Mechanism of uncoupling protein action.

Two competing models of uncoupling protein (UCP) transport mechanism agree that fatty acids (FAs) are obligatory for uncoupling, but they disagree about which ion is transported. In Klingenberg's model, UCPs conduct protons. In Garlid's model, UCPs conduct anions, like all members of this gene family. In the latter model, UCP transports the anionic FA head group from one side of the membrane to the other, and the cycle is completed by rapid flip-flop of protonated FAs across the bilayer. The head groups of the FA analogues, long-chain alkylsulphonates, are translocated by UCP, but they cannot induce uncoupling, because these strong acids cannot be protonated for the flip-flop part of the cycle. We have overcome this limitation by ion-pair transport of undecanesulphonate with propranolol, which causes the sulphonate to deliver protons across the membrane as if it were an FA. Full GDP-sensitive uncoupling is seen in the presence of propranolol and undecanesulphonate. This result confirms that the mechanism of UCP uncoupling requires transport of the anionic FA head group by UCP and that the proton transport occurs via the bilayer and not via UCP.

Identification and properties of a novel intracellular (mitochondrial) ATP-sensitive potassium channel in brain.

Protection of heart against ischemia-reperfusion injury by ischemic preconditioning and K(ATP) channel openers is known to involve the mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)). Brain is also protected by ischemic preconditioning and K(ATP) channel openers, and it has been suggested that mitoK(ATP) may also play a key role in brain protection. However, it is not known whether mitoK(ATP) exists in brain mitochondria, and, if so, whether its properties are similar to or different from those of heart mitoK(ATP). We report partial purification and reconstitution of a new mitoK(ATP) from rat brain mitochondria. We measured K(+) flux in proteoliposomes and found that brain mitoK(ATP) is regulated by the same ligands as those that regulate mitoK(ATP) from heart and liver. We also examined the effects of opening and closing mitoK(ATP) on brain mitochondrial respiration, and we estimated the amount of mitoK(ATP) by means of green fluorescence probe BODIPY-FL-glyburide labeling of the sulfonylurea receptor of mitoK(ATP) from brain and liver. Three independent methods indicate that brain mitochondria contain six to seven times more mitoK(ATP) per milligram of mitochondrial protein than liver or heart.

Alkylsulfonates as probes of uncoupling protein transport mechanism. Ion pair transport demonstrates that direct H(+) translocation by UCP1 is not necessary for uncoupling.

The mechanism of fatty acid-dependent uncoupling by mitochondrial uncoupling proteins (UCP) is still in debate. We have hypothesized that the anionic fatty acid head group is translocated by UCP, and the proton is transported electroneutrally in the bilayer by flip-flop of the protonated fatty acid. Alkylsulfonates are useful as probes of the UCP transport mechanism. They are analogues of fatty acids, and they are transported by UCP1, UCP2, and UCP3. We show that undecanesulfonate and laurate are mutually competitive inhibitors, supporting the hypothesis that fatty acid anion is transported by UCP1. Alkylsulfonates cannot be protonated because of their low pK(a), consequently, they cannot catalyze electroneutral proton transport in the bilayer and cannot support uncoupling by UCP. We report for the first time that propranolol forms permeant ion pairs with the alkylsulfonates, thereby removing this restriction. Because a proton is transported with the neutral ion pair, the sulfonate is able to deliver protons across the bilayer, behaving as if it were a fatty acid. When ion pair transport is combined with UCP1, we now observe electrophoretic proton transport and uncoupling of brown adipose tissue mitochondria. These experiments confirm that the proton transport of UCP-mediated uncoupling takes place in the lipid bilayer and not via UCP itself. Thus, UCP1, like other members of its gene family, translocates anions and does not translocate protons.

Cardioprotection by ischemic preconditioning preserves mitochondrial function and functional coupling between adenine nucleotide translocase and creatine kinase.

M. N. Laclau, S. Boudina, J. B. Thambo, L. Tariosse, G. Gouverneur, S. Bonoron-Adèle, V. A. Saks, K. D. Garlid and P. Dos Santos. Cardioprotection by Ischemic Preconditioning Preserves Mitochondrial Function and Functional Coupling Between Adenine Nucleotide Translocase and Creatine Kinase. Journal of Molecular and Cellular Cardiology (2001) 33, 947-956. This study investigates the effect of ischemic preconditioning on mitochondrial function, including functional coupling between the adenine nucleotide translocase and mitochondrial creatine kinase, which is among the first reactions to be altered in ischemia. Three groups of Langendorff-perfused rat hearts were studied: a control group, a group subjected to 30 min ischemia followed by 15 min reperfusion, and a group subjected to ischemic preconditioning prior to 30 min ischemia and 15 min reperfusion. Ischemic preconditioning significantly delayed the onset and amplitude of contracture during ischemia, decreased enzymatic release, and improved the recovery of heart contractile function after reperfusion. Mitochondrial function was assessed in permeabilized skinned fibers. The protective effect of preconditioning was associated with preservation of mitochondrial function, as evidenced by maintenance of the high K(1/2)for ADP in regulation of mitochondrial respiration and V(max)of respiration, the near absence of respiratory stimulation by exogenous cytochrome c, and preservation of functional coupling between mitochondrial creatine kinase and adenine nucleotide translocase. These data suggest that ischemic preconditioning preserves the structure-function of the intermembrane space, perhaps by opening the mitochondrial ATP-sensitive K(+)channel. The consequence is preservation of energy transfer processes from mitochondria to ATP-utilizing sites in the cytosol. Both of these factors may contribute to cardioprotection and better functional recovery of preconditioned hearts.

Pharmacologic characterization of BMS-191095, a mitochondrial K(ATP) opener with no peripheral vasodilator or cardiac action potential shortening activity.

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.

Bioenergetic consequences of opening the ATP-sensitive K(+) channel of heart mitochondria.

There is an emerging consensus that pharmacological opening of the mitochondrial ATP-sensitive K(+) (K(ATP)) channel protects the heart against ischemia-reperfusion damage; however, there are widely divergent views on the effects of openers on isolated heart mitochondria. We have examined the effects of diazoxide and pinacidil on the bioenergetic properties of rat heart mitochondria. As expected of hydrophobic compounds, these drugs have toxic, as well as pharmacological, effects on mitochondria. Both drugs inhibit respiration and increase membrane proton permeability as a function of concentration, causing a decrease in mitochondrial membrane potential and a consequent decrease in Ca(2+) uptake, but these effects are not caused by opening mitochondrial K(ATP) channels. In pharmacological doses (<50 microM), both drugs open mitochondrial K(ATP) channels, and resulting changes in membrane potential and respiration are minimal. The increased K(+) influx associated with mitochondrial K(ATP) channel opening is approximately 30 nmol. min(-1). mg(-1), a very low rate that will depolarize by only 1-2 mV. However, this increase in K(+) influx causes a significant increase in matrix volume. The volume increase is sufficient to reverse matrix contraction caused by oxidative phosphorylation and can be observed even when respiration is inhibited and the membrane potential is supported by ATP hydrolysis, conditions expected during ischemia. Thus opening mitochondrial K(ATP) channels has little direct effect on respiration, membrane potential, or Ca(2+) uptake but has important effects on matrix and intermembrane space volumes.

The mitochondrial potassium cycle.

The mitochondrial K+ cycle consists of influx and efflux pathways for K+ and anions. Net movement of K+ salts across the inner membrane causes changes of matrix volume, so regulation of the cycle is vital for maintaining the structural integrity of the organelle. The mitochondrial K+ cycle also appears to play important roles in cellular pathophysiology in vivo. Opening the mitochondrial ATP-sensitive K+ channel (mitoK(ATP)) prior to ischemia protects the heart from ischemia-reperfusion injury. MitoK(ATP) is an important player in the cell signaling pathways for ischemic protection and also for gene transcription, roles that appear to depend on the ability of mitoK(ATP) opening to trigger increased mitochondrial production of reactive oxygen species. MitoK(ATP) opening during both ischemia and reperfusion and during the high work state is found to preserve the structure of the intermembrane space and thereby maintains the normally low outer membrane permeability to adenine nucleotides. This review discusses the properties of the mitochondrial K+ cycle that help to understand the basis of these effects.

Opening mitochondrial K(ATP) in the heart--what happens, and what does not happen.

There is considerable evidence that opening the mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) is cardioprotective in ischemia-reperfusion. Two prominent questions surround the role of mitoK(ATP) in the cardiomyocyte: How does opening mitoK(ATP) protect? What is the normal physiological role of mitoK(ATP) in the heart? Before these questions can be addressed, it is necessary to agree on the bioenergetic consequences of opening mitoK(ATP), and this distills down to a single question--does opening mitoK(ATP) cause significant uncoupling or not? The evidence strongly indicates that it does not and that reports of uncoupling and inhibition of Ca2+ uptake are the result of using toxic concentrations of K(ATP) channel openers. Thus, opening mitoK(ATP) results in increased K+ flux that is sufficient to change mitochondrial volume but is insufficient to cause significant depolarization of membrane potential. The volume changes, however, have significant bioenergetic consequences for energy coupling in the cell.

How do uncoupling proteins uncouple?

According to the proton buffering model, introduced by Klingenberg, UCP1 conducts protons through a hydrophilic pathway lined with fatty acid head groups that buffer the protons as they move across the membrane. According to the fatty acid protonophore model, introduced by Garlid, UCPs do not conduct protons at all. Rather, like all members of this gene family, they are anion carriers. A variety of anions are transported, but the physiological substrates are fatty acid (FA) anions. Because the carboxylate head group is translocated by UCP, and because the protonated FA rapidly diffuses across the membrane, this mechanism permits FA to behave as regulated cycling protonophores. Favoring the latter mechanism is the fact that the head group of long-chain alkylsulfonates, strong acid analogues of FA, is also translocated by UCP.

ATP-Sensitive potassium channels: a review of their cardioprotective pharmacology.

ATP-sensitive potassium channels (K(ATP)) have been thought to be a mediator of cardioprotection for the last ten years. Significant progress has been made in learning the pharmacology of this channel as well as its molecular regulation with regard to cardioprotection. K(ATP)openers as a class protect ischemic/reperfused myocardium and appear to do so by conservation of energy. The reduced rate of ATP hydrolysis during ischemia exerted by these openers is not due to a cardioplegic effect and is independent of action potential shortening. Compounds have been synthesized which retain the cardioprotective effects of first generation K(ATP)openers, but are devoid of vasodilator and cardiac sarcolemmal potassium outward currents. These results suggest receptor or channel subtypes. Recent pharmacologic and molecular biology studies suggest the activation of mitochondrial K(ATP)as the relevant cardioprotective site. Implications of these results for future drug discovery and preconditioning are discussed.

The state of water in biological systems.

This paper addresses the issue of how the aqueous cytoplasm is organized on a macroscopic scale. Mitochondria were used as the experimental model, and a unique experimental approach was used to probe the properties of water in the mitochondrial matrix. The results demonstrate aqueous phase separation into two distinct phases with different osmotic activity and different solute partition coefficients. The larger phase, designated "normal water," is osmotically active and behaves in every respect like a bulk, dilute salt solution. The smaller phase, designated "abnormal water," is osmotically inactive and comprises the water of hydration of matrix proteins. It is, nevertheless, solvent water, with highly selective partition coefficients, and behaves like a Lewis base.

Transport function and regulation of mitochondrial uncoupling proteins 2 and 3.

Uncoupling protein 1 (UCP1) dissipates energy and generates heat by catalyzing back-flux of protons into the mitochondrial matrix, probably by a fatty acid cycling mechanism. If the newly discovered UCP2 and UCP3 function similarly, they will enhance peripheral energy expenditure and are potential molecular targets for the treatment of obesity. We expressed UCP2 and UCP3 in Escherichia coli and reconstituted the detergent-extracted proteins into liposomes. Ion flux studies show that purified UCP2 and UCP3 behave identically to UCP1. They catalyze electrophoretic flux of protons and alkylsulfonates, and proton flux exhibits an obligatory requirement for fatty acids. Proton flux is inhibited by purine nucleotides but with much lower affinity than observed with UCP1. These findings are consistent with the hypothesis that UCP2 and UCP3 behave as uncoupling proteins in the cell.

Existence of uncoupling protein-2 antigen in isolated mitochondria from various tissues.

Antibodies against Escherichia coli-expressed uncoupling protein-2 (UCP2) and uncoupling protein-3 (UCP3) were raised by operating the blotted proteins into the spleen of minipigs. The antisera reacted more intensively with the recombinant UCP2 and UCP3 than with uncoupling protein-1 (UCP1) isolated from brown adipose tissue. Moreover, anti-UCP2 and cross-reacting anti-UCP3 antibodies identified the presence of the UCP2/3 antigen in isolated mitochondria from rat heart, rat kidney, rat brain, rabbit epididymal white adipose tissue, hamster brown adipose tissue, and rabbit skeletal muscle. It has been concluded that UCP2 is expressed in these tissues (UCP3 in skeletal muscle); however their existence in mitochondria had not previously been demonstrated.

Mammalian mitochondrial uncoupling proteins.

The mammalian uncoupling protein (UCP-1) from the gene family of mitochondrial carriers is a dimer of identical 33 kDa subunits, each containing six membrane-spanning alpha-helices. Its expression, restricted to brown fat, occurs upon birth, cold acclimation and overfeeding. UCP-1 dissipates redox energy and thereby provides heat to the animal. Two additional isoforms have recently been discovered, 59% homologous UCP-2, widely expressed (heart, kidney, lung, placenta, lymphocytes, white fat); and UCP-3 (57% homologous), found in brown fat and skeletal muscle. Their physiological roles are unknown, but may include the regulation of body weight and energy balance, muscle nonshivering thermogenesis, fever, and defense against generation of reactive oxygen species. Consequently, great pharmacological potential is expected in revealing their biochemical and hormonal regulators. UCP-1 mediates a purine-nucleotide-sensitive uniport of monovalent unipolar anions, including fatty acids, that lead to fatty acid cycling and uncoupling. UCP-2 and UCP-3 are expected to share a similar mechanism.

The mechanism of proton transport mediated by mitochondrial uncoupling proteins.

The effort to understand the mechanism of uncoupling by UCP has devolved into two models - the fatty acid protonophore model and the proton buffering model. Evidence for each hypothesis is summarized and evaluated. We also evaluate the obligatory requirement for fatty acids in UCP1-mediated uncoupling and the question of fatty acid affinity for UCP1. The structural bases of UCP transport function and nucleotide inhibition are discussed in light of recent mutagenesis studies and in relationship to the sequences of newly discovered UCPs.

Fatty acid cycling mechanism and mitochondrial uncoupling proteins.

We hypothesize that fatty acid-induced uncoupling serves in bioenergetic systems to set the optimum efficiency and tune the degree of coupling of oxidative phosphorylation. Uncoupling results from fatty acid cycling, enabled by several phylogenetically specialized proteins and, to a lesser extent, by other mitochondrial carriers. It is suggested that the regulated uncoupling in mammalian mitochondria is provided by uncoupling proteins UCP-1, UCP-2 and UCP-3, whereas in plant mitochondria by PUMP and StUCP, all belonging to the gene family of mitochondrial carriers. UCP-1, and hypothetically UCP-3, serve mostly to provide nonshivering thermogenesis in brown adipose tissue and skeletal muscle, respectively. Fatty acid cycling was documented for UCP-1, PUMP and ADP/ATP carrier, and is predicted also for UCP-2 and UCP-3. UCP-1 mediates a purine nucleotide-sensitive uniport of monovalent unipolar anions, including anionic fatty acids. The return of protonated fatty acid leads to H+ uniport and uncoupling. UCP-2 is probably involved in the regulation of body weight and energy balance, in fever, and defense against generation of reactive oxygen species. PUMP has been discovered in potato tubers and immunologically detected in fruits and corn, whereas StUCP has been cloned and sequenced froma a potato gene library. PUMP is supposed to act in the termination of synthetic processes in mature fruits and during the climacteric respiratory rise.

State-dependent inhibition of the mitochondrial KATP channel by glyburide and 5-hydroxydecanoate.

The mitochondrial KATP channel (mitoKATP) is hypothesized to be the receptor for the cardioprotective effects of K+ channel openers (KCO) and for the blocking of cardioprotection by glyburide and 5-hydroxydecanoate (5-HD). Studies on glyburide have indicated that this drug is inactive in isolated mitochondria. No studies of the effects of 5-HD on isolated mitochondria have been reported. This paper examines the effects of glyburide and 5-HD on K+ flux in isolated, respiring mitochondria. We show that mitoKATP is completely insensitive to glyburide and 5-HD under the experimental conditions in which the open state of the channel is induced by the absence of ATP and Mg2+. On the other hand, mitoKATP became highly sensitive to glyburide and 5-HD when the open state was induced by Mg2+, ATP, and a physiological opener, such as GTP, or a pharmacological opener, such as diazoxide. In these open states, glyburide (K1/2 values 1-6 microM) and 5-HD (K1/2 values 45-75 microM) inhibited specific, mitoKATP-mediated K+ flux in both heart and liver mitochondria from rat. These results are consistent with a role for mitoKATP in cardioprotection and show that different open states of mitoKATP, although catalyzing identical K+ fluxes, exhibit very different susceptibilities to channel inhibitors.

Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP-sensitive K+ channels. Possible mechanism of cardioprotection.

Previous studies showed a poor correlation between sarcolemmal K+ currents and cardioprotection for ATP-sensitive K+ channel (KATP) openers. Diazoxide is a weak cardiac sarcolemmal KATP opener, but it is a potent opener of mitochondrial KATP, making it a useful tool for determining the importance of this mitochondrial site. In reconstituted bovine heart KATP, diazoxide opened mitochondrial KATP with a K1/2 of 0.8 mumol/L while being 1000-fold less potent at opening sarcolemmal KATP. To compare cardioprotective potency, diazoxide or cromakalim was given to isolated rat hearts subjected to 25 minutes of global ischemia and 30 minutes of reperfusion. Diazoxide and cromakalim increased the time to onset of contracture with a similar potency (EC25, 11.0 and 8.8 mumol/L, respectively) and improved postischemic functional recovery in a glibenclamide (glyburide)-reversible manner. In addition, sodium 5-hydroxydecanoic acid completely abolished the protective effect of diazoxide. While-myocyte studies showed that diazoxide was significantly less potent than cromakalim in increasing sarcolemmal K+ currents. Diazoxide shortened ischemic action potential duration significantly less than cromakalim at equicardioprotective concentrations. We also determined the effects of cromakalim and diazoxide on reconstituted rat mitochondrial cardiac KATP activity. Cromakalim and diazoxide were both potent activators of K+ flux in this preparation (K1/2 values, 1.1 +/- 0.1 and 0.49 +/- 0.05 mumol/L, respectively). Both glibenclamide and sodium 5-hydroxydecanoic acid inhibited K+ flux through the diazoxide-opened mitochondrial KATP. The profile of activity of diazoxide (and perhaps KATP openers in general) suggests that they protect ischemic hearts in a manner that is consistent with an interaction with mitochondrial KATP.

Identification by site-directed mutagenesis of three arginines in uncoupling protein that are essential for nucleotide binding and inhibition.

Primary regulation of uncoupling protein is mediated by purine nucleotides, which bind to the protein and allosterically inhibit fatty acid-induced proton transport. To gain increased understanding of nucleotide regulation, we evaluated the role of basic amino acid residues using site-directed mutagenesis. Mutant and wild-type proteins were expressed in yeast, purified, and reconstituted into liposomes. We studied nucleotide binding as well as inhibition of fatty acid-induced proton transport in wild-type and six mutant uncoupling proteins. None of the mutations interfered with proton transport. Two lysine mutants and a histidine mutant had no effect on nucleotide binding or inhibition. Arg83 and Arg182 mutants completely lost both the ability to bind nucleotides and nucleotide inhibition. Surprisingly, the Arg276 mutant exhibited normal nucleotide binding, but completely lost nucleotide inhibition. To account for this dissociation between binding and inhibition, we propose a three-stage binding-conformational change model of nucleotide regulation of uncoupling protein. We have now identified three nucleotides by site-directed mutagenesis that are essential for nucleotide interaction with uncoupling protein.

The nucleotide regulatory sites on the mitochondrial KATP channel face the cytosol.

The mitochondrial KATP channel (mitoKATP) is richly endowed with regulatory sites for metabolites and drugs, but the topological location of these sites is unknown. Thus, it is not known whether ATP, GTP and acyl CoA esters regulate mitoKATP from the matrix or cytosolic side of the inner membrane, nor whether they all act from the same side. The experiments reported in this paper provide an unambiguous answer to these questions. Electrophysiological experiments in bilayer membranes containing purified mitoKATP showed that current is blocked asymmetrically by ATP. K+ flux experiments using proteoliposomes containing purified mitoKATP showed that mitoKATP is unipolar with respect to regulation by Mg2+, ATP, GTP, and palmitoyl CoA and that all of these ligands react on the same pole of the protein. This demonstration was made possible by the new finding that mitoKATP is 85-90% oriented inward or outward in liposomes, depending on the presence or absence of Mg2+ in the reconstitution buffer. K+ flux experiments in respiring rat liver mitochondria showed that mitoKATP was inhibited by palmitoyl CoA and activated by GTP when these agents were added to the external medium. Given that the inner membrane is impermeant to these ligands and that mitoKATP is unipolar with respect to nucleotide regulation, it follows that the regulatory sites on mitoKATP face the cytosol.