©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
An Electrophysiological Study of Yeast Mitochondria
EVIDENCE FOR TWO INNER MEMBRANE ANION CHANNELS SENSITIVE TO ATP (*)

(Received for publication, February 24, 1995; and in revised form, April 19, 1995)

Cristina Ballarin M. Catia Sorgato (§)

From the Dipartimento di Chimica Biologica, Università di Padova, Via Trieste 75, 35121 Padova, Italy

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

A novel aspect of mitochondrial physiology has been disclosed by the application of electrophysiological techniques to the inner (IM) (^1)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.


MATERIALS AND METHODS

Preparation of Yeast Mitoplasts and of Proteoliposomes for Patch Clamp Recordings

The wild type yeast strains of S. cerevisiae D273-10B (kindly given by Dr. W. Neupert, University of München, Germany) and HR125-2B and the mutant bearing the deletion of the VDAC gene, M22-2 (kindly given by Dr. M. Forte, Oregon University), were grown under standard aerobic conditions. Spheroplasts were obtained by use of zymolyase, from which mitochondria were isolated according to (8) , except for the presence of EDTA (1 mM) in the homogenization medium and for the final centrifugation at 3000 g to pellet mitochondria. Mitochondria were suspended (at a concentration of approximately 10-20 mg of protein/ml) in 0.6 M mannitol, 10 mM Tris-HCl, 1 mM phenylmethylsulfonyl fluoride, pH 7.4. Having observed that the protocol for mammalian mitoplasts (3) led to destruction of yeast mitochondria, two alternative milder hypotonic procedures were used. In one, the initial step was followed (swelling of mitochondria in 110 mosm) for the isolation of mitochondrial intermembrane components described in (8) . 2-5 µl of the resulting pellet, suspended in the assay buffer (see below), were then placed on the patch clamp dish and covered with approximately 1 ml of the same medium. At the optical microscope, among vesicles of different size, some were observed with a diameter (1-2.0 µm) larger than that of parent mitochondria. These vesicles closely resembled mammalian mitoplasts for the presence of the cap region and thus, by analogy, were selected for electrophysiological analysis. Even if fresh mitoplasts were prepared approximately every 1 h, good seals formed only within a very limited period of time, probably because the high osmotic stress rendered the IM fragile (the tonicity of the swelling medium (110 mosm) being around one-sixth that of a yeast cell). However, vesicles with similar appearance but with a slightly more resistant membrane formed in approximately 5-10 min by diluting, directly in the patch clamp dish, few microliters of the mitochondrial stock suspension in the assay buffer (of around 350 mosm) (see below). Patch clamp results were identical with either preparation. Hence, despite the lower yield, the second preparation was that more often preferred.

Using approximately 200 µg of mitochondrial proteins, large proteoliposomes were prepared as described in (7) and (9) .

Patch Clamp Technique

Patch clamp experiments (10) were performed at 18-22 °C using, unless otherwise specified, symmetrical solutions of 150 mM KCl, 20 mM Hepes (pH 7.2 with KOH), 0.1 mM CaCl(2). Compared to liver mitoplasts, with the yeast IM giga seals were more difficult to obtain (in less than 10% of the attempts), probably because of the small dimensions of the vesicles and the sticky texture of the membrane. Moreover, as several of the good seals broke within a few seconds (35%) or were silent (14%), analyzable data were obtained in only 5% of the attempts. Sylgard-coated electrodes (Kimax, Kimble Division, Vineland, NJ) with resistance higher than 10 M were used. When needed, suitable amounts of EGTA were added to yield a free Ca final concentration of 10M. Except for selectivity measurements or with proteoliposomes, the given sign of the potential refers to that found inside the vesicles. Ion selectivity was determined on excised patches from the reversal potential value (E) measured under asymmetrical KCl; the bath contained the 150 mM KCl solution, while the pipette had 30 mM KCl, 20 mM Hepes, 0.1 mM CaCl(2), 240 mM sucrose, pH 7.2. By necessity, E was sometimes calculated after application of voltage ramp protocols (see ``Results'').

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.

Chemicals

Zymolyase 20T (Arthrobacter luteus) was purchased from Seikagaku Corp. (Tokyo). Phospholipids for liposomes preparation(7) , nucleotides, and all inhibitors tested were purchased from Sigma, except for bongkrekic acid (kindly given by Dr. F. Dabbeni-Sala, University of Padova, Italy) and adenylylimidodiphosphate obtained from Boehringer (Mannheim, Germany). Sylgard (RTV 615) was from General Electric.


RESULTS

Patch Clamping of the Native Yeast IM

The patch clamping of yeast mitoplasts obtained from wild type and VDAC-less strains showed the presence of two distinct channels readily distinguishable on the basis of the intensity of the current flowing through them. Thus, they will be referred to as the small and large channels according to their conductance.

Small Channel

A reproducible kinetic behavior pertained to the small channel, observed alone in 28% of the patches (n = 46). As evident from the current-voltage relationships (Fig. 1, leftpanels), in all strains the conductance was maximal at positive and slightly negative potentials (51 ± 2.4 pS (A) and 45 ± 0.3 pS (B) for the wild type D273-10B and HR125-2B strains, respectively, and 44 ± 2 pS for the VDAC-less strain (C)). The channel exhibited a mean open time () whose value and voltage dependence varied also rather uniformly in all strains (Fig. 1, rightpanels). Incidentally, an increased flickering activity combined with a shorter length of the events was responsible for the lower at positive potentials. Data of Fig. 1were obtained from records presumably showing the activity of only one channel (see ``Materials and Methods''). In fact, at any imposed voltage the channel opened with low frequency; for instance, 15-20 events per min were typical at -40 mV, although sometimes only 5 events per min were counted. These events were too sparse to allow the meaningful evaluation of the open probability (P(o)) and of the of a (putative) single channel so that these parameters were calculated from records with a higher frequency of openings, i.e. when at least two channels were clearly present (Fig. 2). The higher P(o) found at negative voltages for the channel of the HR125-2B strain (Fig. 2A) was consistent with the decrease of the (Fig. 2B) and with the increase of the (Fig. 1B) in the same voltage range. These parameters varied uniformly also in all other strains (not shown).


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(2), 20 mM Hepes-KOH, pH 7.2, were used.




Figure 2: Variation with voltage of the P(o) (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(o) 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(K) 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(K) 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(o), 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(o) of only 50%. 2 mM Mg, added to excised patches, did not affect the kinetics nor the P(o) 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).

Large Channel

This second type of channel was found alone with a higher incidence (44%, n = 71). However, it was not unusual for both channels to be observed in the same membrane patch (in 22 out of 161 patches) with unchanged characteristics. The large channel displayed a much larger conductance and a marked voltage dependence. Voltage affected the large channel in two ways. On the one hand it modified the conductance, which varied from approximately 800 pS, at 40 mV, to around 400 pS, at -40 mV (Fig. 5A). The other effect was on the gating mechanism. From traces recorded after imposition of a voltage ramp protocol (Fig. 5B) or voltage pulses (Figs. 6, A and B), it is evident that the channel P(o) diminished sharply at negative potentials. Traces of Fig. 6A reveal a different kinetics associated with the closure and opening process, the former being much faster than the latter. We also observed that the higher the voltage the higher the number of openings and the shorter the time needed for all steps to take place (not shown).


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 10M 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 10M 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(o) 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(o) = [(a - b)/1 + (k/[ATP])] + b, where a and b represent the maximum and minimum P(o) values, k the ATP concentration causing 50% increase of P(o), 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(K) 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.

Patch Clamping of Proteoliposomes Containing Both Mitochondrial Membranes

Most of the records, obtained from proteoliposomes with mitochondrial membranes of yeast, were too complicated for analysis. On the rare patches with more discrete activity, two conductances were, however, identified. Fig. 8, A-C, contains traces and data analysis of records of a proteoliposome patch with membranes from the VDAC-less strain. The channel of Fig. 8C resembled for the conductance value the small channel described in the native IM. On the contrary, the other conductance most frequently detected (of around 500 pS) behaved differently than the large channel of mitoplasts (Fig. 8A). Indeed, there was no sign of current rectification (compare Fig. 8B, upperplot, with Fig. 5A) nor of the tendency to close at negative potentials (compare the lowerplot of Fig. 8B with Fig. 5B and 6). Additionally, selectivity was cationic (P(K)/P = 4.85) (not shown). Finally, voltage ramp protocols imposed to a proteoliposome patch with mitochondrial membranes of a wild type strain (Fig. 8D) revealed similar large conductances, although the computed average current (tracea) demonstrated a different voltage dependence than that displayed by the 500-pS channel of the mutant strain membranes in proteoliposomes.


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(0), C(1), and C(2) 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(0) (*), C(1) (), and C(2) (bullet) are shown. Slope conductances, computed by linear regression, had the following values: 1.286 ± 0.01 nS (bullet), 0.801 ± 0.013 nS (), 0.295 ± 0.021 nS (*). In the lowerplot, voltage dependence of the P(o) of each current level, C(0) (*), C(1) (), and C(2) (bullet), is shown. P(o) 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.




DISCUSSION

Functional Properties of the Yeast IMM Cl Channels

Despite the difficulty in obtaining mitoplasts from S. cerevisiae amenable to patch clamp analysis (see ``Materials and Methods''), their appearance was so close to mammalian mitoplasts (see (11) ) that, most likely, the patch clamp experiments reported here have been carried out on the expanded IM. The finding, at the molecular level, of high conductance pathways in the native IM of yeast mitochondria thus definitively establishes that channels are a fundamental property of the eukaryotic IM. Two distinct channels have been identified; a few times they also appeared in the same patch (Fig. 7A, inset), which further supports their belonging to the same membrane. Although having in common a slight preference for anions and an outwardly rectifying current-voltage relationship (Fig. 1, 3, and 5), the two channels were clearly physically and behaviorally independent. One of the two, defined small channel, was detected also after reconstitution in proteoliposomes (Fig. 8C). It had a conductance of around 45 pS (in symmetrical 150 mM KCl), was marginally sensitive to voltage, and its activity was completely blocked by mM matrix ATP (Fig. 1, 2, and 4). Conversely, the other channel (defined large) had at least a 10-fold higher conductance, opened predominantly at positive potentials, and matrix ATP locked it in the open conformation (Fig. 5-7).

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) .

Interaction with ATP

Physiological concentrations of matrix ATP (Mg free) modified both channels but with a different mode of action. Importantly, the reported effects were detected in those excised (inside out) patches where each channel displayed the physiological behavior (i.e. a higher flickering of the small channel and a higher P(o) of the large channel at positive potentials). Data of Fig. 4show the profound inhibitory effect of the nucleotide on the small channel. Conversely, ATP provided a switch in the gating mechanism of the large channel, which immobilized the channel in the open state also at physiological (negative) values of the mitochondrial membrane potential (Fig. 7).

Physiological Significance of the Yeast IMM Cl Channels

The electric characteristics and the particular response to physiological compounds allow to exclude the identity of both Cl channels to other known IM proteins of yeast and mammals. For example, contrary to what is reported in this paper, the so called anion channel, described spectrophotometrically in yeast and mammals(12, 13) , and, in mammals, probably also electrophysiologically(14) , is controlled by matrix Mg. Likewise, the lack of effect of bongkrekic acid argues against the candidacy of the adenine nucleotide translocator. Recently, swelling experiments with yeast mitochondria have allowed to identify in the IM another poorly specific anion uniport whose activity was greatly enhanced by mM ATP(15) . Some of the features reported for this protein do resemble closely those of the large channel described here. However, the location of the binding site for ATP on the external side of the IM prevents, for the time being, the identity of the two proteins.

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.


FOOTNOTES

*
This work was supported by grants from the Italian Consiglio Nazionale delle Ricerche and Ministero dell` Universita e della Ricerca Scientifica e Tecnologica. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Fax: 39-49-807-3310.

(^1)
The abbreviations used are: IM, inner membrane; IMM, inner mitochondrial membrane; OM, outer membrane; pS, picosiemens; nS, nanosiemens; mCS channel, mitochondrial centum picosiemens channel; VDAC, voltage-dependent anion channel; P(o), open probability.


ACKNOWLEDGEMENTS

We are indebted to Dr. Oscar Moran for helpful discussions.


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