Andrew D. BeavisS and Keith D. Garlid. From the Department of ...... Selwyn, M. J., Dawson, A. P., and Fulton, D. V. (1979) Biochem. Soc. 12. Warhurst, I. W.
THEJOURNALOF BlOLOGlCAL CHEMISTRY
Vol. 263,No. 16, Issue of June 5, pp. 7574-7580,1988 Printed in U.S.A.
0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of the Mitochondrial InnerMembrane Anion Channel by Dicyclohexylcarbodiimide EVIDENCE FOR A SPECIFICTRANSPORT PATHWAY* (Received for publication, November 17,
Andrew D. BeavisS and Keith D. Garlid From the Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43699
Electrophoretic uniport of anions through the inner wide variety of anions andthat itis also regulated by protons. mitochondrial membrane can be activated byalkaline These latterfindings led us to propose that thepH- and M2+pH or by depleting the matrix of divalent cations. It regulated’ pathways are identical (8,9). has also been suggestedthat, in the presence ofvalinA question still unanswered is whether the change in permeomycin and potassium, respiration can also activate ability induced by M2+depletion and/or alkaline pH repreanion uniport.We have proposed thatsingle a pathway sents anopening of a specific pore or a change in the permeis responsible forall three of these transport processes ability properties of the lipid bilayer. In recent years, Selwyn’s (Garlid, K. D., and Beavis, A.D. (1986) Biochim. group (10) has demonstrated that at elevated pH electrophoBiophys. Acta 863, 187-204). We now present evi- retic transport of bicarbonate takes place in mitochondria but dence that like the “pH-dependent” pore the divalent notin liposomes and has suggested that a specific “pHcation-regulatedporeandthe“respiration-induced” pore are blocked by N,N’-dicyclohexylcarbodiimide dependent anion-conducting pore” exists in mitochondria (11).Furthermore, they have presented evidence that this (DCCD). Moreover, the kinetics of inhibition of the latter two pathways are identical and exhibit a second pore is blocked by N,N’-dicyclohexylcarbodiimide (12). DCCD is a hydrophobic alkylating agent which can react order rate constant of 2.6 % lo-’ (nmol DCCD/mg)-’. min-’. DCCD inhibits the uniport of C1-, phosphate, with a number of different groups on proteinsand lipids malate, and other lipophobic anions completely, but it including thiols and carboxyl groups. In thepast few years it has no effect on the classical electroneutral phosphate has proved to be a very useful tool in the study of a number anddicarboxylate carriers. In Mg+-depleted mito- of bioenergetically important proteinsof the inner mitochonchondria DCCD partiallyinhibitsthetransport of drial membrane including the F,Fo-ATPase (13), complex I11 SCN-; however, in Mg2+-containing mitochondria and(14-16), complex IV (17), the transhydrogenase (18, 19), and at low pH, no inhibition is observed. Furthermore, in the K’/H+ antiporter (20-22). Thus, although the finding DCCD-treated mitochondria,even following depletion that DCCD inhibitsanionuniport is consistent with the of M 8 + , the transport of SCN- is independent of pH. existence of a proteinpore, by itself this finding does not rule These results lead us to conclude that two pathways out the possibility that transport occurs through the lipid for anion uniport exist: a specific, regulated pathway bilayer. In fact, Jung et al. (23) have reported that DCCD whichcanconductawide variety ofanionsanda blocks monovalent cation uniport through the inner memnonregulated pathway through the lipid bilayer which brane of heart mitochondria and have attributed this effect only conducts lipid-soluble ions. to blockage of a nonspecific leak. Wehave now investigated the effect of DCCD on the divalent-cation-regulated anion uniport pathway and a numNormal, freshly isolated mitochondria possess a very low ber of other transportprocesses in mitochondria. The results electrophoretic permeability to most anions, with the excep- indicate that DCCD completely blocks anion uniport through tion of anions which are capable of delocalizing their electric the putative pore buthas no effect on uniport of SCNcharge such as SCN- and C10;. Twenty years ago, however, through the lipid bilayer or electroneutral phosphateand the groups of Azzone (1-4) and Brierley (5-7) demonstrated dicarboxylate transport via the classical anion carriers. These that at elevated pH the inner membranes of liver and heart findings provide further supportfor the proposal that the pHmitochondria, respectively, become permeable to many differ- dependent pore and the divalent cation-regulated pathway ent inorganic and organic anions. Recently, we presented are identical and suggest that these transport processes do evidence that an anion uniport pathway can be opened also not reflect leakage through the lipid bilayer. Some of these at pH 7.4 by depleting the matrix of divalent cations (8). In data have been presented elsewhere in a preliminary report addition, we demonstrated that this pathway can transport a (24).
* This work wassupported by National Institutes of Health Grants GM 31086 andHL 36573 awarded by the National Institute of General Medical Sciences and the National Heart, Lung and Blood Institute, UnitedStates Public Health Service, Department of Health and Human Services and also by a grantfrom the Northwestern Ohio Chapter of the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘‘Oduertkemnt’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ T o whom correspondence should be addressed Dept. of Pharmacology, Medical College of Ohio, C.S. 10008,Toledo, OH 43699.
Rat liver mitochondria were prepared as previously described (25). Respiratory control was determined in a medium containing the K+ ‘The abbreviations used are: M2’, divalent cation; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; CCCP, carbonyl cyanide m-chlorophenylhydrazone; TES, N-tris[hydroxymethyll methyl-2-aminoethanesulfonicacid L.S., light scattering; mosm, milliosmolal; nosm, nanoosmolal; DCCD, N,N’-dicyclohexylcarbodiimide.
Anion Uniport inMitochondria salts of Cl(120 mM), TES (5 mM), succinate (5 mM), Pi (2 mM), and EGTA (0.5mM) plus rotenone (1 pglmg). Mitochondria were usedat about 2 mgof protein/ml. Anion uniport was assayed by following swelling, which accompanies net salt transport, using the light scattering technique as described indetail elsewhere (25, 26). Usingthis technique we generate a light scatteringvariable, 8, which normalizes P reciprocalabsorbanceformitochondrialproteinconcentration, (milligrams/ml), accordingto the formula
1 4 ,,,_,_.,,.._..
0.2 - A23187
where a is a machine constant and P. (equals 1mg/ml) is a constant introduced to make B dimensionless. Note that B increases as the mitochondrial matrixvolume increases. The rate of salt transport is calculated from the rate of change of 6 according to the formula (26)
P (A-' - a )