108-pS Channel in Brown Fat Mitochondria Might Be Identical to the Inner Membrane Anion Channel*

(Received for publication, February 27, 1997, and in revised form, May 27, 1997)

Jirí Borecký Dagger §, Petr Jezek Dagger and Detlef Siemen par **

From the Dagger  Department of Membrane Transport Biophysics, Institute of Physiology, Academy of Sciences of Czech Republic, CZ-14220 Prague 4, Czech Republic and the par  Zoologisches Institut, Biologiezentrum, Christian-Albrechts-Universität zu Kiel, D-24118 Kiel, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

Single-channel and whole-mitoplast patch-clamp recordings were employed to characterize the 108-pS (Cl-) channel in brown fat mitochondrial mitoplasts. We demonstrated the ability of this channel to conduct di- and trivalent anions, such as sulfate, phosphate, and benzenetricarboxylates, and its blockage by propranolol, 1,4-dihydropyridine-type Ca2+ antagonists, and Cibacron blue. Moreover, we have revealed its pH dependence for the first time. As a basis for the characteristic potential dependence of the whole-mitoplast current, we identified an open probability, increasing with depolarizing (positive) potentials, Eh, and being almost zero in the hyperpolarizing range. Events at negative Eh exhibit a short flickering behavior, whereas at positive Eh, they become much longer. This voltage dependence is influenced by pH in such a way that, at acidic pH, the 108-pS channel possesses a low open probability throughout the observed potential range, whereas at alkaline pH, the channel switches to long openings, even at a negative potential. All these properties lead us to conclude that the inner membrane anion channel, which has been characterized only by light scattering studies, and the 108-pS inner membrane channel, which has been characterized electrophysiologically, are one and the same process.


INTRODUCTION

The 108-pS channel was the first ion channel to be discovered in mitochondria (1), nevertheless its function remains unknown, and it has not been identified with any known mitochondrial transport activity. It has been also referred as the inner mitochondrial membrane channel (2) or mitochondrial centum picosiemens channel (3). The 108-pS channel has been detected in patch-clamp experiments on mitoplasts from liver (1, 3-5), heart (5), brain (6), and brown adipose tissue (BAT; 7)1 and was characterized as pH-insensitive (1). First attempts to inhibit this channel with known channel blockers failed (5). In BAT mitochondria, the 108-pS channel displays infrequent substates, the main state having a conductance about half that of the fully open state (7), and was reported to be partially blocked by purine nucleotides (7). In mitochondria from other tissues, antimycin (8), protoporphyrin IX, and ligands of the mitochondrial benzodiazepine receptor (9) block the channel. Amiodarone and propranolol decrease the open probability, while increasing the conductance of the channel (10). Sorgato (1) and others (2) recognized the possibility that the 108-pS channel may reflect conductance through the inner membrane anion channel (11, 12); however, many observations appeared to conflict with this identification (1, 2).

Anion uniport in Mg2+-depleted mitochondria was attributed to an inner membrane anion channel (IMAC) by Garlid and Beavis in 1986 (13). The transport properties of IMAC have subsequently been characterized in detail by Beavis and co-workers (12, 14-19). IMAC is inhibited by Mg2+ and greatly activated at alkaline pH. It is nonselective for anions and conducts mono-, di-, and trivalent anions (15, 16, 20). Known inhibitors include dicyclohexylcarbodiimide (15), propranolol (17), organotin compounds (18), sulfhydryl reagents (12, 19), and dyes (12), such as Cibacron blue 3GA (21). IMAC is also inhibited by Ca2+ channel (L-type) antagonists of the DHP class such as niguldipine (22) and of phenylalkylamine and benzothiazepine classes (22). Ca2+ antagonists bind to specific binding sites in mitochondria, presumably on a putative IMAC protein (22). Although no inhibition of IMAC with nucleotides has been found, this as yet unidentified protein might also bear a structure similar to a nucleotide-binding site (21), because its inhibitor Cibacron Blue is known to bind to such sites.

Beavis (12) has proposed that the physiological function of IMAC is to contribute to the contractile phase of volume homeostasis, but this hypothesis has not been tested. Characterization of IMAC has been entirely phenomenological, relying almost exclusively on light scattering (matrix swelling) studies. The protein or gene responsible for the phenomenology has not been identified, nor has the process been reconstituted into liposomes. It is not known, in fact, whether IMAC is a channel at all, as had been proposed (13).

For molecular characterization of IMAC, it would be very useful to identify its corresponding channel activity. To this end, we decided to re-examine the possibility that the 108-pS channel reflects IMAC activity by applying patch-clamp to mitoplasts of BAT mitochondria, which have been shown to contain IMAC (23). We found that the 108-pS channel exhibits the salient properties of IMAC, including substrate specificity, inhibitor specificity, and pH dependence. We conclude that the 108-pS channel is responsible for IMAC activity.2


EXPERIMENTAL PROCEDURES

Chemicals purchased from Merck (Darmstadt, Germany) were: CaCl2, NaCl, KCl, K2SO4, and sucrose; from Sigma (Deisenhofen, Germany): Cibacron blue 3GA, 1,2,3-benzenetricarboxylic acid, 1,3,5-benzenetricarboxylic acid, EGTA, GDP, HEPES, and propranolol; and from Research Biochemicals International (Natick, MA): nifedipine and niguldipine.

Mitochondria were prepared from interscapular brown adipose tissue from 7-21-week-old male hamsters (Mesocricetus auratus), cold-adapted at 6 °C for 4 weeks. Mitochondria from 1 g of BAT were prepared (24) in a medium containing 250 mM sucrose, 5 mM K-HEPES, 1 mM K-EGTA, and 0.1% bovine serum albumin (pH 7.2). The final two washes were performed in 150 mM KCl, 20 mM K-HEPES, 1 mM K-EGTA, and 0.1% bovine serum albumin at pH 7.2. Mitochondria were resuspended in 1 ml of hypertonic medium (750 mM KCl, 100 mM K-HEPES, 1 mM K-EGTA (pH 7.2)) and stored on ice for up to 48 h. Mitoplasts were obtained by hyposmotic shock: removal of the outer membranes of the shrunken ice-cold mitochondria was carried out by sonication (5 × 2-s intervals). Subsequently, the ionic strength was lowered 10-fold by addition of a hypotonic medium (5 mM K-HEPES, 1 mM K-EGTA (pH 7.2)). After a further 1-2-min incubation at room temperature, isotonicity was restored by the addition of the hypertonic medium described above. Mitoplasts were kept on ice for a maximum of 12 h.

Patch-clamp experiments followed the method of Hamill et al. (25). Borosilicate glass pipettes were polished to give a resistance of 9-15 MOmega and were filled by a pipette solution of 150 mM KCl, 20 mM K-HEPES, 1 mM K-EGTA (pH 7.2). Bath solutions were applied by inserting the pipette tip into a pipe of a peristaltic pump-driven "sewer-pipe flow system." Free-floating mitoplasts were "chased" by means of an electrically driven micromanipulator and moved to their final position on the pipette tip by gentle suction. Gigaseals of 5-20 GOmega formed spontaneously in about 80% of the cases, thus improving the method previously reported (7). Signals were recorded by a L/M-EPC 7 patch-clamp amplifier, filtered by a 4-pole Bessel filter at a corner frequency 1 kHz, and sampled at 2.5 kHz. Data recorded during the slow voltage ramps were sampled at 16 Hz. Processing of the data was carried out by means of the pCLAMP6 program package (Axon Instruments, Foster City, CA). Experiments were performed at room temperature (22-27 °C). Various blockers were added by using the described flow system, which gave a constant flow of the blocker around the mitoplasts, and records were repeated every 2-3 min, thus monitoring the approach to the steady-state equilibrium, which was reached usually after 10-13 min. Measurements in symmetrical sulfate were made by using an Ag|AgCl|KCl salt bridge (150 mM KCl in 1% agar) in the patch-clamp pipette interior and with mitoplasts washed solely in sucrose medium.

Data were analyzed by employing plots of integral conductance G = I/Eh versus holding potential Eh. With BAT mitoplasts, these plots showed a sigmoidal shape. The curves represented the transition from an ensemble of mostly closed channels to an ensemble in which the open channels dominated. This allowed the conductance to be determined around 0 mV (which is inaccessible in I-Eh plots) and, the upper limit of the leak conductance GL determined as G near -40 mV. The plots were fitted by using the modified equation of Bräu et al. (26),
G=<FR><NU>I</NU><DE>E<SUB><UP>h</UP></SUB></DE></FR>=<FENCE>G<SUB><UP>L</UP></SUB>+G<SUB><UP>max</UP></SUB> <FR><NU>1</NU><DE>1+e<SUP>&kgr; · (E<SUB><UP>M</UP></SUB><UP>−</UP>E<SUB><UP>h</UP></SUB>)</SUP></DE></FR></FENCE> (Eq. 1)
where Eh is the holding potential (in millivolts), GL is the leak conductance, Gmax is an integral conductance at saturation, EM is a potential at half-saturation, and kappa  is a steepness factor, reflecting the steepness of the transition. kappa  is reciprocal to the Bräu's factor k (26).


RESULTS

"Whole-cell" Current versus Single-channel Events in BAT Mitoplasts

Typical current-voltage (I-Eh) characteristics as obtained in the "whole-mitoplast mode" (by analogy to the "whole-cell mode") in symmetrical 150 mM KCl medium (pH 7.2) with voltage ramps between -40 and 40 mV are illustrated in Fig. 1a. To avoid time hysteresis, the ramps had to be very slow (2 min). A typical feature of the I-Eh characteristics was their asymmetry in the voltage range. The current was one to 2 orders of magnitude lower at negative potentials than at positive potentials. The transition between these domains was observed in the region of -10 to 35 mV, with inflection occurring between -10 and 16 mV (n = 30) as apparent from plots of an integral conductance G versus Eh (I/Eh versus Eh plots, Fig. 1b). A low G in the range below -20 mV reflected a state in which most of the channels were closed, whereas above 35 mV nearly all channels were open as indicated by saturation of the integral conductance.


Fig. 1. Whole-cell Cl- currents in brown adipose tissue mitoplasts. a, current-voltage, I-Eh, characteristics. The patch-clamp pipette was attached to a single osmotically swollen BAT mitoplast, and the membrane patch sealing the opening was broken. The total current (I)-voltage (Eh, holding potential) characteristics were recorded at 26.5 °C by using ramps from -40 to 40 mV. The medium in the pipette and bath contained 150 mM KCl, 20 mM K-HEPES, 1 mM K-EGTA (pH 7.2). b G versus Eh characteristics. The trace shown in a was recalculated to obtain an integral conductance G = I/Eh, which was plotted versus holding potential Eh. It allows the upper limit of the leak, taken as G near -40 mV, to be estimated, and the conductance around 0 mV to be resolved. Saturation of G is observed with increasing Eh above 35 mV. The overall transition in G reveals the transition from an ensemble of mostly closed channels to an ensemble where open states prevail. An example of the observed single channel opening with a single-channel conductance of 113 pS is encircled (at -18 mV). In the illustrated experiment, at least 43 channels contributed to the observed total current.
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The reason for the observed asymmetry was a changing pattern of channel fluctuations as detected by the single-channel recordings in the mitoplast-attached mode (Fig. 2, a and c) and a changing pattern of the observable channel events in the whole-mitoplast mode (Fig. 2, b and d). The former yielded statistically reproducible conductances of 109.6 ± 0.7 pS (S.D., n = 5, 24 °C) in 150 mM KCl. The derived temperature dependence (Fig. 3) yielded a value of 1.2 pS·K-1, explaining the scatter in values of the 108-pS channel conductances reported in the literature (2). A distinct marker property of the 108-pS channel (Figs. 2, a and b, and 4a) was a flickering behavior, i.e. bursts of frequent short openings, at more negative (hyperpolarizing) potentials (clearly visible in Fig. 4a and between -30 and -10 mV in Fig. 2c) and long time openings at positive (depolarizing) potentials. This behavior seemed to be the main cause for the observed asymmetry of the I-Eh characteristics. Consequently, the Cl- uptake into the mitoplast was much higher in comparison with the Cl- efflux. This asymmetry was a distinct electrophysiological marker of the 108-pS channel.3 Those G-Eh plots, in which the single-channel conductance was visible (Fig. 2, c and d), demonstrated the constancy of the apparent channel conductance at all voltages applied. Moreover, infrequent substates were observed, corresponding to channel openings at about <FR><NU>1</NU><DE>3</DE></FR> (Fig. 2c), 1/2, and <FR><NU>2</NU><DE>3</DE></FR> of the main full-open amplitude.


Fig. 2. Single-channel events in mitoplast-attached mode (a, c) and in whole-mitoplast mode (b, d) in the brown adipose tissue mitoplasts. a, I-Eh characteristics in the mitoplast-attached mode. Only two levels of channel openings are present (ramp duration 2 s, 25.5 °C). The distinct behavior of the channel at negative and positive potential is pronounced. Between -40 and -37 mV, both channels were closed, whereas between -37 and -24 mV, first channel was open, and above -24 to 40 mV both channels were open (with the exception of the 17 to 23 mV region). Note that flickering is dense at negative potentials, whereas it is almost absent at positive Eh. b, I-Eh characteristics of whole-mitoplast current under conditions with only a small number of accessible channels. This was achieved by choosing smaller mitoplasts to distinguish the single-channel events, even in the whole-mitoplast mode (ramp duration 2 s, 25 °C). The three levels of channel openings are visualized by solid lines. Other conditions are as described for a. Only one level was open between -40 and -27 mV and between -23 and 17 mV, whereas this level was closed e.g. between -27 and -23 mV. One level (up to 18 mV), two levels (up to 28 mV), and three levels (above 28 mV) were open at the positive Eh. c, G versus Eh characteristics in the mitoplast-attached mode constructed from the data of Fig. 2a. Transitions between closed and open states are notable. The derived single-channel conductance amounted to 113.6 pS. Note a <FR><NU>1</NU><DE>3</DE></FR> substate at -24 mV. d, G versus Eh characteristics constructed from the data of Fig. 2b. The distance between the solid lines shows the single-channel conductance 112 pS.
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Fig. 3. Temperature dependence of the single-channel conductance of 108-pS channel. Single-channel conductance (g) derived from seven experiments. Data were measured as described in the legend to Fig. 2a. The derived temperature dependence yielded a value of 1.2 pS·K-1. Data at 25 °C agreed well with the values reported previously (1).
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Fig. 4. Inhibitory effect of propranolol. a, single-channel recordings. Time courses of 108-pS channel activity at various Eh (amplitudes are indicated for each trace) are illustrated before, during, and after the external application of 50 µM propranolol to the mitoplast. At all voltages applied, propranolol blocked the channel activity completely. An almost complete recovery of this block (right panel) suggests the reversibility of the propranolol inhibition, not only the activity, but also the typical distinct fluctuation pattern at negative and positive Eh being recovered. The arrows and dotted lines indicate the closed state of the 108-pS channel. b, normalized integral conductance G in the whole-mitoplast mode at various concentration of propranolol. The plots of G/Gmax GL versus Eh are displayed in the absence (top trace) or in the presence of 50 µM propranolol (bottom trace). The normalized G/Gmax + GL versus Eh plots of the other records at 1, 10, and 20 µM propranolol (as indicated by numbers right of the traces) are shown for comparison. Normalization was performed by dividing each conductance G by the maximal conductance in the absence of propranolol (Gmax) for each experiment, assuming that Gmax was unchanged by propranolol. The leak conductance GL of each experiment was normalized to be the same as in the absence of the blocker. The drug was added by using the constant flow system. I-Eh characteristics were measured every 2-3 min by using 2-min voltage ramps, thus monitoring the block as it slowly approached a steady-state equilibrium, which was usually reached after 10-13 min. Other conditions are the same as described in the legend to Fig. 1. Data were fitted as described under "Experimental Procedures" (solid lines), yielding the parameters EM and "steepness factors" kappa . With progression of the propranolol blockage, EM is shifted to more positive potentials, whereas kappa  is almost unchanged. EM values (kappa  values in parentheses) were: 8.8, 8.5, and 16 mV (0.10, 0.06, and 0.10 mV-1) for controls and 34, 48, 62, and 63 mV (0.05, 0.04, 0.05, and 0.07 mV-1) for 1, 10, 20, and 50 µM propranolol, respectively.
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Propranolol, Cibacron Blue, and DHP Ca2+ Antagonist Blockage of the 108-pS Channel

The single-channel events after transferring the patches into a medium containing 50 µM propranolol, an amphiphilic amine better known as a beta -receptor antagonist, are illustrated in Fig. 4a. At all voltages applied, propranolol blocked the 108-pS channel completely. Moreover, the block was reversible, as apparent when flickering behavior (at negative voltages) or long openings (at positive voltages) were restored after washing. Unlike Antonenko et al. (10), we did not observe an increase in the single-channel conductance with propranolol.

In the whole-mitoplast mode and after 10-13-min incubation in the presence of saturating amounts (constant flow) of propranolol (Fig. 4b), Cibacron blue 3GA (Fig. 5a), or the DHP Ca2+ antagonist nifedipine (Fig. 5b), the typical current in depolarizing potentials was suppressed close to the magnitude of the leak current (Fig. 4b and insets in Fig. 5). Another DHP Ca2+-antagonist, niguldipine (35 µM, Fig. 5c), caused only partial inhibition (43%). Moreover, intermediate concentrations of propranolol (Fig. 4b) lead to a partial suppression of the integral conductance G, whereas the half-maximum parameter EM of the theoretical fit (cf. Eq. 1) increased with increasing propranolol concentration. With 50 µM propranolol, the threshold was shifted from -20 mV in controls to 10 mV, and EM increased to around 63 mV (Fig. 4b). In addition, the G versus Eh plots indicated that the current at negative (hyperpolarizing) potentials was also reduced. The dose-response curves constructed from G values at 35 mV (Eh at which G is saturated in controls without blocker) and corrected for the leak conductance are illustrated in Fig. 6. Thus, the 108-pS channel in the whole-mitoplast mode was inhibited by propranolol (Fig. 6a) and Cibacron blue (Fig. 6b) with apparent Ki values of 2.8 µM and 0.15 µM, respectively.


Fig. 5. Whole-mitoplast current in the presence of blockers. a, 100 µM Cibacron blue 3GA ("+CiB"); b, 50 µM nifedipine ("+nif"); c, 35 µM niguldipine ("+ngl"). Whole-mitoplast currents were measured as described in Fig. 1, first without blockers and then at the same patch in the presence of the blockers after a delay of about 10 min. Insets, G versus Eh characteristics constructed from corresponding data. Fits according to Equation 1 (solid lines) yielded the Eh values (kappa  values in parentheses) of 8.5, 7.8, and 10 mV (0.09, 0.09, and 0.07 mV-1) for controls in a, b, and c, 130 mV (0.02 mV-1) for 100 µM Cibacron blue, 260 mV (0.01 mV-1) for 50 µM nifedipine, and 38.5 mV (0.06 mV-1) for 35 µM niguldipine.
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Fig. 6. Dose-responses for inhibition of the whole-mitoplast current by propranolol (a) and Cibacron Blue 3GA (b). Dose-response curves constructed from the integral conductance, G, at 35 mV (saturating Eh for control), obtained from fits of G versus Eh plots corrected for the leak conductance. Measurements were performed as described in the legends to Figs. 1 and 4b for a series of doses of each drug (13-min wash-in). The inhibition was calculated by subtracting the remaining activity in the presence of the blockers from the corresponding control values (both taken as fitted G at 35 mV corrected for leak conductance). Solid lines represent the fits to the Hill equation. Ki values (nH values in parentheses) obtained by linear regression of the Hill plots were: 2.8 µM (0.6) for propranolol and 0.15 µM (0.9) for Cibacron blue.
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Sulfate, Phosphate, and Benzenetricarboxylate Conduction by the 108-pS Channel

Single-channel recordings in symmetrical 100 mM (Fig. 7a) and 150 mM potassium sulfate media demonstrated channel events with a similar pattern of fluctuation in negative and positive Eh as those found in KCl medium; they exhibited unit conductances of 34 and 56 pS, respectively. In the whole-mitoplast mode (Fig. 7b), sulfate currents also exhibited the same asymmetrical I-Eh relationship and were sensitive to propranolol, which inhibited at higher concentration than that in KCl medium (Fig. 7b). Therefore, we concluded that the same channel was responsible for both sulfate and Cl- conductances.


Fig. 7. Sulfate currents via 108-pS channels in BAT mitoplasts. a, single-channel events in symmetrical 100 mM K2SO4. A pipette was attached to a single osmotically swollen BAT mitoplast in medium containing 100 mM K2SO4, 20 mM K-HEPES, 1 mM K-EGTA (pH 7.2) (24 °C). The single-channel events were recorded at various holding potentials Eh (indicated by numbers right of each trace). A unit conductance was derived as 34 pS. The dotted lines indicate the closed state of the 108-pS channel. b, whole-mitoplast mode in K2SO4 and blockage of sulfate current by propranolol. The experiment was performed as described in the legend to Fig. 1 in symmetrical K2SO4 medium (same as in Fig. 7a, 24 °C) in the absence and presence of 200 µM propranolol. The fits (see insets) yielded the EM values (kappa  values in parentheses) of -7.8 mV (0.08 mV-1) for the control and 18.5 mV (0.08 mV-1) for 200 µM propranolol.
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Only noise was observed at positive Eh when a potassium gluconate medium replaced the previous anions (7). Small (10% with regard to Cl- at 20 mV) and intermediate (20% of Cl-) currents were observed when the external KCl was substituted by potassium salts of 1,3,5-benzenetricarboxylate (BTC, Fig. 8a) and phosphate (Fig. 8b), respectively. However, a different behavior was found in the presence of external 1,2,3-BTC, when the I-Eh characteristics had completely lost their asymmetry in the whole-mitoplast mode (Fig. 8a, trace 1). At negative voltages, where the Cl- efflux mainly contributed to the total current, one could observe an increase in leak current between -40 and -25 mV. Between -20 and 0 mV, smaller absolute values of current than those in symmetrical KCl medium were apparent, indicating a possible trans-inhibition of the Cl- efflux by the external 1,2,3-BTC. At positive voltages, the I-Eh plot showed the true characteristics of the 1,2,3-BTC current. The single-channel conductance carried by 1,2,3-BTC was apparently lower than that carried by sulfate, if one assumed the same number of channels to be open.


Fig. 8. Whole-mitoplast currents in external 1,3,5- and 1,2,3-BTC (a) and in phosphate (b). Experiments were performed as described in the legend to Fig. 1 with mitoplasts swollen in KCl medium. Afterward, the patches were transferred to external media where 150 mM KCl was replaced by K+ salts of a: trace 1, 75 mM 1,2,3-BTC or trace 2, 1,3,5-BTC; b: trace 4, 125 mM phosphate. Other compounds and pH were unchanged (see "Experimental Procedures"). Trace 3, controls in symmetrical KCl medium.
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Increase in 108-pS Channel Activity at Alkaline Extramitoplast pH

Transference of mitoplast-attached patches to KCl media of variable pH during single-channel recording (Fig. 9) clearly demonstrated that pH affected the fluctuation pattern of the 108-pS channel. At pH 6.0, the long channel openings were rare, even at high positive potentials, whereas at pH 7.2, shorter events at negative Eh and long channel openings (or clusters, as they were interrupted by short closures) at positive Eh were predominant, as illustrated above (Fig. 2a). At pH 8.5, the channel was so active that, at hyperpolarizing (negative) potentials, the open probability was high, and activity appeared as a dense flickering. At positive Eh, the clusters of channel openings were slightly prolongated. Thus, contrary to the report of Sorgato et al. (1), we observed that the 108-pS channel was pH-dependent. The pH effect was however reversible, since on transferring patches back to the original medium at pH 7.2, the characteristic fluctuation pattern described for pH 7.2 (Figs. 4a and 9) was completely restored.


Fig. 9. pH dependence of 108-pS channel in the single-channel recordings. Measurements were performed first as described in the legend to Fig. 4a in KCl medium of pH 7.2 (middle panel) at holding potentials, Eh, indicated above each trace (left panel), and afterward patches were transferred into a medium of pH 6.0 (left panel) or pH 8.5 (right panel). Arrows and dotted lines indicate the closed state of the 108-pS channel. Deactivation of the 108-pS channel in pH 6.0 and its activation in pH 8.5 is notable.
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Moreover, whole-mitoplast recordings at various pH (Fig. 10, a and b) confirmed that the asymmetry of the I-Eh characteristics was pH-dependent. At pH 6.0, the whole-mitoplast current was lower than at neutral pH within the complete range of applied voltages up to 32 mV (Fig. 10b). At pH 8.5, the absolute value of current at negative Eh was higher than at pH 7.2, whereas at positive Eh, the current was slightly lower. However, when the mitoplast was transferred back to pH 7.2, the current at positive Eh did not return to its original value. Hence, the marginal decrease in current at pH 8.5 was not attributable to the function of the 108-pS channel. Plots of integral conductance versus potential (G versus Eh plots) illustrated the pH effect more clearly (insets in Fig. 10). At pH 6.0, both the threshold of transition and EM were shifted by about 10 mV to positive voltages, whereas at pH 8.5, the G versus Eh characteristic was shallower and began at much higher G, and G gradually increased to saturation over the whole Eh range. This corresponded to single-channel recordings, where 108-pS channels were predominantly open (Fig. 9). One could assume saturation of G at Eh higher than 80 mV.


Fig. 10. pH dependence of 108-pS channel in the whole-mitoplast mode. a, whole-mitoplast currents measured at external pH 7.2 and 8.5; b, at pH 6.0. Measurements were performed first as described in the legend to Fig. 1 at pH 7.2. Afterward, the pipette was transferred into media of different pH. Insets, corresponding G versus Eh plots of the same data. Fits of the G versus Eh plots yielded the EM values (kappa  values in parentheses) of 2.8 mV (0.07 mV-1) for pH 7.2 and 36.3 mV (0.04 mV-1) for pH 8.5 (a) and 12.4 mV (0.12 mV-1) for pH 6.0 (b).
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DISCUSSION

With several exceptions (2, 27), no channel phenomena have been correlated up to date with the known ion transport proteins in mitochondria, i.e. the described uniporters. On the contrary, when inserted into giant liposomes and patch-clamped, the known purified anion carriers, such as the ADP/ATP carrier (28), the Pi carrier (29, 30), and uncoupling protein (31), display completely different properties, namely switching to the uniport mode and translocating Cl- and other nonphysiological substrates (28-30). In this work, we provide the evidence that the 108-pS channel is identical with the phenomenon of IMAC.

We have further characterized the 108-pS channel in BAT mitoplasts and demonstrated clearly its ability to conduct di- and trivalent anions and its blockage by DHP-type Ca2+ antagonists and by Cibacron blue; we have also shown its pH dependence for the first time. Moreover, we have identified a basis for the asymmetry of "whole-mitoplast" currents to which it mostly contributes, i.e. the voltage sensitivity of channel openings, which are almost absent at hyperpolarizing (negative) potentials. Their frequency increases, however, with increasing voltage, so that at positive voltages, the open states are "packed" into single long openings. Moreover, this voltage dependence is influenced by pH, as we found contrary to Sorgato et al. (1) in the mitoplast-attached mode: at acidic pH, the 108-pS channel exhibits very little activity with a fluctuation pattern identical to that found at negative potentials at neutral pH. At neutral pH and positive potentials, the channel switches to long openings as it does over the whole potential range at alkaline pH. Such activation of the 108-pS channel at alkaline pH resembles the phenomenon of alkaline pH-dependent swelling of mitochondria first described by Azzi and Azzone (32) and involves a dicyclohexylcarbodiimide-sensitive anion pore (15, 33, 34). This has subsequently been attributed to the phenomenon of the IMAC, which has been studied biochemically by Beavis and co-workers (12, 14-19). They have pointed out the dependence of the IMAC on internal pH. Nevertheless, in the mitoplast-attached mode (single-channel recording), external pH could influence a putative internal protonation site via a leak pathway. In the whole-mitoplast mode, when intramitoplast (matrix) pH is controlled by the buffer of the pipette solution, the external pH effect is analogous, but much less pronounced, than that in the mitoplast-attached mode. Thus, the sensitivity of the channel to changes in the matrix pH is the first important property that the 108-pS channel and IMAC have in common, suggesting their possible identity.

Respiring mitochondria possess a negative potential, which will tend to drive anions outward through IMAC. On the other hand, most measurements on IMAC were made at nearly zero potential, at which anions move inward (16). To compare ion selectivities with those of the 108-pS channel, we must compare currents of our patch-clamp study taken as the positive voltages approach zero. Under these conditions, the estimated order of conductances Cl- > SO42- > Pi congruent  1,2,3-BTC > 1,3,5-BTC for the 108-pS channel is almost identical to the order of rates reported for IMAC (16). Thus, in addition to Cl-, the 108-pS channel conducts divalent anions, such as sulfate and HPO42-, and trivalent 1,2,3-BTC. The low currents observed in 1,2,3-BTC at positive potentials could partly result from trans-inhibition by Cl- from the mitoplast interior, as Cl- was replaced only externally in this case. On the other hand, Cl- efflux (current at negative Eh) was trans-inhibited by external 1,2,3-BTC. In conclusion, the observed anion pattern seems to be similar for the 108-pS channel and IMAC, thus again suggesting their identity.

The third piece of evidence for the identity of the 108-pS channel and IMAC comes from the finding of a similar pattern of blockers. Pharmacological determination of channels is a classic paradigm, but previously, only propranolol, amiodarone (10), and benzodiazepines have been found to be blockers that the 108-pS channel and IMAC have in common (2). Now, we have discovered two new types of common blockers of the 108-pS channel and IMAC and have demonstrated more clearly the effect of propranolol. For each drug tested, its potency in inhibiting the 108-pS channel was found to be in the same concentration range as its reported potency for inhibiting IMAC (2, 12). Thus, the DHP-type Ca2+ antagonist nifedipine (cf. Fig. 5b and Ref. 22) and the amphiphilic amine propranolol (Figs. 4, a and b, 6a, 7b and Ref. 10 versus Ref. 12) inhibit in a micromolar range, whereas the antraquinone dye Cibacron blue 3GA (Fig. 5a versus Ref. 34) inhibits even in the submicromolar range. Unlike previous studies (10), our measurements show a complete block of the 108-pS channel activity by propranolol, besides a complete block by Cibacron blue and nifedipine. The nature of the blocker action corresponds to noncompetitive inhibition of transport, since we have observed shifts in the EM of G transition, i.e. shifts in the saturation of the integral conductance in the whole-mitoplast mode toward higher voltages with all blockers tested. Hence, with a blocker at low or intermediate concentration, a low opening probability is manifested only at high positive voltage. Thus, the blockers act in a similar way as acidic pH. The effect resembles the reported antimycin-induced shift of curves relating the open probability to Eh (8).

The only (as yet unexplained) discrepancy concerns the block by nucleotide di- and triphosphates (7), which have never been shown to inhibit IMAC in biochemical studies (16) or BAT mitochondria (34). In patch-clamp studies, a partial block of 44-84% was reported by Klitsch and Siemen (7) with BAT mitoplasts from warm-adapted rats, whereas there is only an occasional block in our recent experiments with BAT mitoplasts from cold-adapted hamster.4 Nevertheless, since nucleotides prevent the noncompetitive binding of DHP Ca2+-antagonists (22), their effects have to be studied further.

The three lines of evidence described above lead us to the conclusion that the 108-pS channel is identical to the biochemically observed phenomenon of the IMAC. This is not only because mitochondriologists have called it "a channel" (13), but because the similarities shown above are pronounced. To our knowledge, this is the first positive correlation of the single-channel measurements of the 108-pS channel with biochemical IMAC studies. Moreover, when single-channel measurements were attempted with mitoplasts loaded with 1 mM MgCl2, no channels with 108-pS characteristics were found.4 This is in accordance with IMAC inhibition by matrix Mg2+.

One may compare the conductance of the 108-pS channel in KCl and the maximum rate found for Cl- uniport by the IMAC as follows. Vmax of 1.35 µmol·min-1·(mg of protein)-1 was reported at [H+] limiting to zero (12). To reach 108 pS at 10 mV, i.e. to reach the current of 1.08 pA that accounts for a turnover of 6.75·106 s-1. Taking these data, the channel content is equal to 3.3 fmol/mg of mitochondrial protein. Further adjustment of the open probability and different potentials would yield the estimated content of 10-100 fmol/mg of protein, which appears reasonable for a channel abundance.

Identification of the 108-pS channel with IMAC does not exclude the possibility that this activity represents an altered state of a known carrier.5 Each integral membrane transport protein, biochemically defined as a carrier and physiologically mediating the electroneutral symport or antiport, can exhibit the behavior of an anion channel, while switching to a nonselective uniport mode. Such behavior has been reported for the ADP/ATP carrier (28), Pi carrier, glutamate/aspartate carrier (29, 30), and the uncoupling protein (31). Although all but the uncoupling protein do not physiologically allow for the uniport mode, they exhibit channel behavior in giant liposomes, conducting nonstandard substrates such as Cl-. The uniport mode of these carriers, which can be inhibited from the trans-side by other anions, has also been induced by mercurials, namely by HgCl2 (29, 30). For a definitive answer of this question, the protein responsible for both the biochemical IMAC phenomenon and the 108-pS channel must be found.


FOOTNOTES

*   This work was supported by Deutsche Forschungsgemeinschaft Grant Si310/5-1.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   Visiting scientist at the Institute of Zoology, University of Kiel, supported by Deutsche Forschungsgemeinschaft Grant Si310/5-1.
   Supported by Grant 301/95/0620 from the Grant Agency of the Czech Republic. E-mail: jezek{at}sun1.biomed.cas.cz.
**   To whom correspondence should be addressed: Physiologisches Institut I, Universitaet Tuebingen, Gmelinstr. 5, D-72076 Tuebingen, FRG. Fax: 49-7071-29-3073; E-mail: detlef.siemen{at}uni-tuebingen.de.
1   The abbreviations used are: BAT, brown adipose tissue; 1,2,3-BTC, 1,2,3-benzenetricarboxylate; 1,3,5-BTC, 1,3,5-benzenetricarboxylate; DHP, 1,4-dihydropyridine; IMAC, inner membrane anion channel (biochemically defined); S, siemens.
2   A preliminary report of these results has been published in the Abstract of 9th European Bioenergetics Conference (1996) Eur. Bioenerg. Conf. Rep. 9, pp. 120.
3   We have occasionally observed conductances other than 108 pS, which may represent other mitochondrial channels (2).
4   J. Borecký and D. Siemen, unpublished data.
5   Note, however, that the inherent feature of the single-channel recording is that it preferentially selects the most active proteins, which might behave differently from the whole ensemble. A heterogeneity in function could exist in the given preparation either naturally or because of experimental manipulation. Consequently, single-channel measurements are unique and do not have a counterpart in biochemical studies.

ACKNOWLEDGEMENT

Stimulating discussions with Prof. Dr. Reinhard Krämer (Institute of Biotechnology, Jülich, Germany) are gratefully acknowledged.


REFERENCES

  1. Sorgato, M. C., Keller, B. U., and Stühmer, W. (1987) Nature 330, 498-500 [CrossRef][Medline] [Order article via Infotrieve]
  2. Zoratti, M., and Szabó, I. (1994) J. Bioenerg. Biomembr. 26, 543-553 [Medline] [Order article via Infotrieve]
  3. Ballarin, C., and Sorgato, M. C. (1995) J. Biol. Chem. 270, 19262-19268 [Abstract/Free Full Text]
  4. Sorgato, M. C., Lippe, G., Keller, B. U., and Stühmer, W. (1988) in Integration of Mitochondrial Function (Lemasters, J. J., Hackenbrock, C. R., Thurman, R. G., and Westerhoff, H. V., eds), pp. 305-311, Plenum Press, New York
  5. Sorgato, M. C., Moran, O., DePinto, V., Keller, B. U., and Stühmer, W. (1989) J. Bioenerg. Biomembr. 21, 485-506 [Medline] [Order article via Infotrieve]
  6. Moran, O., Sandri, G., Panfili, E., Stühmer, W., and Sorgato, M. C. (1990) J. Biol. Chem. 265, 908-913 [Abstract/Free Full Text]
  7. Klitsch, T., and Siemen, D. (1991) J. Membr. Biol. 122, 69-75 [Medline] [Order article via Infotrieve]
  8. Campo, M. L., Kinnally, K. W., and Tedeschi, H. (1992) J. Biol. Chem. 267, 8123-8127 [Abstract/Free Full Text]
  9. Kinally, K. W., Zorov, D. B., Antonenko, Yu. N., Snyder, A. H., McEnerry, M. W., and Tedeschi, H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1374-1378 [Abstract]
  10. Antonenko, Yu. N., Kinnally, K. W., Perini, S., and Tedeschi, H. (1991) FEBS Lett. 285, 89-93 [CrossRef][Medline] [Order article via Infotrieve]
  11. Selwyn, M. J. (1987) Nature 330, 424-425 [Medline] [Order article via Infotrieve]
  12. Beavis, A. D. (1992) J. Bioenerg. Biomembr. 24, 77-90 [Medline] [Order article via Infotrieve]
  13. Garlid, K. D., and Beavis, A. D. (1986) Biochim. Biophys. Acta 853, 187-204 [Medline] [Order article via Infotrieve]
  14. Beavis, A. D., and Vercesi, A. E. (1992) J. Biol. Chem. 267, 3079-3087 [Abstract/Free Full Text]
  15. Beavis, A. D., and Garlid, K. D. (1988) J. Biol. Chem. 263, 7574-7580 [Abstract/Free Full Text]
  16. Beavis, A. D., and Garlid, K. D. (1987) J. Biol. Chem. 262, 15085-15093 [Abstract/Free Full Text]
  17. Beavis, A. D. (1989) J. Biol. Chem. 264, 1508-1515 [Abstract/Free Full Text]
  18. Powers, M. F., and Beavis, A. D. (1991) J. Biol. Chem. 266, 17250-17256 [Abstract/Free Full Text]
  19. Beavis, A. D. (1989) Eur. J. Biochem. 185, 511-519 [Abstract]
  20. Jezek, P., and Garlid, K. D. (1990) J. Biol. Chem. 265, 19303-19311 [Abstract/Free Full Text]
  21. Murray, A. G., Halle-Smith, S. C., and Selwyn, M. J. (1988) Eur. Bioenerg. Conf. Rep. 5, 206
  22. Zernig, G., Graziadei, I., Moshammer, T., Zech, C., Reider, N., and Glossmann, H. (1990) Mol. Pharmacol. 38, 362-369 [Abstract]
  23. Jezek, P., Beavis, A. D., DiResta, D. J., Cousino, R. N., and Garlid, K. D. (1989) Am. J. Physiol. 257, C1142-C1148 [Abstract/Free Full Text]
  24. Cannon, B., and Lindberg, O. (1979) Methods Enzymol. 15, 65-78
  25. Hamill, O. P., Marty, A., Neher, E., Sakman, B., and Sigworth, F. J. (1981) Pflügers Arch. 391, 85-100 [Medline] [Order article via Infotrieve]
  26. Bräu, M. E., Dreyer, F., Jonas, P., Repp, H., and Vogel, W. (1990) J. Physiol. (Lond.) 420, 365-385 [Abstract]
  27. Pau&cgrave;ek, P., Mironova, G., Mahdi, F., Beavis, A. D., Woldegiorgis, G., and Garlid, K. D. (1992) J. Biol. Chem. 267, 26062-26069 [Abstract/Free Full Text]
  28. Tikhonova, I. M., Andreyev, A. Yu., Antonenko, Yu. N., Kaulen, A. D., Komrakov, A. Yu., and Skulachev, V. P. (1994) FEBS Lett. 337, 231-234 [CrossRef][Medline] [Order article via Infotrieve]
  29. Herick, K., Stappen, R., and Krämer, R. (1995) in Thirty Years of Progress in Mitochondrial Bioenergetics and Molecular Biology (Palmieri, F., ed), pp. 83-87, Elsevier Science Publishers B. V., Amsterdam
  30. Stappen, R., and Krämer, R. (1993) Biochim. Biophys. Acta 1149, 40-48 [Medline] [Order article via Infotrieve]
  31. Huang, S.-G., and Klingenberg, M. (1996) Biochemistry 35, 16806-16814 [CrossRef][Medline] [Order article via Infotrieve]
  32. Azzi, A., and Azzone, G. F. (1967) Biochim. Biophys. Acta 131, 468-478 [Medline] [Order article via Infotrieve]
  33. Warhurst, I. W., Dawson, A. P., and Selwyn, M. J. (1982) FEBS Lett. 149, 249-252 [CrossRef][Medline] [Order article via Infotrieve]
  34. Jezek, P., and Borecký, J. (1996) Int. J. Biochem. Cell Biol. 28, 659-666 [CrossRef][Medline] [Order article via Infotrieve]

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