Determination of the rate of K movement through potassium channels in isolated rat heart and liver mitochondria

Both ATP-regulated (mitoK_ATP) and large conductance calcium-activated (mitoBK_Ca) potassium channels have been proposed to regulate mitochondrial K⁺ influx and matrix volume and to mediate cardiac ischaemic preconditioning (IP). However, the specificity of the pharmacological agents used in these studies and the mechanisms underlying their effects on IP remain controversial. Here we used increasing concentrations of K⁺-ionophore (valinomycin) to stimulate respiration by rat liver and heart mitochondria in the presence of the K⁺/H⁺ exchanger nigericin. This allowed rates of valinomycin-induced K⁺ influx to be determined whilst parallel measurements of light scattering (A₅₂₀) and matrix volume (³H₂O and [¹⁴C]-sucrose) enabled rates of K⁺ influx to be correlated with increases in matrix volume. Light scattering readily detected an increase in K⁺ influx of < 5 nmol K⁺ min^− 1 per mg protein corresponding to < 2% mitochondrial matrix volume increase. In agreement with earlier data no light-scattering changes were observed in response to any mitoK_ATP channel openers or blockers. However, the mitoBK_Ca opener NS1619 (10-50 µM) did decrease light scattering slightly, but this was also seen in K⁺-free medium and was accompanied by uncoupling. Contrary to prediction, the mitoBK_Ca blocker paxilline (10-50 µM) decreased rather than increased light scattering, and it also slightly uncoupled respiration. Our data argue against the presence of significant activities of either the mitoK_ATP or the mitoBK_Ca channel in rat liver and heart mitochondria and provide further evidence that preconditioning induced by pharmacological openers of these channels is more likely to involve alternative mechanisms.     

The internal concentration of K+ in isolated rat liver mitochondria

A simple osmotic method has been developed to determine the internal K+ concentration of mitochondria by determining the concentration of external K+ at constant osmotic pressure at which metabolically inhibited mitochondria neither shrink nor swell. This concentration has been found to correspond to approx. 80-85 mM in freshly isolated mitochondria and considerably lower after additional centrifugation procedures. Since mitochondria are in osmotic equilibrium with the suspending medium (in this case, 0.32 osmolal), and K+ is the primary exchangeable internal ion, a significant proportion of the internal osmotic pressure must be exerted by the sucrose. Results for experiments determining internal K+ after centrifuging mitochondria at various G values confirm the reports of Sitaramam et al. (Sitaraman, V. and Sarma, M.K.J. (1981) Proc. Natl. Acad. Sci. USA 78, 3441-3445 and Sambasivarao, D. and Sitaramam, V. (1983) Biochim. Biophys. Acta 722, 256-270) that centrifugation induces the entry of sucrose in mitochondria isolated in a sucrose medium.     

Water movement from intracristal spaces in isolated liver mitochondria

When analyzing mitochondria isolated in a sucrose medium that had been embedded for thin sectioning according to one low denaturation embedding technique, large intracristal spaces were present in close to 90% of the mitochondria. The two crista membranes were closely apposed in only 40% of all cristae. When the mitochondria were transferred to an incubation medium, the percentage of mitochondria with intracristal spaces was reduced to 40%. About 90% of all cristae were lacking any space separating the two crista membranes. The presence of inorganic phosphate in the medium was required for the closing of the intracristal spaces. The percentage of cristae lacking an intracristal space remained the same after addition of substrate for respiration (state 4) and of ADP (state 3). Inhibition or uncoupling of respiration led to an increase in the percentage of intracristal spaces, showing that oxidative phosphorylation is required to maintain the crista membranes closely apposed. The appearance and disappearance of the intracristal spaces was an indication of water movements across the crista membranes. The mean volume of the mitochondria increased 33% when they were transferred from the sucrose medium to the incubation medium, showing that the removal of water from the cristae was not caused by a passive osmotic effect. Addition of substrate made the volume decrease by 28%. After further addition of ADP, the volume decreased another 23%. No change in volume was associated with inhibition or uncoupling of respiration. The observations revealed that water can move into or out of the cristae independently of water movement out from the entire mitochondrion. Therefore, the water moving out from or into the cristae is translocated across the cristae membrane. The observations are interpreted to reveal the presence of a mechanism that actively prevents water from accumulating in the crista membrane. This mechanism allows for a low water activity to be maintained within the membrane. The variations in the frequency of intracristal spaces occurred without any simultaneous changes in the width of the space appearing between the two surface membranes after isolation of the mitochondria. The observations, therefore, do not agree with the concept that there is an outer compartment that communicates freely with intracristal spaces.     

Ammonia formation in isolated rat liver mitochondria

Studies of isolated rat liver mitochondria were undertaken in order to evaluate the importance of glutamate transport, oxidation reduction state, and product inhibition on the rates of formation of ammonia from glutamate. Uptake and efflux of glutamate across the mitochondrial membrane were measured isotopically in the presence of rotenone. Efflux was stimulated by H+ in the mitochondrial matrix and was found to be first order with respect to matrix glutamate except when the matrix pH was unphysiologically low. The data suggest that the Km of matrix glutamate for efflux is decreased by H+. Matrix H+ also appeared to stimulate glutamate uptake, but the effect was to increase both the Km of medium glutamates and Vmax. Mitochondria were incubated at 15 and 28 degrees C with glutamate and malonate. Under these conditions, glutamate was metabolized only by the deamination pathway. Flux was evaluated by assay of ammonia formation. Oxidation reduction state was varied with ADP and uncoupling agents. Matrix alpha-ketoglutarate was varied either by the omission of malonate from the incubation media or by adding alpha-ketoglutarate to the external media. Influx and efflux of glutamate could be calculated from previously determined transport parameters. The difference between calculated influx and efflux was found to be equal to ammonia formation under all conditions. It was, therefore, possible to evaluate the relative contributions of oxidation reduction state, transport, and product inhibition as effectors of ammonia formation. The contribution of transport was relatively small while oxidation reduction state exerted a large influence. alpha-Ketoglutarate was found to be a potent competitive inhibitor of ammonia production and glutamate dehydrogenase. Inhibition of glutamate dehydrogenase by alpha-ketoglutarate was judged to be a potentially important modulator of metabolic fluxes.     

Subcellular localization of the delayed rectifier K(+) channels KCNQ1 and ERG1 in the rat heart

In the heart, several K(+) channels are responsible for the repolarization of the cardiac action potential, including transient outward and delayed rectifier K(+) currents. In the present study, the cellular and subcellular localization of the two delayed rectifier K(+) channels, KCNQ1 and ether-a-go-go-related gene-1 (ERG1), was investigated in the adult rat heart. Confocal immunofluorescence microscopy of atrial and ventricular cells revealed that whereas KCNQ1 labeling was detected in both the peripheral sarcolemma and a structure transversing the myocytes, ERG1 immunoreactivity was confined to the latter. Immunoelectron microscopy of atrial and ventricular myocytes showed that the ERG1 channel was primarily expressed in the transverse tubular system and its entrance, whereas KCNQ1 was detected in both the peripheral sarcolemma and in the T tubules. Thus, whereas ERG1 displays a very restricted subcellular localization pattern, KCNQ1 is more widely distributed within the cardiac cells. The localization of these K(+) channels to the transverse tubular system close to the Ca(2+) channels renders them with maximal repolarizing effect.     

Two mechanisms of K(+)-dependent potentiation in Kv2.1 potassium channels

Elevation of external [K(+)] potentiates outward K(+) current through several voltage-gated K(+) channels. This increase in current magnitude is paradoxical in that it occurs despite a significant decrease in driving force. We have investigated the mechanisms involved in K(+)-dependent current potentiation in the Kv2.1 K(+) channel. With holding potentials of -120 to -150 mV, which completely removed channels from the voltage-sensitive inactivated state, elevation of external [K(+)] up to 10 mM produced a concentration-dependent increase in outward current magnitude. In the absence of inactivation, currents were maximally potentiated by 38%. At more positive holding potentials, which produced steady-state inactivation, K(+)-dependent potentiation was enhanced. The additional K(+)-dependent potentiation (above 38%) at more positive holding potentials was precisely equal to a K(+)-dependent reduction in steady-state inactivation. Mutation of two lysine residues in the outer vestibule of Kv2.1 (K356 and K382), to smaller, uncharged residues (glycine and valine, respectively), completely abolished K(+)-dependent potentiation that was not associated with inactivation. These mutations did not influence steady-state inactivation or the K(+)-dependent potentiation due to reduction in steady-state inactivation. These results demonstrate that K(+)-dependent potentiation can be completely accounted for by two independent mechanisms: one that involved the outer vestibule lysines and one that involved K(+)-dependent removal of channels from the inactivated state. Previous studies demonstrated that the outer vestibule of Kv2.1 can be in at least two conformations, depending on the occupancy of the selectivity filter by K(+) (Immke, D., M. Wood, L. Kiss, and S. J. Korn. 1999. J. Gen. Physiol. 113:819-836; Immke, D., and S. J. Korn. 2000. J. Gen. Physiol. 115:509-518). This change in conformation was functionally defined by a change in TEA sensitivity. Similar to the K(+)-dependent change in TEA sensitivity, the lysine-dependent potentiation depended primarily (>90%) on Lys-356 and was enhanced by lowering initial K(+) occupancy of the pore. Furthermore, the K(+)-dependent changes in current magnitude and TEA sensitivity were highly correlated. These results suggest that the previously described K(+)-dependent change in outer vestibule conformation underlies the lysine-sensitive, K(+)-dependent potentiation mechanism.     

Oxidation of lactate in isolated rat heart mitochondria

In unwashed mitochondria the oxidation of L-lactate (with NAD+) proceeds in presence of the added lactate dehydrogenase. The respiration is characterized by the high rate in state 4 and is stimulated by ADP. This process takes place in unwashed mitochondria and homogenate of the heart in absence of added lactate dehydrogenase. Oxidation of lactate with NAD+ is inhibited by rotenone. It has been also revealed that the oxidation of glutamate is insufficiently altered in presence of lactate (with NAD+) in unwashed mitochondria as compared with the washed ones. It is supposed that the stimulating effect of lactate with NAD+ on the mitochondria respiration is not so much a result of the membrane-damaged action as a result of oxidation of lactate dehydrogenase reaction products: phosphorylative oxidation of pyruvate and nonconjugated oxidation of NADH. Utilization of these products takes place in the main respiratory chain, including its first stage.     

Osmotically-induced alterations in volume and ultrastructure of mitochondria isolated from rat liver and bovine heart

Detailed studies correlating changes in mitochondrial optical density, packed volume, and ultrastructure associated with osmotically-induced swelling were performed. Various swelling states were established by incubating mitochondria (isolated in 0.25 M sucrose) at 0°C for 5 min in series of KCl and sucrose solutions ranging in tonicity from 250 to 3 milliosmols. Reversibility of swelling was determined by examining mitochondria exposed to 250 milliosmols media after they had been induced to swell. Swelling induced by lowering the ambient tonicity to approximately 130 (liver mitochondria) and 90 (heart mitochondria) milliosmols involves primarily swelling of the inner compartment within the intact outer membrane. Decreasing the ambient tonicity beyond this level results in rupture of the outer membrane and expansion of the inner compartment through the break. The maximum extent of swelling, corresponding with complete unfolding of the cristae and an increase in over-all mitochondrial volume of approximately 6-fold (liver mitochondria) and 11-fold (heart mitochondria), is reached at approximately 15 (liver mitochondria) and 3 (heart mitochondria) milliosmols. Exposure of liver mitochondria to media of lower tonicity results in irreversibility of inner compartment swelling and escape of matrix material. These changes appear to result from increased inner membrane permeability, possibly due to stretching.     

Subunit composition of ATP-sensitive potassium channels in mitochondria of rat hearts

Mitochondrial ATP-sensitive potassium (mitoKATP) channels play a pivotal role in early and late ischemic preconditioning, but the subunit composition of mitoKATP channels remains unclear. In this study, we investigated the subunit composition of mitoKATP channels in rat hearts using confocal microscopy, immunofluorescence, and Western blot analysis. The green fluorescent probe glibenclamide-BODIPY was colocalized with the red fluorescent mitochondrial marker MitroTracker Red in isolated ventricular myocytes and in ventricular myocyte mitochondria, indicating the presence of sulfonylurea receptors (SURs) in the mitochondria. Anti-Kir6.1, anti-Kir6.2, and anti-SUR2 immunofluorescence was colocalized with that of MitoTracker Red in isolated mitochondria, suggesting that Kir6.1, Kir6.2, and SUR2 subunits are present in the mitochondria. Similarly, Kir6.1 (approximately 46 kDa), Kir6.2 (approximately 46 and approximately 40 kDa), and SUR2 (approximately 140 kDa) proteins were found to be expressed in mitochondria using Western blot analysis. By contrast, SUR1 was not present in mitochondria. These results suggest that mitoKATP channels in rat hearts might comprise a combination of Kir6.1, Kir6.2, and SUR2 subunits.     

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.     

K(+)-p-nitrophenylphosphatase activity in rat brain and liver

K(+)-p-nitrophenylphosphatase (K(+)pNPPase) is the enzyme, which is considered to be involved in K(+)-dependent hydrolysis of the phosphoenzyme in the reaction cycle of Na(+), K(+)ATPase. The aim of our present study was to characterize some features of K(+)pNPPase in homogenates of the rat brain and liver. We determined p-nitrophenylphosphatase (pNPPase) activity in the presence of various ion combinations (Mg(2+)+ K(+), Mg(2+), K(+)). We found a higher total pNPPase activity in the brain (0.8+/-0.079 nkat/mg protein) than in the liver (0.08+/-0.01 nkat/mg protein). Contrary to the liver, the main part of the total brain activity was K(+)-dependent. The activity of K(+)pNPPase was significantly higher in cerebral cortex homogenates (0.86+/-0.073 nkat/mg protein) in comparison to those of the whole brain (0.57+/-0.075 nkat/mg protein). The specific K(+)pNPPase activity was two times higher in the isolated pellet fraction (0.911+/-0.07 nkat/mg protein), rich in synaptosomes, compared to the whole brain homogenate (0.57+/-0.075 nkat/mg protein). Our results demonstrate the high activity of K(+)pNPPase in the brain tissue and its distribution mainly into the pellet fraction, what might indicate a possible role of K(+)pNPPase in specific structures of the brain, e.g. in synaptosomes.     

Amino acid production in isolated rat liver mitochondria

Amino acid analyses of mitochondrial membranes are compared with the amino acid composition of whole mitochondria (Alberti, 1964) and found to be very similar except in the cystine content. The composition of the endogenous amino acids found in freshly prepared mitochondria has been established and shown to differ considerably from the amino acid composition of membranes or whole mitochondria. The amino acids produced during anaerobic incubation of mitochondria at pH7.4, on the other hand, resemble the membrane in composition, supporting the view that neutral proteinase activity is responsible for their appearance. Aerobic incubation produces a similar pattern of amino acids except that amino acids such as proline, serine, asparagine, glutamic acid and glutamine, which can be metabolically utilized under aerobic conditions, are present to a smaller extent. The presence of large relative concentrations of endogenous taurine, cysteic acid and oxidized glutathione and the accumulation of taurine during incubation is found. The selective retention of taurine and cysteic acid within the mitochondria is established. It is proposed that the first step in the degeneration of isolated mitochondria results from lipid hydroperoxide accumulation caused by the lack of glutathione reductase in isolated mitochondria.     

The role of the ATP-sensitive potassium channel in the activation of the K+ cycle in rat liver mitochondria

It is known that the mitochondrial ATP-sensitive potassium channel (mitoK_ATP) plays a key role in protecting myocardium during ischemia. We have suggested that the mechanism of this protection is associated with the potassium cycle in mitochondria. In this paper, for the first time, a direct proof was obtained of the existence of a cycle of potassium ions in rat liver mitochondria that are associated with the functioning of mitoK_ATP. Activation of the cycle was recorded by optical density changes of mitochondrial suspension in the form of two or three swelling-contraction waves of the organelles. Using activators and inhibitors of mitoK_ATP we showed that a significant role in the potassium cycle belongs to the channel. It was found that in vitro sildenafil has a direct effect on mitoK_ATP, being its activator. The results obtained indicate that the cardioprotective effect of sildenafil observed previously is associated with the activation of mitoK_ATP. In order to study the structure and volume changes of mitochondria in various stages of the cycle in the presence of potassium channel modulators, the electron microscopy studies of mitochondria preparations were carried out. A correlation between the optical density decrease of mitochondrial suspension and the swelling of mitochondria was revealed. The data obtained in this study suggest participation of mitoK_ATP in the protection of tissues from hypoxic damage.