Evidence for two distinct chloride uniport pathways in brown adipose tissue mitochondria.

Chloride permeability of the inner membrane of brown adipose tissue mitochondria was analyzed by monitoring mitochondrial swelling in KCl salts in the presence of K+ ionophores. The results indicate that the high anion conductance observed in these mitochondria is due to the presence of two separate pathways: 1) a Cl-conducting pathway that is inhibited by guanosine 5'-diphosphate (GDP) but neither by N,N'-dicyclohexylcarbodiimide (DCCD) nor by amphiphilic amines and that is found uniquely in brown adipose tissue mitochondria and 2) an inner membrane anion uniport channel that is inhibited both by DCCD and by amphiphilic amines but not by GDP and that is opened either by depletion of matrix Mg2+ or by alkalinization of the matrix.

K+-H+ exchange and volume homeostasis in brown adipose tissue mitochondria.

K+-H+ exchange activity in hamster brown adipose tissue mitochondria is activated following depletion of matrix Mg2+ with the divalent cation ionophore A23187. Quinine inhibits K+-H+ exchange reversibly with an I50 of 22 microM, whereas mild treatment with N,N'-dicyclohexylcarbodiimide (DCCD) inhibits this activity irreversibly. In an attempt to label and identify the K+-H+ antiporter protein, brown adipose tissue mitochondria were incubated with [14C]DCCD and subjected to denaturing polyacrylamide gel electrophoresis and fluorography. We observed a labeled band of relative mol wt, 78,000, which satisfies criteria established in rat liver mitochondria for the identification of this carrier (W. H. Martin et al., J. Biol. Chem. 259: 2062-2065, 1984). Thus Mg2+ and quinine each protect the K+-H+ exchanger against both inhibition and binding by DCCD. Volume homeostasis in brown adipose tissue mitochondria, as in other mitochondria, requires a balance between K+ influx and efflux. We propose that regulation of the K+-H+ antiporter, the primary K+ efflux mechanism, plays a major role in this process.

Kinetics of inhibition and binding of dicyclohexylcarbodiimide to the 82,000-dalton mitochondrial K+/H+ antiporter.

Inhibition of K+/H+ antiport by N,N'-dicyclohexylcarbodiimide in Mg2+ depleted mitochondria follows first order kinetics, exhibiting a half-time of 13 min when mitochondria are incubated with 50 nmol/mg inhibitor at 0 degrees C. 14C radiolabeled N,N'-dicyclohexylcarbodiimide binds to the 82,000-dalton protein, and the second order rate constant for binding is found to be approximately the same as the second order rate constant for inhibition. These findings provide additional confirmation of the identification of this porter with the 82,000-dalton protein and permit us to estimate that rat liver mitochondria contain about 8 pmol/mg of K+/H+ antiporter with a turnover number of 700 s-1. The K+/H+ antiporter of rat liver mitochondria is protected from N,N'-dicyclohexylcarbodiimide inhibition and binding by quinine and by endogenous Mg2+. An 82,000-dalton, [14C]N,N'-dicyclohexylcarbodiimide-binding protein is also observed in rat liver submitochondrial particles, establishing this as an integral protein of the inner membrane. Submitochondrial particles, presumed to be inverted in membrane orientation, are protected from radiolabeling by external Mg2+, supporting the contention that the Mg2+ binding site is localized to the matrix side of the K+/H+ antiporter.

On the mechanism by which dicyclohexylcarbodiimide and quinine inhibit K+ transport in rat liver mitochondria.

Passive uptake of potassium acetate into the mitochondrial matrix can be induced by nigericin, a K+/H+ antiporter, or by A23187, a Mg2+/2H+ antiporter. The latter process is thought to reflect operation of the Mg2+-dependent, endogenous K+/H+ antiporter, but there is ambiguity with respect to the mechanism of K+ transport in this assay (Nakashima, R.A., and Garlid, K.D. (1982) J. Biol. Chem. 257, 9252-9254). Kinetic analysis of potassium acetate transport provides verification that Mg2+ depletion 1) unmasks the K+/H+ antiporter, 2) opens up an intrinsic anion uniporter, 3) has no effect on acetic acid transport, and 4) does not induce high K+ uniport conductance. Mg2+-dependent uptake of potassium acetate is thereby shown to be mediated specifically by operation of the endogenous K+/H+ antiporter, as previously proposed. An extension of this analysis confirms that N,N'-dicyclohexylcarbodiimide and quinine block potassium acetate uptake via specific action on the K+/H+ antiporter. These findings support those of a previous study (Martin, W.H., Beavis, A.D., and Garlid, K.D. (1984) J. Biol. Chem. 259, 2062-2065) in which binding of [14C]N,N'-dicyclohexylcarbodiimide to membrane proteins under selective conditions was used to identify an 82,000-dalton band as the protein responsible for K+/H+ antiport in mitochondria.