspect of mitochondrial physiology has been disclosed by
the application of electrophysiological techniques to the inner (IM) ()and outer (OM) membranes of mitochondria. In particular,
the direct analysis of the native membranes with a patch clamp
electrode has provided strong evidence of the presence of high
conductance ion channels (reviewed in (1) ). By applying this
technique to the IM of mitoplasts (obtained from mitochondria by
removal of the OM), at least three types of channels have been found.
One has a conductance of around 10 pS (in 100 mM salt) and is
distinguishable for the selective permeability toward potassium ions.
It is not regulated by voltage but is sensitive to a number of drugs
and physiological effectors, including matrix ATP, which acts as
inhibitor in the mM range(2) . Conversely, the main
feature of the other channel, slightly anionic and with a conductance
of around 100 pS (in 150 mM KCl) (mitochondrial centum
picosiemens (mCS) channel), is the voltage dependence(3) . At
positive (unphysiological) matrix potentials the channel is mainly
open, whereas the open probability decreases as the voltage is made
negative. Interestingly, the current-voltage relationship of the entire
IM shows an identical behavior, thus suggesting a major role of mCS
channels in the electric activity of this membrane(1) . The
third type of channel, frequently referred to as mitochondrial
megachannel, is notable for the peak conductance of 1-1.3 nS (in
150 mM KCl) and the multiple
substates(4, 5) . For the similar response to a
variety of negative and positive effectors, the megachannel has been
tentatively identified with the mitochondrial permeability transition
pore and/or with the mitochondrial benzodiazepine receptor (for this
topic, see (6) ). On the other hand, the electric features
displayed in planar bilayers by voltage-dependent anion channel (VDAC),
the most abundant OM protein, have not been detected in integral
mitochondria analyzed with a patch clamp electrode, by which at least
three other channels were identified(7) .
Undoubtedly, much has been learned by tackling mitochondria with electrophysiological tools. A number of unknowns, however, remain (for example, the physiological significance of the presence of high conductive pathways in the mitochondrial membranes, in particular of those in the IM, or their structural identity). Until now, almost all electrophysiological studies concerning the native inner mitochondrial membrane (IMM) have been carried out using mammalian tissues. In the light of the very similar physiology shared by this membrane in yeast and mammalian cells, it seemed of value to compare their electric properties. Indeed, were mammalian channels absent in yeast, this would imply that they perform a function specific to higher eukaryotes. Conversely, should they be present, then this result would indicate that channels are a fundamental property of all mitochondria. A long term aim of this study relates to the molecular understanding of IM channels, given that use of yeast mutants is the faster route to unraveling the structure and function of proteins.
We report here the patch clamping of yeast mitoplasts isolated from two wild type strains of Saccharomyces cerevisiae and from a mutant lacking the VDAC gene. In all cases, channel activity was detected, but the overall electric behavior of the IM diverged from that observed in mammals. The electrophysiological analysis of proteoliposomes containing both mitochondrial membranes is also presented.
Using approximately 200 µg of mitochondrial proteins, large proteoliposomes were prepared as described in (7) and (9) .
Channel activity was recorded and stored
unfiltered on videotape using a pulse code modulator A/D VCR adapter
(VR10A, Instrutech Corp., Great Neck, NY). Either an Axopatch-200 (Axon
Instruments, Inc., Foster City, CA) or a L/M-PC (List-Medical,
Darmstadt, Germany) amplifier was used for voltage clamping and current
amplification. Records were replayed, low-pass filtered with an
eight-pole Bessel 902 filter (Frequency Devices, Inc., Haverhill, MA)
at a corner frequency of 1 kHz, and transferred to an Atari computer
using a ITC-16 interface (Instrutech Corp.) and a M2 LAB Atari data
acquisition system at a sample rate of 5 kHz. Data were analyzed either
with an M2-Analysis software for Atari computers (Instrutech Corp.) or
with a MAC-TAC program for Macintosh MAC II computers (Instrutech
Corp.). Unless otherwise stated, event duration analysis was performed
on records containing a single channel, i.e. when the
simultaneous opening of two channels was not observed during the course
of the experiment. However, due to the rare openings typical of the low
conductance channel (see ``Results''), in this case such
criterion may not have been necessarily valid. For the sparse events
and in view of the fast (flickering) closures present only at positive
voltages, calculated mean closed times () refer
to silent periods between two consecutive events. Additions (of a
maximal volume of 10% that of the bath) were usually made with an
automatic pipette, and the dish volume was measured at the end of the
experiment. No effect was detected if equal volumes of the experimental
medium alone were added. In the case of ATP (1.2 mM), its
effect on the small channel was also checked by perfusing the chamber
with the nucleotide in the assay medium. Perfusion, however, was
generally avoided because most patches broke under this condition. Na
salts of the nucleotides (pH 7.2) were used.
Figure 1:
Current-voltage relationships (left) and variation with voltage of (right) of the small channel of yeast mitoplasts.
Excised or mitoplast-attached patches were obtained from the wild type
yeast strains D273-10B (A) and HR125-2B (B) and from the VDAC-less M22-2 strain (C).
Data points of the current-voltage relationships were derived from
current amplitude histograms at the given potentials. Each straightline shows the best fit to the data; the corresponding
slope conductance values are reported in the figure. Event duration
yielded a single
value. The sign of the potential
refers to that found inside the mitoplast. Mitoplasts were obtained
from mitochondria after removal of the OM by osmotic shock (see
``Materials and Methods''). Symmetrical solutions of 150
mM KCl, 0.1 mM CaCl
, 20 mM Hepes-KOH, pH 7.2, were used.
Figure 2:
Variation with voltage of the P (A) and of
(B) of the small channel of yeast mitoplasts. Data were
collected from a mitoplast-attached patch of the wild type
HR125-2B strain containing at least two channels. Event duration
yielded a dominant
value at all potentials
shown. Each value of P
and
was calculated on 55-s records. For other experimental details,
see the legend to Fig. 1.
Data so far presented argue in favor of a same protein responsible for the activity of the small channel in the various strains. This was further supported by the similar preference for anions (Fig. 3) and, most importantly, by inhibition exerted by ATP (Fig. 4).
Figure 3:
Selectivity measurements of the small
channel of yeast mitoplasts. Inside out patches with a putative single
channel (see ``Materials and Methods''), obtained from yeast
mitoplasts of the wild type HR125-2B strain (A) and of
the VDAC-less M22-2 strain (B), were examined under
asymmetrical salt (150 mM KCl in the bath, 30 mM KCl
in the pipette). From the E values (-19.2
mV (A) and -9.7 mV (B), pipette potentials)
calculated from the linear regression of the experimental points, P
/P
ratios were 3.3 and 1.8
for the wild type and mutant strain, respectively. An identical
experiment with a wild type mitoplast gave a P
/P
of 3.2. For other
experimental details, see the legend to Fig. 1.
Figure 4:
Inhibitory effect of ATP on the activity
of the small channel of yeast mitoplasts (A) and dose response
curve (B). A, uppertraces were
recorded at -50 mV, before and after the addition of 1.2 mM sodium ATP, from a mitoplast of the wild type HR125-2B
strain. Lowertraces were obtained at -30 mV by
applying the same protocol to a VDAC-less mitoplast. In either case,
the patch contained at least two channels. Interruptions indicate a
time span of 4 s, and bars represent the closed state of the
channel. Gain, 50 mV/pA. Filter, 1 kHz. B, dose response curve
for inhibition by ATP, determined in a wild type HR125-2B
mitoplast, at -30 mV. The P, determined in
the first 2 min after the nucleotide addition, was normalized with
respect to the value in the absence of ATP. The shown final ATP
concentrations were achieved by consecutive additions, at 0 mV, of a
concentrated solution of the nucleotide. The curve, fitted by the
computer, yielded an IC
of 0.24 mM. In both A and B, inside out patches were used. For other
experimental conditions, see the legend to Fig. 1.
Fig. 4A shows the instant block
of channel activity occurring when mM concentrations of ATP
were added to an inside out patch of a wild type (uppertrace) or mutant (lowertrace)
mitoplast. Seldom, sparse events reappeared after more than 5 min from
the addition. The dose response curve of Fig. 4B shows
that the inhibitory action was complete at around 1 mM ATP
(IC, 0.24 mM). UTP (1 mM) was equally
inhibitory, whereas adenylylimidodiphosphate (1.25 mM) or cAMP
(1.4 mM) were unable to suppress channel activity.
Interestingly, however, in the presence of 1.4 mM cAMP, an
equal concentration of ATP reduced the channel P
of only 50%. 2 mM Mg
, added to excised
patches, did not affect the kinetics nor the P
of
the channel nor, added before ATP (1 mM), altered the
nucleotide inhibition (data not shown). The small channel was
insensitive to glybenclamide (5 µM), 4-aminopyridine (5
mM), and bongkrekic acid (150 µM).
Figure 5:
Voltage affects the conductance value (A) and the gating mechanism (B) of the large channel
of yeast mitoplasts. A, current-voltage relationships were
constructed from data obtained from excised or mitoplast-attached
patches of the wild type yeast HR125-2B strain (squares and diamonds) and of the VDAC-less M22-2 strain (asterisks). The experimental medium was as described in the
legend to Fig. 1, except for the data marked with diamonds where Ca was 10
M in
both the pipette and the bath. All relationships are clearly non-ohmic.
Each data point was derived from current amplitude histograms. In the
wild type patches, a single channel was present, whereas four channels
were detected in the mutant strain. These latter channels had identical
amplitudes (see also Fig. 6A), thus the reported points
refer to a single channel current. B, superimposition of a
single channel response to 17 identical voltage ramp protocols (from
-30 to 30 mV for a total time of 2 s; holding potential at 0 mV
for 200 ms). The behavior of the channel upon imposition of one such
protocol is shown in bold. Note that negative voltages induce
closure of the channel and also reduce the conductance value (the dashedline indicates the current that would have
flown were the channel in the full conductance state at negative
voltages). The mitoplast-attached patch was obtained from the wild type
HR125-2B strain. The bar indicates the closed state of
the channel. The symmetrical 150 mM KCl medium contained
10
M Ca
. Gain, 10 mV/pA.
Filter, 1 kHz. For other experimental conditions, see the legend to Fig. 1.
Figure 6: Voltage dependence of the large channel of yeast mitoplasts assayed in VDAC-less M22-2 (A) and HR125-2B (B) mitoplast-attached patches. A, a step at -20 mV (for 1 s) was followed by a step at 20 mV (for 1 s) with a 200-ms interval at 0 mV. B, the trace is the outcome mean current of 46 successive pulse protocols consisting of a step at -30 mV followed by one at 30 mV (either 500 ms long), with 200-ms intervals at 0 mV. Continuous recordings of this patch revealed the presence of 9 channels. In either A or B, the gain was 10 mV/pA, and the filter was 2 kHz. Other conditions were as described in the legend to Fig. 1.
ATP, however, changed this behavior
dramatically. Indeed, as reported in Fig. 7A, the
progressive openings and closures of the control (upper)
traces (at 20 and -20 mV, respectively) disappeared upon addition
of the nucleotide (lowertraces) and were substituted
by a constant current. The interpretation of this result envisages that
ATP locked the channels in the open state, although the reduced total
current at -20 mV (by 20-25% with respect to the control),
could also indicate a modification of the structure of the pore at
negative potentials. The dose response curve is shown in Fig. 7B. The kfor ATP depended
upon voltage, i.e. was 0.326 mM at 20 mV and 0.551
mM at -20 mV. Rundown of channel activity in the
presence of the nucleotide was never observed. ATP was equally
effective in all strains (not shown).
Figure 7:
Induction of a permanent open state of the
large channel of yeast mitoplasts by ATP (A) and dose response
curve (B). A, continuous recordings from an inside
out patch of the wild type HR125-2B strain (containing two
channels), at 20 mV (lefttraces) and -20 mV (righttraces), before (-ATP) and after
(+ATP) the addition of 1.4 mM ATP. In the presence of the
nucleotide, the two channels in the patch were kept permanently in the
open state. Both in the absence and in the presence of ATP, 20 mV were
imposed starting from 0 mV, then the potential was switched again to 0
mV (for 1 s, not shown) and finally to -20 mV. Interruptions in
the traces indicate a time span of 3 s (but for the first one in the
upper left trace indicating 30 s), and the bar represents the
closed state. The inset in the upperrightquadrant is an expansion of a piece of the record at
-20 mV, showing the presence of two small channels. Gain, 10
mV/pA. Filter, 1 kHz. B, data on the effect of different ATP
concentrations on the P of the channel at the
shown potentials were collected from the same patch of A.
Records analyzed were at least 30 s long. In the control experiments or
with low ATP, this period of time was sufficient for both channels to
open (at 20 mV) or to close (at -20 mV). After ATP addition, the
protocol detailed in A was imposed. Data were fitted according
to the empirical equation P
= [(a - b)/1 + (k
/[ATP])
]
+ b, where a and b represent the
maximum and minimum P
values, k
the ATP concentration causing 50%
increase of P
, and n the slope parameter.
The final reported ATP concentrations were achieved as described in the
legend to Fig. 4. Other conditions were as described in the
legend to Fig. 1.
The large channel was anionic.
In most patches more than one conductance was present, and thus
selectivity had to be evaluated by imposing a voltage ramp protocol
under asymmetrical salt (see ``Materials and Methods''). The E value of -22 mV (pipette potential)
indicated a P
/P
value of 4.2
(data not shown). Mg
(1-2 mM), added
to excised patches, did not change the behavior of the channel.
Bongkrekic acid (150 µM) was also ineffective.
Figure 8:
A large and a small channel are present in
proteoliposomes containing both membranes of yeast mitochondria. A, continuous recordings at the shown potentials from an
excised patch of proteoliposomes with mitochondrial membranes of the
VDAC-less M22-2 strain. C, C
, and C
are the different current levels observed. B, in the upperplot, current voltage relationships (obtained
from the same patch of A) for the three current levels C
(*), C
(
), and C
(
) are
shown. Slope conductances, computed by linear regression, had the
following values: 1.286 ± 0.01 nS (
), 0.801 ± 0.013
nS (
), 0.295 ± 0.021 nS (*). In the lowerplot, voltage dependence of the P
of
each current level, C
(*), C
(
), and
C
(
), is shown. P
values were
calculated from the ratio of time at the corresponding level over total
time of the records, which was at least 45 s long. C,
expansion of a piece of the record of A (uppertrace)
showing activity of a small channel (below is the relative amplitude
histogram). In D, a is the average current in
response to 50 voltage ramp protocols (from 40 to -40 mV, 500 ms)
imposed to an excised patch with two channels (each with approximately
500-pS conductance) obtained from a proteoliposome containing the
mitochondrial membranes of the HR125-2B strain. In the shown
response to one of such protocols, o is the maximal current
level and c the closed state. The sign of the potential is
that of the pipette. Gain, 10 mV/pA. Filter, 1 kHz. For other
experimental conditions, see ``Materials and Methods'' and
the legend to Fig. 1.
With respect to the voltage sensitivity of the large channel, it is necessary to mention that, after attempting to achieve an inside out patch, the response to voltage was sometimes opposite to that insofar reported. At first, the loss of a modulator during membrane excision seems the most logical interpretation at the basis of these results. Nevertheless, by taking into account the minute size of yeast mitoplasts and the particular texture of the IM, there is no stringent proof of the axiomatic formation of an inside out patch following the standard manipulations from the mitoplast-attached configuration. It is therefore possible that we were examining either a whole mitoplast patch (undetectable from the increase in membrane capacitance) or an outside out patch. However that may be, the physiological response was as shown in Fig. 5B and 6, i.e. open at positive and closed at negative voltages, as there were a number of experiments where the size of the mitoplast was sufficiently big to allow confirmation at the microscope of the cell-attached configuration.
After reconstitution in proteoliposomes of mitochondrial membranes of the wild type or of the VDAC-less strain (Fig. 8), a conductance of around 500 pS was found, which was cationic and lacked the marked voltage dependence that distinguished the large channel in mitoplasts. Therefore, either the reconstitution procedure altered the native protein, or the 500-pS conductance belongs to the OM. Parenthetically, the features reported in Fig. 8D cannot be attributed to VDAC, at least to that behavior displayed in planar bilayers(1) .
To
our knowledge, this is the first detailed description of channels in
the native IM of yeast; thus, comparison with other channels (defined
as such on electrophysiological grounds) is perforce bound to those
present in the homologous mammalian membrane. None of those already
characterized (1) apparently meets all the features reported
here for yeast channels. Despite the selective permeability for
K, the mammalian K
channel, first described in mitoplasts with the patch clamp
technique (2) and afterwards reconstituted in
liposomes(16) , is that which resembles best the yeast small
channel for the low conductance, voltage insensitivity and block by
physiological concentrations of ATP. In fact, these similarities and
the discovery that a K
selective uniport exists also
in yeast mitochondria (13) could suggest that, by working
together, the two channels may control the volume of
mitochondria(16) . As already addressed for the
K
channel(16, 17) , an
unanswered question relates to which mitochondrial factor might be able
to bypass the blockage of the small channel by ATP. In the light of our
findings and of its effect on other Cl
channels of
the plasma membrane(18) , an interesting candidate worth
further investigation is, for example, cAMP.
The behavior of the large channel reveals no clue to its role. Undoubtedly, however, it is mandatory for the channel to be regulated, as a living mitochondrion could hardly tolerate the high ion flux elicited by ATP.
To end, a comment is due to the absence in yeast of the marker mCS channel of the mammalian IMM(1, 3) . It appears unlikely that it escaped our detection. Hence, given the similar physiology of yeast and mammalian mitochondria, the suspect remains that an activator of the mCS channel might get lost during the preparation of yeast mitochondria. On the other hand, at face value, the IMM electric behavior in yeast does differ from that in mammals (see Introduction and (1) ), thus leaving open the possibility that, in low and high eukaryotes, proteins with dissimilar electrophysiological behavior might perform an identical task.