Anion Uniport in Plant Mitochondria Is Mediated by …Anion Uniport in Plant Mitochondria Is Mediated by a Mg2+-insensitive Inner Membrane Anion Channel* (Received for publication,

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 5, Issue of February 15, pp. 3079-3087,1992 Printed in U. S. A.

Anion Uniport in Plant Mitochondria Is Mediated by a Mg2+-insensitive Inner Membrane Anion Channel*

(Received for publication, September 16, 1991)

Andrew D. Beavis and Anibal E. VercesiS From the Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43699-0008 and the Department of Biochemistry, Uniuersidade Estadual de Campinas, Caixa Postal 11 70, 13100 Campinas SP, Brazil

It has long been established that the inner membrane of plant mitochondria is permeable to C1-. Evidence has also accumulated which suggests that a number of other anions such as Pi and dicarboxylates can also be transported electrophoretically. In this paper, we present evidence that anion uniport in plant mitochondria is mediated via a pH-regulated channel related to the so-called inner membrane anion channel (IMAC) of animal mitochondria. Like IMAC, the channel in potato mitochondria transports a wide variety of anions including NO:, C1-, ferrocyanide, 1,2,3-benzene-tricarboxylate, malonate, Pi, a-ketoglutarate, malate, adipate, and glucuronate. In the presence of nigericin, anion uniport is sensitive to the medium pH (pICsO = 7.60, Hill coefficient = 2). In the absence of nigericin, transport rates are much lower and much less sensitive to pH, suggesting that matrix H+ inhibit anion uniport. This conclusion is supported by measurements of H+ flux which reveal that “activation” of anion transport a t high pH by nigericin and at low pH by respiration is associated with an efflux of matrix H+. Other inhibitors of IMAC which are found to block anion uniport in potato mitochondria include propranolol (ICB0 = 14 pM, Hill coefficient = 1.28), tributyltin (ICBo = 4 nmol/ mg, Hill coefficient = 2.0), and the nucleotide analogs Erythrosin B and Cibacron Blue 3GA. The channel in plant mitochondria differs from IMAC in that it is not inhibited by matrix M&+, mercurials, or N,N’-dicyclohexylcarbodiimide. The lack of inhibition by M 8 + suggests that the physiological regulation of the plant channel may differ from IMAC and that the plant IMAC may have functions such as a role in the malate/ oxaloacetate shuttle in addition to its proposed role in volume homeostasis.

In many respects plant mitochondria are very similar to animal mitochondria with respect to anion transport. For example, they possess the classical electroneutral anion exchange carriers for adenine nucleotides, phosphate, dicarboxylates, oxoglutarate, tricarboxylates, and pyruvate (see Wis- kich (l) , Day and Wiskich (2), and Hanson (3) for reviews). There are also a number of interesting differences. Most

* This work was supported by National Institutes of Health Grant HL 36573 awarded by the National Heart, Lung and Blood Institute, United States Public Health Service, Department of Health and Human Services and in part by the American Heart Association, Ohio Affiliate, Columbus, Ohio. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertkement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ TO whom correspondence should be addressed Dept. of Phar- macology, Medical College of Ohio, P. 0. Box 10008, Toledo, Ohio 43699-0008. Tel.: 419-381-4182.

notably, mitochondria isolated from almost all plant sources are found to be permeable to C1- and other halides (4). Thus, these mitochondria swell upon addition of valinomycin or gramicidin when suspended in KC1 (4-14). In more recent years evidence has accumulated which indicates that plant mitochondria are also permeable to other anions. For example, from studies of swelling and contraction cycles in corn mitochondria, Hensley and Hanson (12) obtained evidence that Pi can be transported electrophoretically. Also, Huber and More- land (15) have shown that influx of C1-, SO:- and Pi in mung bean mitochondria can be stimulated by addition of valinomycin even after inhibition of the phosphate carrier. More recently, Zoglowek et al. (16) have shown that a wide variety of dicarboxylates as well as 1,2,3-benzenetricarboxylate are transported electrophoretically in mitochondria isolated from pea leaf, etiolated pea shoots, and potato tubers.

The inner membrane of mitochondria isolated from animal tissues is normally impermeable to the electrophoretic transport of most anions. However, Azzi and Azzone (17-19) demonstrated that the permeability of rat liver mitochondria is markedly increased by raising the pH to 8.8. Brierley (20) demonstrated the same phenomenon in beef heart mitochondria and went on to show that permeability could also be induced at neutral pH by addition of valinomycin to mitochondria respiring in KC1. In more recent years, evidence has accumulated which suggests that anion uniport in heart and liver mitochondria is mediated by a specific pathway which is now referred to as the inner membrane anion channel or IMAC.’ This pathway is inhibited by matrix H+ (21), matrix M$+ (21, 22), cationic amphiphiles such as propranolol (23), the alkylating agent N,N’-dicyclohexylcarbodiimide (24, 25), mercurials (26), triorganotins (27,28), and nucleotide analogs such as Cibacron Blue 3GA (29, 30) and Erythrosin B (30). The inactivity of IMAC in normal isolated mitochondria is a consequence of inhibition by endogenous matrix Mg2+.

Anion uniport in plant mitochondria has been less well characterized. Early studies by Stoner and Hanson (31) and Yoshida (7) showed that C1- uniport is sensitive to pH. However, the C1- permeability of plant mitochondria has been regarded as “non-carrier-mediated” transport (2, 4), despite being recognized as a “constitutive” property of these mitochondria and not a result of membrane damage (2, 14). In view of the many different anions which have now been reported to be transported electrophoretically and the pH dependence reported for C1- uniport, it is reasonable to hy- pothesize that anion uniport in plant mitochondria is me-

’ The abbreviations used are: IMAC, inner membrane anion channel; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; NEM, N-ethylmaleimide; TES, N-tris[hydroxy-methyl]methyl-2-amino- ethanesulfonic acid; LS, light scattering; DCCD, dicyclohexylcarbodiimide; TBT, tributyltin; RLM, rat liver mitochondria; CCCP, car- bonyl cyanide p-chlorophenylhydrazone.

3079

3080 Anion Uniport in Plant Mitochondria

0 .5

0 . 4

0 . 3

0 0 . 1 0 . 2 0.3 0 . 4

t Iminl FIG. 4. Nigericin and CCCP activate C1- uniport in potato

mitochondria. LS kinetics of mitochondria (0.08 mg/ml) suspended in KC1 assay medium are shown. In each trace swelling was induced by the addition of valinomycin (Val, 1.2 nmol/mg) at 0.2 min, all other additions were made at zero time. Trace a, control without BSA; trace b, plus nigericin (0.12 nmol/mg) without BSA; trace c plus nigericin with BSA (0.3 mg/ml); trace d, control with BSA; trace e, plus CCCP (1.4 pM) with BSA. The assay medium contained the K+ salts of C1- (55 mM), TES (5 mM), EGTA (0.1 mM) plus rotenone (4 pg/mg), and was adjusted to pH 7.4 and maintained at 25 “C.

diated by a single pathway related to IMAC. Moreover, as a consequence of the recent extensive characterization of IMAC i t has now become possible to address this hypothesis.

In this paper evidence is presented that plant mitochondria possess an inner membrane anion channel which has many of the characteristics of IMAC from animals. This pathway, however, differs from IMAC in three important properties. Most significantly, the potato IMAC does not appear to be blocked by matrix M e . It is also insensitive to inhibition by DCCD and mercurials.

EXPERIMENTAL PROCEDURES~

RESULTS

Nigericin Actiuates an Anion Uniport Pathway in Potato Mitochondria-Trace a of Fig. 4 illustrates the well established finding that isolated plant mitochondria swell upon addition of valinomycin when they are suspended in solutions of KCI. This reflects the existence of an electrophoretic pathway for the influx of C1-. They also swell in the presence of nigericin alone which suggests that they are also permeable to protons (trace b) . If, however, bovine serum albumin (BSA) is added to the medium the nigericin-induced swelling is essentially abolished (trace c). Unexpectedly, the valinomycin-induced swelling is also substantially inhibited by BSA (trace d ) . This swelling can, however, be markedly stimulated by the addition of nigericin (cf. traces c and d of Fig. 4).

Nigericin has previously been shown to stimulate the activity of IMAC in MP-depleted liver mitochondria, and this has been attributed to a release of inhibition by matrix protons secondary to equilibration of K+/H+ antiport ( 2 1 ) . If a similar phenomenon is responsible for the effect of nigericin in potato mitochondria, then a protonophore should also stimulate valinomycin-dependent swelling since net K+/H’ exchange should then be possible upon subsequent addition of valinomycin. As shown by trace e of Fig. 4, CCCP alone does not induce swelling but is able to stimulate valinomycin-induced swelling to the same rate as nigericin. Note also that in the presence of nigericin, BSA has no inhibitory effect on valinomycin-induced swelling (cf. traces b and c). These data

* Portions of this paper (including “Experimental Procedures” and Figs. 1-3, 8, 9, 11-13) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

suggest that the inhibition by BSA of swelling observed in the absence of nigericin is a result of the binding of an endogenous protonophore. This conclusion is further supported by the finding that respiratory control is relatively poor if BSA is not included in the assay medium (not shown). Consequently, in all subsequent studies we routinely included BSA in the assay media.

pH Dependence of Anion Uniport in Potato Mitochondria- If the activation of C1- uniport by nigericin and CCCP results from a change in matrix pH, the rate of uniport in the presence of nigericin should be pH-dependent. This is found to be the case. The data contained in Fig. 5A, were obtained with mitochondria isolated in mannitol-containing medium. A very strong pH dependence is evident with a pICBo of 7.35. Note that in the absence of nigericin the effect of pH is much smaller as would be expected if the pH-sensitive site were in the matrix. Moreover, the finding that at no pH value is the rate in the absence of nigericin greater than in its presence suggests that the matrix pH of the mitochondria in the stock suspension is low enough to completely inhibit transport. The data contained in Fig. 5B represent Hill plots of these data plus the results of a similar experiment carried out with mitochondria isolated in sucrose. In the latter experiment, the pH dependence was examined in both 40% isotonic (110 mosm) and 100% isotonic (275 mosm) media. For all three sets of data, the Hill plots are very linear and have slopes close to 2.0 reflecting the high pH sensitivity of this transport pathway and suggesting that two protonation sites may be involved. In the sucrose-prepared mitochondria, the pICso values were 7.6 and 7.8 in the 40 and 100% media, respectively. These values are very close to those reported for IMAC-

100 -A

X

E

75 - m

-3 50- M

25 -

05 Y

6

DH

pH FIG. 5. The pH dependence of C1- uniport in potato rnito-

chondria. A, the rate of C1- transport (% Jmax ([H+] + 0)) in mannitol-isolated mitochondria determined from LS kinetics is plotted uersus the pH of the KC1 assay medium. Swelling was induced by the addition of valinomycin (1 nmol/mg). 0, nigericin (1 nmol/mg) was included in the medium. A, no nigericin present. B, Hill plots of pH dependence of C1- uniport. 0, data from panel A (+nigericin), pICBo = 7.35, slope = 1.96; A, data obtained with sucrose-prepared mitochondria in 110 mosm KC1 medium (+ nigericin), PICSO = 7.60, slope = 1.96; ., data obtained with sucrose-prepared mitochondria in 275 mosm KC1 medium (+nigericin), pIC6, = 7.80, slope = 2.01. The assay media were similar to that described under “Experimental Procedures” except the pH was adjusted to the indicated value and the 275 mosm medium contained 145 mM KCI.

Anion Uniport in Plant Mitochondria 3081

mediated C1- transport (21). The lower pICb0 obtained with the mannitol-prepared mitochondria may reflect the existence of a transmembrane ApH resulting from a decreased matrix K+ concentration due to influx of mannitol.

Proton Fluxes during Activation of Anion Uniport in Potato Mitochondria-The data contained in Figs. 4 and 5 suggest that anion uniport is regulated by matrix pH. To investigate this more directly, we examined the proton fluxes associated with activation of anion uniport by nigericin at pH 8.0. In order to do this, we increased the mitochondrial concentration in the assay medium to 1 mg/ml to enable a pH electrode to be used to follow the proton fluxes, and we used a variable path length probe, set at about 2.5 mm, to enable the LS changes to be followed simultaneously. For each experiment, the reading from the pH electrode was allowed to stabilize before the mitochondria were added to initiate data collection.

The data contained in Fig. 6 show that following addition of mitochondria there is a slow efflux of H+ and that addition of nigericin induces a rapid efflux of about 25 nmol H'/mg (trace a). This is accompanied by a small increase in volume (trace b) , presumably due to influx of K+; however, no large amplitude swelling is observed until valinomycin is added. In the absence of nigericin, valinomycin increases the rate of spontaneous H+ efflux (trace c). However, the rate of swelling is only 6% of that in the presence of nigericin (trace d). Subsequent addition of nigericin induces rapid efflux of protons and rapid swelling. Thus, these data provide strong evidence that it is the release of matrix protons, i.e. the elevation of matrix pH which activates anion uniport. These data also suggest that the matrix pH of isolated potato mitochondria is somewhat acid. These properties are in sharp contrast with isolated rat liver mitochondria which have a matrix pH of 8 or higher and in which, under similar conditions, addition of nigericin induces an influx of about 22 nmol H' (42).

If anion uniport is inhibited by matrix H', then as in liver (23) and heart mitochondria (20), it should be possible to induce swelling at pH 7.0 (or lower) by allowing the mitochondria to respire, so that valinomycin-mediated K+ uptake driven by the H+ pumps of the respiratory chain can alkalinize the matrix. As shown by the data contained in Fig. 7 in which succinate was added as a substrate for respiration, this is found to be the case. Addition of valinomycin induces an immediate and rapid ejection of about 16 nmol of H+/mg (trace a). This is followed by rapid swelling (trace b). Note

0 0 . 4 0 . 0 1.2

t ( m i n l FIG. 6. Proton fluxes associated with activation of Cl- uni-

port by nigericin. Simultaneous LS and pH electrode recordings obtained with mitochondria (0.96 mg/ml) suspended in KC1 assay medium at pH 7.8 and 25 "C are shown. Trace a (pH) and trace b (LS) show effects of adding nigericin (Nig, 0.04 nmol/mg) at 0.2 min followed by valinomycin (Val , 0.18 nmol/mg) at 0.45 min. Trace c (pH) and trace d (LS) show effects of adding valinomycin at 0.45 min followed by nigericin at 0.8 min. The assay medium contained the K' salts of Cl- (59 mM), TES (1 mM), EGTA (0.1 mM) plus rotenone (0.35 pglmg), BSA (0.3 mg/ml), MgClz (0.1 mM). The pH electrode recording was calibrated as described under "Experimental Proce- dures."

2

I3

I 0 0.2 0 . 4

t (min l FIG. 7. Proton fluxes associated with activation of anion

uniport at low pH by respiration. Simultaneous LS and pH electrode recordings obtained with mitochondria (1.2 mg/ml) suspended in KC1 assay medium at pH 7.0 are shown. Trace a (pH) and trace b (LS) show the effect of adding valinomycin (Val , 0.15 nmol/ mg) in the presence of succinate (2 mM). Trace c (pH) and trace d (LS) were obtained in the absence of succinate. The assay medium contained the K+ salts of C1- (59 mM), TES (1 mM), and EGTA (0.1 mM) plus MgC12 (0.1 mM), cytochrome c (10 pM), BSA (0.3 mg/ml), and rotenone (0.35 pglmg). The maximum rate of H+ ejection (trace a ) was 430 nmol H+/min. mg.

TABLE I Comparison of substrate selectivity for the anion uniport pathways in

rat liver and potato mitochondria Relative rates of anion transport (C1- = 100) determined using the

LS technique as described under "Experimental Procedures" are shown. In potato mitochondria, nigericin (1 nmol/mg) was added at zero time and valinomycin (1 nmol/mg) was added at 0.2 min to initiate transport. In rat liver mitochondria nigericin was added at zero time, A23187 (10 nmol/mg) at 0.2 min and valinomycin at 0.4 min. All media were equiosmolal with 60 mM KC1. In this experiment the rates of chloride transport were 740 and 280 nmol of Cl-/min. mg for potato and liver mitochondria respectively. Pi uniport was assayed in NEM-treated liver mitochondria (21) and in mersalyl- treated potato mitochondria to block the classical Pi carrier.

K' salt

NO; c1- Fe(CN)Q- 1,2,3-Benzene tricarboxylate3- Malonate2- P, a-Ketoglutarate*- Malate2- Adipate2- Glucuronate-

Relative rates

Potato Rat liver

209 350 100 100 110 81 102 76 62 68 92 64 38 38 28 32 11 3

0.9 0.3

that net H' ejection driven by respiration is almost complete before swelling begins. This is consistent with the interpre- tation that respiration itself is not driving the influx of KC1, but simply activating anion uniport by alkalinizing the matrix. In the absence of succinate, valinomycin does not induce H+ ejection nor is there rapid swelling (traces c and d) . Similar results have been obtained with rat liver mitochondria (23). However in that case the extent of H' ejection approached 54 nmol of H+/mg before IMAC was activated.

Anion Selectivity of the Anion Channel in Potato Mitochon- dria-The findings presented above strongly suggest that C1- transport in potato mitochondria is mediated by a pathway which is very similar to IMAC. If this is the case, then other anions should also be transported. Moreover, they should follow a similar selectivity sequence. In Table I, we compare the relative rates of transport for a number of anions in rat liver and potato mitochondria. All the anions tested from NO; which is the most rapidly transported down to glucuronate which is the most slowly transported were found to be transported electrophoretically in potato mitochondria.


Inhibition of Anion Uniport in Potato Mitochondria by Propranolol-To investigate further the possibility that anion uniport in potato mitochondria is mediated by an IMAC-like channel, we investigated the effect of propranolol which inhibits IMAC with an ICs0 of about 25 p~ (23). Typical LS traces of potato mitochondria suspended in KC1 assay medium are shown in Fig. 8. Nigericin was added at zero time, and transport was initiated by the addition of valinomycin at 0.2 min (trace a). Propranolol (50 p ~ ) added at zero time inhibits swelling 89% (trace b). Inhibition is also observed if propranolol is added after the rate of transport becomes maximal (trace c), thus propranolol does not simply prevent activation of anion uniport. Dose-response curves determined at pH 7.4 and 8.4 are shown in Fig. 9. A t pH 7.4, the ICs0 is 14 p~ which is lower than the value observed in RLM. The Hill slope (1.28), however, is very similar. As with RLM the ICso is found to increase with pH rising to 50 p~ at pH 8.4. Note, however, that this increase is associated with an increase in the Hill slope which approaches 2 as the pH is raised. In liver mitochondria the IC50 for propranolol is increased by pretreatment of the mitochondria with mercurials and N-ethylmaleimide (43). We could, however, find no evidence for such an effect in potato mitochondria.

Inhibition of Anion Uniport in Potato Mitochondria by TET-Recently, a new class of inhibitors of IMAC has been identified, the triorganotins (28). These compounds are fairly specific inhibitors of IMAC. The only other mitochondrial transport process which has been reported to be inhibited by these agents is the FIFo-ATPase H’ pump (44). The data contained in Fig. 10, compare the effects of TBT on uniport of malonate with that on respiration. Malonate uniport is completely inhibited by TBT with an IC50 of about 4 nmol/

\ \ I

25H i ;.2 0 . 2 0.6 1

l o g ( n m o l TBT/mg) FIG. 10. Dose-response curves for effects of TBT on potato

mitochondria. The effects of TBT on rates of malonate uniport (O), state 3 respiration (0), state 4 respiration (U), and uncoupled respiration (A) are shown. Malonate uniport was assayed using the LS technique. Mitochondria (0.08 mg/ml) were added to the malonate assay medium containing rotenone (4 pg/mg) and nigericin (0.12 nmol/mg). TBT was added at 0.1 min followed by valinomycin at 0.2 min. To determine the effect on respiration, the mitochondria were “pre-conditioned” and pretreated with TBT as described under “Ex- perimental Procedures” before 75 ~1 (.37 mg) was transferred to the oxygen-electrode chamber. The state 4 rate was determined and then at 1.5 min ADP (300 nmol) was added to initiate state 3, and at 3 min CCCP (2.3 p ~ ) was added to determine the uncoupled rate. Respiration rates are plotted as a % of the control state 3 rate. Malonate uniport IC,, = 4 nmol TBT/mg, Hill slope = 2.0; state 3 IC,, = 1.3 nmol TBT/mg, Hill slope = 3.8. The assay media are described under “Experimental Procedures.”

mg and a Hill coefficient of 2.0. Over the same dose range, TBT has a negligible effect on the rate of uncoupled (+CCCP) respiration and only slightly stimulates the rate of state 4 respiration. Thus, TBT induces little nonspecific damage. As expected, TBT inhibits state 3 respiration producing its maximum effect at 2.5 nmol/mg.

The doses required to block malonate uniport (10 nmol/ mg) and state 3 completely are 10- and %fold, respectively, greater than the doses required in RLM. Interestingly, however, the inhibitory dose for IMAC can be increased 10-fold or more by low doses of mercurials (10-15 nmol/mg) (28), while the inhibitory dose in potato mitochondria is unaffected even by addition of 0.1 mM mersalyl to the assay medium.

Inhibition of Anion Uniport by Nucleotide Analogs-Al- though IMAC is not inhibited by nucleotides it is inhibited by a number of nucleotide analogs such as Cibacron Blue 3GA and Erythrosin B (29,30). Consequently, we have investigated the effect of these reagents on anion uniport in potato mitochondria. For this study, we looked at the transport of both C1- and malonate, since the maximum extent of inhibition of IMAC differs substantially for these anions (30).

Fig. 1lA contains data obtained with Erythrosin B. Inhi- bition of both malonate and C1- uniport is observed. However, the ICSo for malonate (32 p ~ ) is about one-third that for C1- (107 pM). These data are a little difficult to analyze because there is some indication that the lowest doses used may actually stimulate transport. A similar phenomenon is observed with RLM (30). However, the IC50 values in RLM are about one-tenth those observed with potato mitochondria.

Fig. 11B contains data obtained with Cibacron Blue. With this drug, C1- transport is inhibited maximally by about 20% compared with 60% in RLM, while malonate transport is almost completely inhibited in both. The ICs0 for malonate transport is about 22 p~ which is about 10-fold higher than that observed in RLM.

Anion Uniport in Potato Mitochondria Is Not Regulated by Matrix Mg2”The most obvious difference between anion uniport in potato mitochondria and animal mitochondria is that rapid transport can be observed in potato mitochondria at pH 7.4 without using A23187 to deplete matrix M e . The results of an experiment designed to investigate the role of matrix Mg2+ in the regulation of anion uniport in potato mitochondria are presented in Fig. 12. Comparison of traces a and b reveals that addition of A23187 does not stimulate anion uniport, in fact, A23187 decreases the rate to 70% of the control rate. One possible explanation for the lack of stimulation could be that the potato mitochondria are already depleted of matrix M$+. However, if this were the explanation, it should still be possible to inhibit anion uniport by adding exogenous M e as previously demonstrated in RLM (22). Trace c of Fig. 12 shows the effect of 2 mM M$+. Transport is inhibited about 20%. However, the transport rate is essentially the same in the presence and absence of A23187 suggesting that the inhibitory site is on the outside. We have also directly assayed the M$+ content of five different preparations of potato mitochondria using atomic absorption spectroscopy and have found 51 f 4 nmol of M%+/mg. This value is close to the value of 44 nmol/mg reported for corn mitochondria (4), and the value of 38 nmol/mg reported for liver mitochondria (35). Thus, it appears that unlike IMAC the anion channel in potato mitochondria is not regulated by matrix Mg2+.

Anion Uniport in Potato Mitochondria Is Not Inhibited by Mercurials-Mercurials such as mersalyl inhibit many electroneutral anion transport proteins in mitochondria and they are also found to inhibit IMAC (26). To determine whether


mersalyl can also-inhibit anion uniport in potato mitochondria, we pretreated batches of mitochondria with various doses of mersalyl and then assayed the rate of anion uniport by measuring valinomycin-dependent swelling in C1-, Pi, malate, and malonate assay media. For comparison, we also assayed the classical Pi carrier by measuring nigericin-induced swelling in KPi medium in the absence of valinomycin.

The data contained in Fig. 13 show that the phosphate carrier is blocked by 20 nmol of mersalyl/mg. However, no inhibition of uniport of any of the anions assayed was observed even with doses up to 110 nmol/mg. Thus, we conclude that anion uniport in these mitochondria is insensitive to mercurials.

Anion Uniport in Potato Mitochondria Is Not Inhibited by DCCD-DCCD was one of the first inhibitors of IMAC to be identified (24,25). This agent which alkylates carboxyl groups in a hydrophobic environment blocks completely the transport of all anions through IMAC. Using doses of DCCD which block IMAC in about 30 min, we found no inhibition of anion uniport in potato mitochondria. Thus, we believe that this channel is insensitive to DCCD and, therefore, may lack the putative reactive carboxyl group.

DISCUSSION

In this paper, we have provided evidence that potato mitochondria contain an anion channel which is closely related to IMAC found in animal mitochondria but which differs in several important respects. Although all the studies presented were carried out with potato mitochondria, it is evident from examination of the literature (e.g. 4, 7, 16, 31) that a similar channel is present in plant mitochondria from many different sources.

The most obvious difference between the channels in plants and animals is the absence of inhibition of the plant channel by endogenous M P . Due to the absence of this regulation, it has become a well-established fact that plant mitochondria are permeable to halides; however, this has been regarded as non-carrier-mediated transport (2, 4). All attempts to elimi- nate this transport process by preparing purer and better coupled mitochondria have, however, failed (14, 16). In our own work, we have obtained very well coupled mitochondria with respiratory control equal to that of RLM, which retain the capacity for anion uniport. Thus, we concur with the conclusion (14, 16) that the anion permeability of plant mitochondria is not a consequence of membrane damage. We have, however, obtained evidence that mitochondria prepared in mannitol-containing media have a larger matrix volume than mitochondria prepared in sucrose. We believe that this is a direct result of the influx of mannitol during the preparation. Although, low temperatures are maintained during isolation, in view of the high concentration of mannitol used (up to 0.3 M) (5) and the relatively long duration of exposure (up to 2 h), significant influx would be expected. Despite the fact that this influx appears to have little effect on the respiratory control ratio for better controlled transport studies we prefer to use sucrose for osmotic support during mitochondrial isolation. Similar conclusions have been drawn by Eb- bighausen et al. (45) who use 0.3 M sucrose in their isolation media.

The most compelling evidence that the anion uniport pathway in plant mitochondria is related to IMAC is our finding that it is inhibited by matrix protons. In the 1960s Stoner and Hanson (31) and Yoshida (7) demonstrated that C1- uniport in corn and castor bean mitochondria, respectively, is stimulated by raising the pH. Consistent with these observa- tions, we have shown that in the presence of nigericin anion

uniport in potato mitochondria is very sensitive to pH. The findings that low doses of nigericin are able to stimulate valinomycin-dependent swelling at alkaline pH but not at acid pH and that this activation is associated with the efflux of H+ provide strong evidence that regulatory site is located in the matrix. Interestingly, the H+ flux induced by nigericin is in the opposite direction from that observed in liver mitochondria in which influx of H' is observed even up to external pH values of 8.4 (23). These findings suggest that the matrix pH of nonenergized isolated plant mitochondria is quite low compared with that of liver mitochondria. This conclusion is consistent with the report by Moore et al. (46), that the matrix pH of nonenergized mung bean mitochondria suspended in a pH 7.2 medium is 0.8 units lower than the medium. The reason for this acid matrix, however, has not been established.

The stimulation of anion uniport by nigericin is most evident in the presence of BSA. In the absence of BSA, the rate of transport accelerates spontaneously following the addition of valinomycin. Since this acceleration phase can be essentially eliminated by the addition of nigericin or an uncoupling agent it is most easily explained on the basis of the efflux of inhibitory H' mediated by an endogenous uncoupling agent coupled to valinomycin-mediated K' influx. This inter- pretation is further supported by the findings that BSA must be included in the respiration assay medium to obtain good respiratory control and that nigericin alone can induce swelling in the absence of BSA. In addition, Diolez and Moreau (47) have shown that BSA is necessary to maintain a high membrane potential in plant mitochondria.

The pH dependence of anion uniport in potato mitochondria exhibits a Hill coefficient of 2.0. This is higher than the value reported for rat liver mitochondria (21). However, in RLM the Hill plot is not linear over the whole pH range and the slope increases to values greater than 1 at the low end of the pH range (21, 43). The cause of the non-linearity of the rat liver data is not known, however, the potato data suggest that two protonation sites are involved in the inhibition. The PIC50 values measured under similar conditions are, however, very similar being 7.6 in potato and 7.8 in RLM in 40% KCl. The lower pICsO obtained with mitochondria prepared in mannitol could result from the presence of mannitol in the matrix, which, by diluting the endogenous K+, will result in [K+Ii, < [K'lOut and after addition of nigericin in [H+Ii, < [H'lOut. This would shift the curve if the inhibitory site(s) is in the matrix.

Further evidence that the anion channel in plant mitochondria is related to IMAC comes from its selectivity. All the substrates of IMAC we have tested are also transported by the channel in potato and in all cases transport is regulated by pH. Recently, Zoglowek et al. (16) have reported that mitochondria from pea leaves and pea shoots, as well as potato tubers, are permeable to various dicarboxylates. However, these authors did not consider the possibility that these anions were being transported via the same pathway responsible for C1- transport. It is interesting to note that the transport of Pi via the uniport pathway occurs about 3-fold faster than transport via the classical Pi carrier (Fig. 1B). Using the LS technique to quantitate rates, we obtain a value of about 1 pmol/min. mg for the latter process. This value appears to be a reasonable estimate, since in our assay of oxidative phosphorylation (Fig. 3) the state 3 respiration rate is about 650 nmol O/min.mg, which would require a Pi transport rate of 1.1 wmol/min. mg if the P/O ratio were 1.7. Other properties common to the two channels include inhibition by propranolol, inhibition by TBT, and inhibition by nucleotide analogs such as Erythrosin B and Cibacron Blue 3GA. There are,


however, some differences. While propranolol is slightly more potent in potato mitochondria, TBT and the nucleotide analogs are substantially less potent. These findings suggest there are some differences in the structure of the two channels.

The most important difference in the properties of the two channels is the absence of inhibition by endogenous matrix Mg2+ in plant mitochondria. This cannot be attributed to a lack of matrix M e since direct measurements show that they contain about 51 nmol of M e / m g . Moreover, oxidative phosphorylation occurs at a rapid rate and this requires matrix M e . In contrast to liver mitochondria, we found that swelling in potato mitochondria is slightly inhibited by A23187. The reason for this is unknown, however, it could reflect a decrease in matrix pH subsequent to Mg2+ loss via Mg2+/2H+ exchange. We did observe some inhibition by 2 mM M e . However, since the rate was the same in the presence or absence of A23187, we believe that this reflects an effect of external Mg2+.

Another important difference is the lack of inhibition by mersalyl. Other mercurial effects also appear to be absent including the elevation of the ICso values for both propranolol and TBT. The absence of all these effects could reflect the absence of these putative " S H groups. For example, the mercurial-reactive site responsible for inhibition of IMAC does not appear to be essential for activity since inhibition is only partial for certain anions such as C1- (26, 43). Interest- ingly, a similar phenomenon has been reported by Phelps et al. (48) who have shown that the phosphate carrier from yeast lacks 5 of the 8 cysteines found in the carrier from beef heart, including the one responsible for inhibition by N-ethylmaleimide.

Another finding which could be explained by a single amino acid substitution is the lack of inhibition by DCCD. DCCD may inhibit IMAC by simply blocking a conformational change necessary for transport or by locking the channel in a closed conformation. Absence of this reactive site, which is believed to be accessible from the lipid bilayer (24), could therefore make the channel insensitive to DCCD without significantly affecting its activity.

The precise physiological function of IMAC is unknown. However, its broad selectivity and the fact that in energized mitochondria the normal direction of flux would be out of the matrix suggest that it may be involved in volume homeostasis (see Ref. 41, for review). In animal mitochondria the most likely candidates for physiological regulators are M e and H+. In plant mitochondria, however, the lack of inhibition by matrix Mg2' suggests that another mechanism may be involved and also that in plants the anion channel may have other functions. One important difference between animal and plant mitochondria is the mechanism by which reducing equivalents are transported across the inner membrane. Ani- mals use a mechanism in which the oxoglutarate carrier and the aspartate/glutamate carrier achieve net exchange of malate and oxaloacetate. Plants, on the other hand, appear to use a malate/oxaloacetate shuttle in which oxaloacetate itself is transported (16, 49, 50). The mechanism by which malate is exchanged for oxaloacetate is uncertain. However, on the basis of mitochondrial swelling studies, Zoglowek et al. (16) have suggested that they both may be transported electrophoretically. Whether electrically coupled exchange could take place efficiently in the face of a large membrane potential remains to be established. However, use of the inhibitors described in this paper may help to determine whether IMAC is involved in the operation of the malate/oxaloacetate shuttle.

In conclusion, in view of the similarities in anion uniport

in animal and plant mitochondria, it is likely that plant mitochondria possess a specific inner membrane anion channel related to IMAC. However, since there appear to be significant differences between these channels from plants and animals, we suggest that the channel in plant mitochondria should be referred to as PIMAC.

Acknowledgment-Joel Shiffler is thanked for his expert technical assistance.

1. 2. 3.

4.

5. 6.

7. 8.

9.

10.

11. 12.

13.

14.

15.

16.

17.

18.

19.

20. 21.

22.

23. 24.

25.

26. 27. 28.

29.

30. 31.

32.

33.

34. 35.

36. 37.

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R., and Day, D. A., eds) Vol. 18, pp. 248-280, Springer-Verlag, Berlin

Hanson, J . B., and Koeppe, D. E. (1975) in Ion Transport In Plant Cells and Tissues (Baker, D. A., and Hall, J. L., eds), pp. 79-99, North-Holland, Amsterdam

Bonner, W. D., Jr. (1967) Methods Enzymol. 10, 126-133 Stoner, C . D., Hodges, T. K., and Hanson, J . B. (1964) Nature

Yoshida, K. (1968) J. Fac. Sci. Uniu. of Tokyo III 10, 63-82 Lorimer, G. H., and Muller, R. J . (1969) Plant Physiol. 44, 839-

Muller, R. J., Dumford, W. S., and Koeppe, D. E. (1970) Plant

Nelson, R. H., Dever, J., Harper, W., and Fry, R. (1972) Plant

Kirk, B. I., and Hanson, J. B. (1973) Plant Physiol. 51,357-363 Hensley, J . R., and Hanson, J. B. (1975) Plant Physiol. 56, 13-

Moore, A. L., and Wilson, S. B. (1977) J. Exp. Botany 28, 607-

Day, D. A., and Hanson, J . B. (1977) Plant Science Lett. 11,99-

Huber, S. C., and Moreland, D. E. (1979) Plant Physiol. 64,115-

Zoglowek, C., Kromer, S., and Heldt, H. W. (1988) Plant Physiol.

Azzi, A., and Azzone, G. F. (1966) Bwchim. Biophys. Acta 120,

Azzi, A., and Azzone, G. F. (1967) Bwchim. Biophys. Acta 131,

Azzi, A., and Azzone, G. F. (1967) Biochim. Biophys. Acta 135,

Brierley, G. P. (1970) Biochemistry 9,697-707 Beavis, A. D., and Garlid, K. D. (1987) J. Biol. Chem. 262,

Beavis, A. D., and Powers, M. F. (1989) J. Bwl. Chem. 264,

Beavis, A. D. (1989) J. Biol. Chem. 264,1508-1515 Beavis, A. D., and Garlid, K. D. (1988) J. Biol Chem. 263,7574-

Warhurst, I. W., Dawson, A. P., and Selwyn, M. J . (1982) FEBS

Beavis, A. D. (1989) Eur. J. Biochem. 185,511-519 Powers, M. F., and Beavis, A. D. (1991) Biophys. J. 59,596a Powers. M. F.. and Beavis. A. D. (1991) J. Biol. Chem. 266.

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Powers, M. F., and Beavis, A. D. (1990) Biophys. J . 57, 179a Stoner, C. D., and Hanson, J . B. (1966) Plant Physiol. 41, 255-

266 Neuberger, M. (1985) in Encyclopedia of Plant Physiology (Douce,

R., and Day, D. A., eds) Vol. 18, pp. 7-24, Springer-Verlag, Berlin

Yoshida, K., and Sato, S. (1968) J. Fac. Sci. Uniu. of Tokyo III,

Tedeschi, H. (1959) J. Biophys. Biochem. Cytol. 6, 241-252 Beavis, A. D., Brannan, R. D., and Garlid, K. D. (1985) J. Biol.

Laties, G. (1973) Biochemistry 12,3350-3355 Jung, D. W., and Hanson, J. B. (1973) Arch. Biochem. Biophys.

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Anion Uniport in Plant Mitochondria 3085 38. Raisin, J. K., Laties, G. G., and Crompton, M. (1973) J. Bioenerg.

39. Douce, R., Christensen, E. L., and Bonner, W. D., Jr. (1972)

40. Garlid, K. D., and Beavis, A. D. (1985) J. Biol. Chem. 2 6 0 ,

41. Beavis, A. D. (1992) J. Bwenerg. Bwmembr. 2 4 , 77-90 48. 42. Beavis, A. D., and Garlid, K. D. (1990) J. Bwl. Chem. 265,2538-

2545 49. 43. Beavis, A. D. (1991) Biochim. Bwphys. Acta 1003,111-119 44. Selwyn, M. J. (1976) in Organotin Compounds: New Chemistry 50.

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Moore, A. L., Rich, P. R., and Bonner, W. D. (1978) J. Exp.

Diolez, P., and Moreau, F. (1983) Physiol. Plant 5 9 , 177-182 Phelps, A., Schobert, C. T., and Wohlrab, H. (1991) Biochemistry

Douce, R., and Bonner, W. D. (1972) Biochem. Biophys. Res.

Kromer, S., and Heldt, H. W. (1991) Biochim. Biophys. Acta

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1057,42-50

WQQLEMENTARV HATERIAL TO

ANION UWIPORT I N QLUIT MITOCHONDRIA IS MEDIATED BV A MG*-IWSENSITIVE INNER MEMBRANE ANION W E L

BV ANDREY D. BEAYlS U I D ANIBAL E. VERCESI

v

eepplutipp O f Potato nitprhPnPUn (Solanum tuberorurn) us ing a modif icat ion of the procedure described by 8onner ( 5 ) . Yhi te

pota toes were purchased at 1 rupemrrkrt and used w i t h i n one or two days. The extraction medium

contained I U C ~ O S I (0.25 M ) , the K' s a l t s o f HEPES (10 W) and EGTA ( 2 MI plus cysteine (3 mpI)

and bovine serum albumen ( I n r ~ / m l ) and the pH was adjusted to 8.0 a t I'C. The wash mdium

contained s u c r o ~ e (0 .25 n) , the K' salts of HEPES ( 2 MU) and EGTA (0.1 nEl) and was adjusted t o

pH 7.1 a t I'C.

1 kg potatoes i s peeled and d i ced i n to 1 . 2 m cubes and t rans fer red to 2 1 O f extraction

' - Mitochondria were I so la ted from wh i te pota to tuber r

medium at 4'C and d i s r u p t e d i n a Yarlng Blender It h igh speed f o r 15 I . The b r e i i s then

necessary adjusted up t o pH 7.1. Dis rup t i on o f po ta toes releaser a c i d r h r c h i s b e l i e v e d to be squeezed through about 10 l ayers of cheese c l o t h and the pH o f t h e f i l t r a t e measured and i f

de t r imenta l t o the mr tochondr l r ( 5 , 3 2 ) . Consequently, to avoid exposing the mitochondria to a c i d pH. the pH of the e x t r a c t i o n medium i s Initially ad jus ted t o L high value (pH 8.0) IO t h a t

upon homgen iz r t ion the pH f a l l s c l o s e to the desired value.

mitochondria are t hen co l l ec ted by c e n t r i f u g a t i o n at 10,000 g f o r 15 mi". resuspended ~n about

40 m1 wash medium and then cent r i fuged twice a t 250 g f o r IO m r n and the s t a r c h p e l l e t s

d iscarded a f ter each spin. The mitochondria are then co l lected by cent r i fugat ion at 6,000 g f o r

15 mi". The f i n a l p e l l e t C o n l i s t s o f a firm p e l l e t w i t h a " f l u f f y " l a y e r on top. The " f l u f f y "

The f i l t r a t e i s then cent r i fuged at 1000 g fo r 15 " in and the pe l l e t d i sca rded . The

0 2 3 4 5

t (min) l a y e r i s d i s c a r d e d and t h e p e l l e t resuspended i n about 2 ml of 0.25 sucrose ta y i e l d a stock

r u r p e n r i o n o f about 20-30 W m l . Fig. I Pot ( ton l tPchondl ia are Permeable t o Ma!ukW. LS k i n e t l c r o f p o t a t o m i t o c h o n d r i a

(0.06 mg/ml) suspended i n mannitol ( t race 1) and sucmle media ( t race k ) are shown. The assay

media con ta ined n lnn l ta l (108 *) or ~ucrose (92 M), and the K' salts o f TES ( 5 W), EGTA (0.1 nU) p lus rotenone ( I pg/mg) and 8SA ( 0 . 3 ng/ml) and were ad jus ted t o pH 7.1 and maintained

The main d i f fe rence between t h i s procedure and that descr ibed by 80nner ( 5 ) i s our

w b s t i t u t i o n o f sucrose f o r mannitol. Ye have made many pleparat lons us ing bath manni to l -

con ta in in4 and sucrose-containina media. from there s tud ies i t i s e v i d e n t t h a t i n c l u l i o n of

mannrtol increaser the matr ix volume o f the mitochondr ia. Voshidl and Sat0 (33) observed a

s i m i l a r phenomenon i n t h e p r e p a r a t i o n o f castor bean mitochondria; however, desp i te I repor t by

Tederchi (34) i t has not been general ly recognized t h a t a11 n i tochondna are permab le ta manni to l . The LS traces con ta ined i n f > g . 1 show t h a t pota to mitochondria (sucrose-prepared)

swell at I s i g n i f i c a n t rate when Suspended ~n mannitol ( t race 1) whi le they swel l at a negligible rate i n sucrose ( t race B ) . UIlng the procedure described below. the rate of mannrtol

mannitol d u r i n g i s o l a t i o n , we f i n d t h a t the slopes o f L S Osmotic curves (35) fo r m i tochondr ia entry i n t h i s experiment i s ca lcu la ted to be abaut 50 nmol/min.mg. Cons is ten t w i th the en t ry o f

prepared i n m a n n i t o l - and sucrose-containing media d i f f e r b y about 3 - f o l d (see Fig . 2 ) . Since

the slopes of these curves are p r o p o r t i o n a l t o t h e s o l u t e Content o f t h e matrix (35). these data

i nd i ca te t ha t t he so lu te content of the mannitol-prepared mitochondr ia is about ] - fo ld tha t of

the sucrose-prepared mitochondr ia. Despi te th is d i f ference, hawver, no s i g n i f i c a n t difference i n t h e rater of r e s p i r a t i o n o r r e s p i r a t o r y c o n t r o l r a t i o s was observed.

at 2 5 ' C .


0.65

0.45

0.2E

I I I

10 20 30

1 lOSM Flg. 2 Cornoarison a f Abrorban2p Ormatrc Curves of Potato HltOChondria Isolated in Sucrase

addltjan o f m t a c h o n d r l a (0 08 m)/ml) to KC1 assay medla are plotted v e r l u ~ the reciprocal -. Valuer of the llght rcatterlng parameter B d e t e r m n e d a t 0.3 min following

orlnolalitler of the medla. The rnltochondrla were prepared r n parallel in sucrase-containing medla (0) and ~n nannltol-containing nedla ( I ) . vhlch were identical except for the substitution of mannltal f w rucrore. I$ dercrlbed under "Expermental Procedure$". The same batch of potatoes w a s used and each potato was dlv7ded between the two preparations The slopes. 11.9 m o m and 3.65 m o m for mannitol- and sucrase-prepared mltochandr\a respectively, a l e

proportional to the omotIcally active contents of the matrix (35). The assay media contained the K' salts of TES (5 M), EGTA (0.1 mM) plus C 1 to give the derlred osmolality plus rotenone

2 5 ' C .

(2 p g / n q ) and BSA (0.3 mg/al). The pH was adlusted to 7 .4 and the temperature malntained at

respiratory control. A typlcll trace abtalned I " I sucrose-bared medium I s shown in F?g. 3. As reported by others (4 .36 -38 ) . rapld rerpiratlan I P only observed a f t e r a p e n o d O f "condltianing". Thus. 200 n m l o f A D P is added after the addition of the mltochandrla and then a second pulse (400 m o l ) I S added after 4 m i " . State 3 rates for the second pulse u~ually fall i n the range 550-650 m o l O/mn.mg with the Uncoupled rate sllghtly higher. This s t a t e 3 Pate 1 3 very close to thlt reported by Neuberger (32 ) far "washed" potato mtochendna; however, the RCR which usually lies between 5.0-6.0 I S twice that reported even far Percoll-purlfied mitochondria (32) and hlgher than that reported far sucrase-density gradlent purifled mitochondria (39). These preparatronr are 1110 qu!te stable, a s evidenced by trace p ?n Fig. 3 whlch was abtalned with a 24 hr old preparation.

U s i n g our Irolat?on procedure. we routinely obtaln preparations which exh)bit very good

). X 0 40 &e

-

20 -

h e d u r r for "pre.co"dit- . ,, . ' - In order t0 determine the effect of inhibitors on rerpiratron ~n experiments ~n which we wished to pretreat the mitochondria with

t o o k 200 pl of the respiration medium described ~n F i g . 3 . added 2 pl rotenone (I q/ml). 6 p1 the inhibitor. we developed I procedure for "pre-conditioning" the mitochondria. Briefly. we

After 2.3 m n o f gentle agitation at 2 5 ' C . these mitochondria were able to r e w i l e at the $am BSA (150 mg/nl), 2 p1 AOV (100 M) fallowed by 50 pl of the stock mitochondrial IUIPLIISIOII.

ra te 15 normally conditioned mtachondrlr upon transfer of 75 pl of the pretreatmnt P us pen lion

to the 0,- electrode chamber. Thus. after 2 min of "pre-conditioning" the inhibitor ( e . 9 . TBT) was added and after 1 further minute an aliquot was transferred to the 0,-electrode chamber.

rnltochondrla. The a n l m unlport activity in there "precondltloned' mitochondria #as simi11.r to that in normal

carried out at pH 7 .4 and 25.t ~n assay media which contained the K' salts of C1' (55 nx1) or k- . Unless a t h e r w r e indicated, assays of anion transport were

malonate (37 O M ) or appropriate concentration Of any ether anion. TIS (5 M), and EGTA (0.1 nx1). The cancentratron of each substrate anion UII chosen to ensure that a11 media were of the saw armalallty. Anion transport was arrayed by following rwellrng. which accompanier net salt transpovt, urlng the llght scattering technlpue a s described in detarl elsewhere ( 3 5 . 4 0 ) . Unng thll technlque we generate L light rcattenng variable, b , which nomalizer reciprocal absorbance far mtochondrlal prote ln concentration. P (mill~grm~/ml), according to t h e f o m u l l

J, - ". ( d e ) nb dt

where # 4 1 the medlum osmolality (110 nilliormlal r n most studies reported here). 8, the solute content of the s t o c k preparation of mltochandria which *e shall arrune i s 190 narmol/mg since the ion cantent/mg proteln appears to be errentially the same a s liver mitochondria (3,4,35), b

I S the number of moles of ormatically a c t l v e particles which make up I mole o f

I S the slope of the equilibrium absorbance osmotic c w v e (3.65 m l l ~ o m o l a l from F i g . 2) and n

nmol/mg for potato mtachondrla, whlle it is 1400 nmal/mg for liver mitochondria reflecting the the tranrparted salt. Thus, far our egulpment a t 4 - 110 milliosmolal, (SJb il about 5700

h>gher value of b (I5 m ~ l l o r m o l a l ) (35).

with d 1 cm probe (2 cm llght path). For O p t i m u m renritlvnty we normally use aitochandrirl lo d e t e r m n e rates of solute transport. we use I Bnnkmann P m b e C a l o r i m t e r (Model PC700)

concentrations between 0.06~.1 nq/ml. However, for Itudlel I n which H' fluxes are examined, i t I S necessary to use m t o c h o n d r l a a t about I W m l . To follow 1s changer i n these suspensions we employ a probe wi th I variable path length and adJult the gap to about 2.5 m. It should be noted. however. that these L S data as presented cannot be compared quantitatively with those obtalned w t h the 1 cm probe. Although 8 I S normallzed for mtochandrlal concentration, I t

remalnr dependent on the length of the light path and the system used to determine light rcattenng changer.

Assay Of Proton Flurer - Proton fluxes were followd using an Orion combination g l m pH electrode (Model 81-02) connected to an Orlon pH meter (Hodel 1 O I A ) connected t o I Cyborg 4111 analogjdigital converter and Apple IIe computer. Proton fluxes were quantitated by internal calibration of each trace by addlog 2 p1 volumes of standard HC1 (0.100 H) at the end of each run. Urlng there Callbrations together with the amount of mlterhondrltl prote in used the data are platted ~n units Of ""01 H'/mg. Since only changes in the signill a m Of interest. far convenience, a certa in reading I S chosen as the reference pwnt and assigned the value zero.

extracts urlng atomic absorption spectrompy I S described elsewhere ( 4 1 ) . Determlnatian o f Ha" - Mg" content Of the rnitachandrla vas d e t e n m n e d by a m l y r i r O f

m r t o c h o n d n a were prepared a$ described prevlourly ( 3 5 ) .

D ~ W I and Reaoentx . M o l t drugs v e m obtained from the Sigma Chemical Company. Rat liver

n I I I I

R

t ( m i n ) Fig. 8 Jnh suspended in KC1 assay medium at pH 7.4 are shown. T n c e 3. control, n i g e r i d n (0.12 n m l / q )

l h L t i o n s f Cl Unioort b y Prooranalol. LS kinetics of mitochondria (0.00 mg/ml)

vas added at zem time and vallnomydn (Val. 1.2 nnol/mg) added as Indicated. Trace Q, control plus propranolol (50 ,A) added a t zero time. T n c e €, control plus propranolol (50 bU) added I S

indicated. The array medium I S dercrSbed under 'Ixperimental Procedures".


100 -

75 -

l o g (IJM PROP)

C 1 Un ipor t war fo l lowed using the L S technxque a$ dercrrbed i n F ig . 8 using K C 1 assay media. F ig . 9 D o x - R w o n ~ Curves f o r I n U b i t i o n o f C 1 Unioort bv ProDranolol. I n h i b l t i o n o f

1, Data Obtdlned a t pH 7.4. IC,, - I 4 lm. H l l l s l o p e 1.28 0. Data abtatned at pH 8.4.

IC,, - 50 lm, Hlll slope - 2 .0 . The assay medta were a s described under "Expenmental

PmCedUres" except TAPS replaced TES i n t h e pH 8.4 medium.

I I I I

01, I I I 1

25 50 75 100

E r y t h r o s i n B PM)

100

u a, 75

a Io

50 x

C I I

25 50 I

C i b a c r o n Blue 3 G A PM)

0 0 . 2 0.4 t (min)

F l g . 12 Ef fec t O f A23181 and no" on Anion UnlDort l n Potato n i t o c h a a . 1s k l n e t i c r of

mrtachondria (0.08 w/mll suspended i n KC1 assay medium a t pH 7.4 are shown. For each trace,

Trace 3, p lus EDTA (0 .1 MI; t race b. Plus EDTA w t h A23187 (A23, 12 nml/mg) added at 0.1 n in ;

n i g e r i c l n 1.12 nmol/mgl was added at zero t ime and valinomycin (Val. 1.1 nnal/mg) at 0 . 3 min.

t race L, p lus Ng" ( 2 mM1i trace d. P l u s Mg" ( 2 dl with A23187 added a t 0.1 ai". The array medlun is dercr lbed under "Exper immtal Procedures"

- I - I a

25 50 75 100

M e r s a l y l (nrnol/rng)

Fig. I1 l n h l b t t i o n O f Anion Unioort bv Nucleotide Analoa. Dose-rerponre curves fo r i n h i b l t l a n o f malonate (0) and C 1 ( I ) unipor t by Ery th ror rn B (panel A) and Cibacran Blue 361 (panel 8 ) are shown. The drug and n i g e r i c I n (0 .5 nmal/mg) *ere added a t zero t i m e . l r m l p o r t ra tes rere d e t e r m n e d e r r e n t r a l l y I S described rn Fig . 8 and valinomycin (1 nmol/nq) *as added a t 0.2 n i n . The co lo r imeter W a f f l t t e d w i t h 670 nm and 470 nm f i l t e r s f o p s t u d i e s w i t h

Ery th ros in 8 and Clbacron Blue respect ively. The assay media are descr ibed under "Exper imntsl

Procedures'

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Anion Uniport in Plant Mitochondria Is Mediated by …Anion Uniport in Plant Mitochondria Is Mediated by a Mg2+-insensitive Inner Membrane Anion Channel* (Received for publication,