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Previous work described ATP-sensitive K(+) channel (K(ATP)) openers (e.g., BMS-180448), which retain the cardioprotective activity of agents such as cromakalim while being significantly less potent as vasodilators. In this study, we describe the pharmacologic profile of BMS-191095, which is devoid of peripheral vasodilating activity while retaining glyburide-reversible cardioprotective activity. In isolated rat hearts subjected to 25 min of global ischemia and 30 min of reperfusion, BMS-191095 increased the time to onset of ischemic contracture with an EC(25) of 1.5 microM, which is comparable to 4.7 microM and 3.0 microM for cromakalim and BMS-180448, respectively. Comparisons of cardioprotective and vasorelaxant potencies in vitro and in vivo showed BMS-191095 to be significantly more selective for cardioprotection with virtually no effect on peripheral smooth muscle, whereas cromakalim showed little selectivity. In addition to increasing the time to the onset of contracture, BMS-191095 improved postischemic recovery of function and reduced lactate dehydrogenase release in the isolated rat hearts. The cardioprotective effects of BMS-191095 were abolished by glyburide and sodium 5-hydroxydecanoate (5-HD). BMS-191095 did not shorten action potential duration in normal or hypoxic myocardium within its cardioprotective concentration range nor did it activate sarcolemmal K(ATP) current (or =30 microM). BMS-191095 opened cardiac mitochondrial K(ATP) with a K(1/2) of 83 nM, and this was abolished by glyburide and 5-HD. These results show that the cardioprotective effects of BMS-191095 are dissociated from peripheral vasodilator and cardiac sarcolemmal K(ATP) activation. Agents like BMS-191095 may owe their cardioprotective selectivity to selective mitochondrial K(ATP) activation.

Certain lipophilic cations have been reported to display anticarcinoma activities because of their selective uptake and retention by mitochondria of cancer cells. Thus, these agents may comprise a unique class of agents directed against carcinoma. After screening more than 1000 lipophilic cations, we found that the monovalent lipophilic cation, 2,6-bis(4-amino-phenyl)-4-[4-(dimethylamino)phenyl]thiopyrylium chloride (AA1), displayed remarkable anticarcinoma activity both in vitro and in vivo. Unlike most other lipophilic cations, AA1 is stable and displays minimal light sensitivity. In vitro testing showed that AA1 was 10 times more toxic to the carcinoma cell line CX-1 than to the normal epithelial cell line CV-1. In vivo animal experiments showed that AA1 significantly prolonged the survival of mice implanted with tumors. For C57BL x DBA/2 F1 mice implanted with the mouse bladder carcinoma cell line, MB49, the treated:control ratio was 344%. For Swiss nu/nu mice implanted i.p. with the human melanoma cell line, LOX, the treated:control ratio was 341%. The most significant observation was obtained with Swiss nu/nu mice that were implanted i.p. with the human ovarian cell line, OVCAR-III. The treated:control ratio in this situation was greater than 450%. In all these tumor models, AA1 produced minimal toxicities. AA1 exhibited little inhibition of electron transport in isolated rat liver mitochondria; however, it inhibited mitochondrial ATPase with 50% inhibitory concentration of 6 microM. Compared with previously reported anticarcinoma lipophilic cations such as rhodamine 123 and dequalinium chloride, AA1 appeared to display more effective in vivo anticarcinoma activity. Thus, AA1 could be considered for further clinical development as a candidate for anticarcinoma chemotherapy.

The transport properties of mitochondria are such that net potassium flux across the inner membrane determines mitochondrial volume. It has been known that K+ uptake is mediated by diffusive leak driven by the high electrical membrane potential maintained by redox-driven, electrogenic proton ejection and that regulated K+ efflux is mediated by an 82-kDa inner membrane K+/H+ antiporter. There is also long-standing suggestive evidence for the existence of an inner membrane protein designed to catalyze electrophoretic K+ uptake into mitochondria. We report reconstitution of a highly purified inner membrane protein fraction from rat liver and beef heart mitochondria that catalyzes electrophoretic K+ flux in liposomes and channel activity in planar lipid bilayers. The unit conductance of the channel at saturating [K+] is about 30 pS. Reconstituted K+ flux is inhibited with high affinity by ATP and ADP in the presence of divalent cations and by glibenclamide in the absence of divalent cations. The mitochondrial ATP-dependent K+ channel is selective for K+, with a Km of 32 mM, and does not transport Na+. K+ transport depends on voltage in a manner consistent with a channel activity that is not voltage-regulated. Thus, the mitochondrial ATP-dependent K+ channel exhibits properties that are remarkably similar to those of the ATP-dependent K+ channels of plasma membranes.

Small mammals, including human infants, rely on nonshivering thermogenesis for a substantial portion of their body heat during exposure to cold. This thermogenesis is mediated in large part by the uncoupling protein, which is found exclusively within the inner membrane of brown adipose tissue mitochondria. The sole function of uncoupling protein is to provide a regulated transport pathway for electrophoretic back-flux of H+ ions into the mitochondrial matrix, thereby dissipating the protonmotive force and producing heat. Thus, uncoupling protein is unique with respect to both its physiological role and its tissue expression. We have now achieved high level expression of rat uncoupling protein in yeast, with import into yeast mitochondria at levels, 70-100 micrograms/mg of mitochondrial protein, similar to those observed in brown adipose tissue mitochondria from cold-adapted rats. When the expressed protein was purified and reconstituted into liposomes, the proteoliposomes exhibited GDP-sensitive proton and chloride uniports that were inhibited by GDP with Ki values similar to those obtained with native protein. Moreover, the molecular activities of the expressed protein with respect to Cl- and H+ transport were indistinguishable from those of native protein. The availability of unlimited amounts of functional, expressed uncoupling protein will now permit application of site-directed mutagenesis to the many intriguing aspects of uncoupling protein structure and function.

In bovine cardiac sarcolemmal vesicles, an outward H+ gradient stimulated the initial rate of amiloride-sensitive uptake of 22Na+, 42K+, or 86Rb+. Release of H+ from the vesicles was stimulated by extravesicular Na+, K+, Rb+, or Li+ but not by choline or N-methylglucamine. Uptakes of Na+ and Rb+ were half-saturated at 3 mM Na+ and 3 mM Rb+, but the maximal velocity of Na+ uptake was 1.5 times that of Rb+ uptake. Na+ uptake was inhibited by extravesicular K+, Rb+, or Li+, and Rb+ uptake was inhibited by extravesicular Na+ or Li+. Amiloride-sensitive uptake of Na+ or Rb+ increased with increase in extravesicular pH and decrease in intravesicular pH. In the absence of pH gradient, there were stimulations of Na+ uptake by intravesicular Na+ and K+ and of Rb+ uptake by intravesicular Rb+ and Na+. Similarly, there were trans stimulations of Na+ and Rb+ efflux by extravesicular alkali cations. The data suggest the existence of a nonselective antiporter catalyzing either alkali cation/H+ exchange or alkali cation/alkali cation exchange. Since increasing Na+ caused complete inhibition of Rb+/H+ exchange, but saturating K+ caused partial inhibitions of Na+/H+ exchange and Na+/Na+ exchange, the presence of a Na(+)-selective antiporter is also indicated. Although both antiporters may be involved in pH homeostasis, a role of the nonselective antiporter may be in the control of Na+/K+ exchange across the cardiac sarcolemma.

Under highly selective conditions, pretreatment of rat liver mitochondria with N,N'-dicyclohexylcarbodiimide (DCCD) results in 95% inhibition of K+/H+ antiport. This inhibition is irreversible. The K+/H+ antiporter is reversibly inhibited by physiological (Mg2+ and H+) and pharmacological (quinine and propranolol) inhibitors. Each of these inhibitors protects the K+/H+ antiporter against inhibition by DCCD. DCCD is without effect on Na+/H+ antiport under all conditions tested, confirming our contention that rat liver mitochondria possess two different alkali cation/proton antiporters (Nakashima, R. A., and Garlid, K. D. (1982) J. Biol. Chem. 257, 9252-9254). The selective nature of irreversible inhibition by DCCD has enabled us to label and identify the protein responsible for K+/H+ antiport. This protein, the first cation antiporter so identified, migrates on sodium dodecyl sulfate-polyacrylamide gels with a molecular weight of 82,000.

The active potential difference across bovine corneal endothelium was measured in vitro at different temperatures, pH, osmolality and salt compositions. The measurements were made using either identical or different solutions on each side of the membrane. The experimental results are consistent with a model in which sodium is actively transported into the intercellular cleft. We propose that Na+ re-enters the cell electroneutrally by coupled co-transport with carbonate, derived from bicarbonate in the solution.

Mitochondria contain two Na+/H+ antiporters, one of which transports K+ as well as Na+. The physiological role of this non-selective Na+/H+ (K+/H+) antiporter is to provide mitochondrial volume homeostasis. The properties of this carrier have been well documented in intact mitochondria, and it has been identified as an 82,000-dalton inner membrane protein. The present studies were designed to solubilize and reconstitute this antiporter in order to permit its isolation and molecular characterization. Proteins from mitoplasts made from rat liver mitochondria were extracted with Triton X-100 in the presence of cardiolipin and reconstituted into phospholipid vesicles. The reconstituted proteoliposomes exhibited electroneutral 86Rb+ transport which was reversibly inhibited by Mg2+ and quinine with K0.5 values of approximately 150 and 300 microM, respectively. Incubation of reconstituted vesicles with dicyclohexylcarbodiimide resulted in irreversible inhibition of 86Rb+ uptake into proteoliposomes. Incubation of vesicles with [14C]dicyclohexylcarbodiimide resulted in labeling of an 82,000-dalton protein. These properties, which are also characteristic of the native Na+/H+ (K+/H+) antiporter, lead us to conclude that this mitochondrial carrier has been reconstituted into proteoliposomes with its known native properties intact.

1. Ionophore A23187 depletes the endogenous Mg2+ of beef heart mitochondria to 4 ng ions . mg-1 (or less) and Ca2+ to less than 2 ng ions . mg-1 in a reaction which does not require metabolic energy. Ionophore A23187 also promotes the near quantitative extrusion of endogenous K+ from heart mitochondria to a K+-free medium, but this reaction requires respiration and is sensitive to uncouplers. Respiring mitochondria which extrude K+ in the presence of A23187 maintain high membrane potentials and do not swell. The K+ depletion has the properties of a K+/H+ exchange reaction occurring at the expense of delta pH of respiration. 2. The rate and extent of K+ efflux are dependent on the removal of endogenous divalent cations by A23187, but are otherwise independent of the amount of A23187 present. It is concluded that K+ efflux results from activation of an endogenous K+/H+ exchange component and that A23187 does not function directly as a K+/H+ exchanger in mitochondria.

1. A23187 and ionomycin cause the release of Ca2+, Mg2+, and K+ from the mitochondrial matrix. The electroneutral K+ efflux does not reflect direct transport by these ionophores but, rather, results from release of the endogenous K+/H+ exchanger from inhibition by divalent cations. 2. A23187 and ionomycin differ in their affinities for CA2+ and Mg2+, having relative affinity ratios (Ca2+/Mg2+) in respiring mitochondria of 3 and 35, respectively. 3. This difference in Ca2+/Mg2+ affinities was exploited in dose-response studies designed to determine which cation exerts primary control over K+/H+ exchange activity. The results of such studies demonstrate that removal of Ca2+ has no effect on K+ efflux, and that K+ efflux follows Mg2+ efflux whether induced by ionomycin or A23187. 4. K+ steady states, in which K+ uniport equals K+/H+ antiport, were induced by adding low levels of valinomycin to respiring mitochondria. The addition of Ca2+ perturbed the steady state in the direction of increased K+/H+ exchange. 5. Addition of Ca2+ to respiring mitochondria resulted in electroneutral K+ efflux which was strongly affected by the anion composition of the medium. We propose that Ca2+ turns on the K+/H+ exchanger indirectly, through a decrease in matrix [Mg2+] secondary to swelling and uptake of chelating anions. This phenomenon may be of physiological importance as a temporary protection against excessive swelling due to Ca2+ uptake. 6. These studies support the contention that Mg2+, not Ca2+, is the inhibitory cation responsible for the physiological regulation of mitochondrial K+/H+ exchange.

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.

Respiring mitochondria drive the electrophoretic uptake of K+ and other cations. In the presence of permeant acids this transport leads to mitochondrial swelling if it is not compensated by electroneutral K+/H+ exchange mediated by the K+/H+ antiporter. The mechanism of influx has yet to be established; however, evidence is accumulating that in addition to leak pathways a specific K+ channel or uniporter may be involved. We examine some of the properties of K+ uniport which are consistent with the existence of a specific ATP-regulated K+ channel. In contrast to the K+/H+ antiporter, K+ uniport shows little dependence on pH. K+ uniport is, however, very sensitive to inhibition by adenine nucleotides. The maximum percent inhibition is increased from 40 to 60% by treatment of mitochondria with N-ethylmaleimide (30 nmol/mg) which stimulates K+ uniport 3.6-fold. N-Ethylmaleimide, however, has no effect on the IC50 values which are 0.5, 2.3, and 8 microM for ADP, ATP, and AMP, respectively. GDP has no effect, while carboxyatractyloside is found to inhibit. The nucleotide analogs Cibacron blue 3GA and erythrosin B exhibit three effects on K+ uniport. Low doses partially inhibit K uniport (I50 = 0.13 microM Cibacron Blue), while higher doses stimulate (EC50 = 13 microM Cibacron Blue). Stimulation is especially apparent in N-ethylmaleimide-treated mitochondria. These analogs also antagonize inhibition by ATP. Since the EC50 values for this antagonism for these two drugs are similar, while the IC50 values for inhibition of ATP transport differ by a factor of five, we suggest that inhibition of K+ uniport by ATP is not mediated via the adenine nucleotide translocase. These data are consistent with the existence of an ATP-regulated K+ channel in the inner mitochondrial membrane.

1. Rat liver mitochondria undergo a spontaneous, respiration-dependent K+ extrusion which is accelerated by citrate. This K+ efflux is electroneutral and is considered to occur on an endogenous K/H exchanger. The spontaneous efflux, but not nigericin-induced K/H exchange, is always preceded by a lag phase, suggesting that the lag phase is a characteristic property of the endogenous exchange reaction. 2. K+ extrusion induced by ionophore A23187 also has the characteristics of K/H exchange. The rate of K+ efflux is faster and the lag time is shorter when compared to endogenous K+ efflux. The effects of A23187 on the lag phase suggest that the ionophore acts by unmasking the endogenous exchanger. This conclusion is supported by the finding that K+ efflux rates reach a maximum which cannot be exceeded by increasing the dose of A23187 but is exceeded by adding nigericin. 3. Steady state perturbation studies were carried out on respiring mitochondria in which electrophoretic K+ influx was balanced by electroneutral K+ efflux. These steady states were appropriately shifted in opposite directions by additions of nigericin or valinomycin. In contrast, addition of A23187 had no effect. It is concluded that A23187 is incapable of transporting K+ in rat liver mitochondria. 4. These results are consistent with a model in which free matrix Mg2+ acts as a K/H carrier "brake." The proposed role of this carrier-brake mechanism is to provide volume homeostasis with minimal energy expenditure. According to this model, both citrate and A23187 stimulate K/H exchange by reducing Mg2+ activity within the matrix. Citrate acts by complexation of Mg2+, while A23187 acts by transporting Mg2+ out of the matrix.

We describe purification of three different states of the 82-kDa K+/H+ antiporter from rat liver mitochondria. The denatured 82-kDa protein, identified by its selective labeling with [14C]dicyclohexylcarbodiimide (DCCD), was purified by preparative two-dimensional gel electrophoresis. This purified product was used to raise and immunopurify monospecific polyclonal antibodies. Western blot analysis showed that the [14C] DCCD-labeled 82-kDa protein is not a DCCD-crosslinked product. The native, [14C]DCCD-labeled, 82-kDa protein was purified by (NH4)2SO4 fractionation and column chromatography, using 14C labeling and gel electrophoresis to track the protein. The native, non-DCCD-labeled 82-kDa protein was purified by similar procedures, using immunopurified antibodies to track the protein. DCCD binding had no effect on chromatographic behavior of the antiporter protein. This protocol resulted in purification of the 82-kDa protein to apparent homogeneity. The purified, native 82-kDa protein was reconstituted into proteoliposomes and assayed for K+ transport with the new fluorescent probe, PBFI. K+ transport was electroneutral and was inhibited by DCCD, Mg2+, and timolol. The turnover number for K+ transport was about 1000 s-1, very similar to the value previously estimated in intact mitochondria.

The amount of light scattered by a mitochondrial suspension depends on matrix volume (Tedeschi, H., and Harris, D.L. (1955) Arch. Biochem. Biophys. 58, 52-67), a correlation which has been extensively exploited for qualitative studies of solute transport across the inner membrane. To obtain reliable, quantitative estimates of solute transport, it is first necessary to characterize the factors determining mitochondrial light scattering. We show that the dependence of absorbance on mitochondrial concentration can be linearized, resulting in an intrinsic light scattering parameter which is independent of the concentration and source of mitochondria. We show that the absorbance osmotic curve is segmentally linear, exhibiting discontinuities which disappear irreversibly following preswelling. In contrast, direct measurements reveal matrix volume to be reversibly and linearly dependent on inverse osmolality. This divergence is a consequence of the fact that the optical technique samples total particle volume, including contributions from folded membranes and trapped medium. These contributions are minimized by structural components, such as intermembrane connections and the outer membrane, which contribute to efficient packaging of the mitochondrion. When these structures are broken, the mitochondrion cannot return to its native state. We observe that the swelling-induced, irreversible transition from efficient packaging to a random packing state begins at a matrix volume of 1.9 microliter/mg and is complete at 3.1 microliter/mg. These findings complicate the interpretation of light scattering results but do not appear to present an insurmountable obstacle to the quantitative application of this technique to transport kinetics.

The uncoupling protein is one of a family of mitochondrial transport proteins involved in energy metabolism. It dissipates oxidative energy to generate heat, either by catalyzing proton transport directly or by catalyzing fatty acid anion transport, thus enabling fatty acids to act as cycling protonophores. This transport process is tightly regulated by purine nucleotides. We have expressed uncoupling protein in yeast and examined its proton transport activity after its reconstitution into proteoliposomes. A directed change of Arg276 to Leu or Gln completely abolished nucleotide inhibition of protonophoretic action of the reconstituted mutant uncoupling proteins without affecting the transport process. Arg276 is the first residue of functional importance to be identified in uncoupling protein. Mutation of the homologous residue in the yeast ADP/ATP translocator prevented the growth of yeast on a nonfermentable carbon source, presumably by interfering with nucleotide exchange (Nelson, D. R., Lawson, J. E., Klingenberg, M., and Douglas, M. G. (1993) J. Mol. Biol. 230, 1159-1170). Demonstration of the essential role of a single homologous residue in protein-nucleotide interaction within these two transporters is the first direct evidence that uncoupling protein and the ADP/ATP translocator belong to the same gene family.

The mitochondrial Na+/Ca2+ antiporter plays a key role in the physiological regulation of intramitochondrial Ca2+, which in turn attunes mitochondrial enzymes to the changing demands of the cell for ATP. We have now purified the Na+/Ca2+ antiporter from beef heart mitochondria by assaying detergent-solubilized chromatography fractions for reconstitutive activity. Na+ and Ca2+ transport were assayed using the fluorescent probes, sodium-binding benzofuran isophthalate and Fura-2, respectively. This approach enabled us to identify Na+/Ca2+ exchange activity with a 110-kDa inner membrane protein that catalyzed Na(+)-dependent Ca2+ transport and Ca(2+)-dependent Na+ transport. A new finding was that the Na+/Ca2+ antiporter also catalyzed Na+/Li+ exchange in the absence of Ca2+. All modes of transport were electroneutral and were inhibited by diltiazem and tetraphenylphosphonium cation. Monospecific polyclonal antibodies to the 110-kDa protein inhibited Na+/Ca2+ and Na+/Li+ exchange in the reconstituted system and recognized 110-kDa proteins in mitochondrial membranes isolated from rat heart, liver, and kidney.

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.