KChAP/Kvbeta1.2 interactions and their effects on cardiac Kv channel expression.

KChAP and voltage-dependent K+ (Kv) beta-subunits are two different types of cytoplasmic proteins that interact with Kv channels. KChAP acts as a chaperone for Kv2.1 and Kv4.3 channels. It also binds to Kv1.x channels but, with the exception of Kv1.3, does not increase Kv1.x currents. Kvbeta-subunits are assembled with Kv1.x channels; they exhibit "chaperone-like" behavior and change gating properties. In addition, KChAP and Kvbeta-subunits interact with each other. Here we examine the consequences of this interaction on Kv currents in Xenopus oocytes injected with different combinations of cRNAs, including Kvbeta1.2, KChAP, and either Kv1.4, Kv1.5, Kv2.1, or Kv4.3. We found that KChAP attenuated the depression of Kv1.5 currents produced by Kvbeta1.2, and Kvbeta1.2 eliminated the increase of Kv2.1 and Kv4.3 currents produced by KChAP. Both KChAP and Kvbeta1.2 are expressed in cardiomyocytes, where Kv1.5 and Kv2.1 produce sustained outward currents and Kv4.3 and Kv1.4 generate transient outward currents. Because they interact, either KChAP or Kvbeta1.2 may alter both sustained and transient cardiac Kv currents. The interaction of these two different classes of modulatory proteins may constitute a novel mechanism for regulating cardiac K+ currents.

Ischemic injury to mitochondrial electron transport in the aging heart: damage to the iron-sulfur protein subunit of electron transport complex III.

The aging heart sustains greater injury during ischemia and reperfusion compared to adult hearts. Aging decreases oxidative function in interfibrillar mitochondria (IFM) that reside among the myofibers, while subsarcolemmal mitochondria (SSM), located beneath the plasma membrane, remain unaltered. Aging decreases complex III activity selectively in IFM via alteration of the cytochrome c binding site. With 25 min of global ischemia, complex III activity decreases in SSM and further decreases in IFM in the aging heart. Ischemia leads to a marked decrease in the electron paramagnetic resonance signal of the iron-sulfur protein (ISP) in both SSM and IFM, despite a preserved content of ISP peptide. Thus, ischemia results in a functional decrease in the iron-sulfur center in ISP without subunit peptide loss. In the aging heart, at the onset of reperfusion, IFM contain two tandem defects in the path of electron flow through complex III, providing a likely mechanism for enhanced oxidant production and reperfusion damage.

Aging decreases electron transport complex III activity in heart interfibrillar mitochondria by alteration of the cytochrome c binding site.

Aging alters cardiac physiology and structure and enhances damage during ischemia and reperfusion. Aging selectively decreases the rate of oxidative phosphorylation in the interfibrillar population of cardiac mitochondria (IFM) located among the myofibers, whereas subsarcolemmal mitochondria (SSM) located beneath the plasma membrane remain unaffected. Aging decreased the rate of oxidative phosphorylation using durohydroquinone, an electron donor to complex III, in IFM only. Complex III activity was decreased in IFM, but not SSM. Aging did not alter the content of catalytic centers of complex III (cytochromes b and c(1)and iron-sulfur protein). Complex III activity measured at physiologic ionic strength in IFM from aging hearts was decreased by 49% compared to IFM from adults, whereas activity measured at low ionic strength was unchanged, localizing the aging defect to the cytochrome c binding site of complex III. Subunits VIII and X of the cytochrome c binding site were present in complex III with the aging defect, indicating that loss of subunits did not occur. Study of aging damage to complex III will help clarify the contribution of altered electron transport in IFM to increased oxidant production during aging, formation of the aging cardiac phenotype, and the relationship of aging defects to increased damage following ischemia.

KChAP as a chaperone for specific K(+) channels.

The concept of chaperones for K(+) channels is new. Recently, we discovered a novel molecular chaperone, KChAP, which increased total Kv2.1 protein and functional channels in Xenopus oocytes through a transient interaction with the Kv2.1 amino terminus. Here we report that KChAP is a chaperone for Kv1.3 and Kv4.3. KChAP increased the amplitude of Kv1.3 and Kv4.3 currents without affecting kinetics or voltage dependence, but had no such effect on Kv1.1, 1.2, 1.4, 1.5, 1.6, and 3.1 or Kir2.2, HERG, or KvLQT1. Although KChAP belongs to a family of proteins that interact with transcription factors, upregulation of channel currents was not blocked by the transcription inhibitor actinomycin D. A 98-amino acid fragment of KChAP binds to the channel and is indistinguishable from KChAP in its enhancement of Kv4.3 current and protein levels. Using a KChAP antibody, we have coimmunoprecipitated KChAP with Kv2.1 and Kv4.3 from heart. We propose that KChAP is a chaperone for specific Kv channels and may have this function in cardiomyocytes where Kv4.3 produces the transient outward current, I(to).

Direct inhibition of mitochondrial respiratory chain complex III by cell-permeable ceramide.

Ceramide is a lipid second messenger that mediates the effects of tumor necrosis factor alpha and other agents on cell growth and differentiation. Ceramide is believed to act via activation of protein phosphatase, proline-directed protein kinase, or protein kinase C. Tumor necrosis factor alpha-induced common pathway of apoptosis is associated with an early impairment of mitochondria. Herein, we demonstrate that ceramide can directly inhibit mitochondrial respiratory chain function. In isolated mitochondria, a rapid decline of mitochondrial oxidative phosphorylation occurs in the presence of N-acetylsphingosine (C2-ceramide), a synthetic cell-permeable ceramide analog. An investigation of the site of ceramide action revealed that the activity of respiratory chain complex III is reduced by C2-ceramide with half-maximum effect at 5-7 microM. In contrast, N-acetylsphinganine (C2-dihydroceramide), which lacks a functionally critical double bond and is ineffective in cells, did not alter mitochondrial respiration or complex III activity. We suggest that these in vitro observations may set the stage for identifying a novel mechanism of regulation of mitochondrial function in vivo.

Permeability transition pore of the inner mitochondrial membrane can operate in two open states with different selectivities.

Prooxidants induce release of Ca2+ from mitochondria through the giant solute pore in the mitochondrial inner membrane. However, under appropriate conditions prooxidants can induce Ca2+ release without inducing a nonspecific permeability change. Prooxidant-induced release of Ca2+ is selective. Presumably, this is the result of the operation of a permeability pathway for H+ coupled to the reversal of the Ca2+ uniporter, the latter generating the selectivity. The solute pore and prooxidant-induced Ca2+-specific pathways exhibit common sensitivities to a set of inhibitors and activators. It is proposed that the pore can operate in two open states: (1) permeable to H+ only and (2) permeable to solutes of M(r) < 1500. Under some conditions, prooxidants induce the H+-selective state which, in turn, collapses the inner membrane potential and permits selective loss of Ca2+ via the Ca2+ uniporter.

The peptide mastoparan is a potent facilitator of the mitochondrial permeability transition.

Mastoparan facilitates opening of the mitochondrial permeability transition pore through an apparent bimodal mechanism of action. In the submicromolar concentration range, the action of mastoparan is dependent upon the medium Ca2+ and phosphate concentration and is subject to inhibition by cyclosporin A. At concentrations above 1 microM, pore induction by mastoparan occurs without an apparent Ca2+ requirement and in a cyclosporin A insensitive manner. Studies utilizing phospholipid vesicles show that mastoparan perturbs bilayer membranes across both concentration ranges, through a mechanism which is strongly dependent upon transmembrane potential. However, solute size exclusion studies suggest that the pores formed in mitochondria in response to both low and high concentrations of mastoparan are the permeability transition pore. It is proposed that low concentrations of mastoparan influence the pore per se, with higher concentrations having the additional effect of depolarizing the mitochondrial inner membrane through an action exerted upon the lipid phase. It may be the combination of these effects which allow pore opening in the absence of Ca2+ and in the presence of cyclosporin A, although other interpretations remain viable. A comparison of the activities of mastoparan and its analog, MP14, on mitochondria and phospholipid vesicles provides an initial indication that a G-protein may participate in regulation of the permeability transition pore. These studies draw attention to peptides, in a broad sense, as potential pore regulators in cells, under both physiological and pathological conditions.

Magnesium ion modulates the sensitivity of the mitochondrial permeability transition pore to cyclosporin A and ADP.

Regulation of the mitochondrial permeability transition pore has been investigated following the release of matrix solutes which normally participate in pore regulation. Under these conditions, neither cyclosporin A nor ADP induces pore closure, as judged by restoration of delta psi, unless Mg2+ is also added. Mg2+ alone is ineffective. In liver mitochondria, the Mg2+ effect is expressed over a 0 to 0.5 mM concentration range with higher concentrations inhibiting repolarization. In heart mitochondria, the inhibitory action of high Mg2+ is not seen and it can be shown that the Mg2+ effect on repolarization increases progressively up to a concentration of 5 mM. In liver mitochondria, when the pore is closed by maximally effective concentrations of Mg2+ plus cyclosporin A or Mg2+ plus ADP, reopening occurs upon the addition of carboxyatractyloside. The latter compound, however, fails to reopen the pore when Mg2+, cyclosporin A, and ADP are present simultaneously. In heart mitochondria, where higher Mg2+ concentrations can be employed, Mg2+ plus cyclosporin A or Mg2+ plus ADP produces pore closure in a carboxyatractyloside insensitive manner. Titration experiments support the adenine nucleotide translocase as the site at which carboxyatractyloside acts to regulate the pore. However, the action of ADP appears to involve a translocase-independent site. In intact mitochondria the action of carboxyatractyloside on pore opening is counteracted by oligomycin, apparently through inhibition of the F1F0 ATP synthase, with a consequent increase in the matrix space ADP/ATP ratio. It is concluded that the permeability transition pore induced by Ca2+ plus P(i) is not formed from the adenine nucleotide translocase although the translocase conformation is one of several factors which regulate the pore. The matrix Mg2+ concentration is also one of these factors. Formation of the pore by a Ca2+ and ADP binding protein is one model which is consistent with the present data.

Stimulation of respiration in rat thymocytes induced by ionizing radiation.

The effect of X irradiation on the respiration of rat thymocytes was studied. An increase in the rate of O2 uptake was observed 1 h after cells were irradiated with doses of 6-10 Gy. The radiation-induced increase in respiration could be blocked by oligomycin, an inhibitor of mitochondrial ATP synthase, suggesting control by increased cytoplasmic ATP turnover. The stimulation of respiration was not associated with changes in the activity of mitochondrial electron transfer enzymes or permeability of the inner membrane. Several inhibitors of processes which used ATP were screened for their effects on the basal respiration rate and on the radiation response. In irradiated thymocytes, an enhancement of inhibition of respiration by ouabain, La3+ and cycloheximide was observed. These results indicate that the radiation-induced stimulation of respiration is due to changes in ion homeostasis and protein synthesis. The effect of X irradiation was shown to be independent of the redox status of nonprotein thiols and was not associated with detectable changes in some products of lipid peroxidation. The radiation-induced decrease in activity of superoxide dismutase suggests free radical involvement in deleterious effects of radiation.

The permeability transition in heart mitochondria is regulated synergistically by ADP and cyclosporin A.

Heart mitochondria respiring in a sucrose medium containing P(i) show a permeability transition when challenged with Ca2+ and an oxidant such as cumene hydroperoxide. The transition results from the opening of a Ca(2+)-dependent pore and is evidenced by loss of membrane potential (delta psi) and osmotic swelling due to uptake of sucrose and other solutes. In the absence of oxidant, high concentrations of Ca2+ (100-150 microM) are necessary to induce loss of delta psi and initiate swelling. Cyclosporin A delays the loss of delta psi but enhances swelling under these conditions, apparently by promoting better retention of accumulated Ca2+. Cyclosporin A and ADP together restore delta psi in respiring mitochondria that have undergone the permeability transition at levels that are not effective when either is added alone. When the state of the Ca(2+)-dependent pore is assessed using passive osmotic contraction in response to polyethylene glycol (Haworth, R. A., and Hunter, D. R. (1979) Arch. Biochem. Biophys. 195, 460-467), cyclosporin A is found to be a partial inhibitor of solute flow through the open pore. Cyclosporin A decreases the Vmax of passive contraction and increases the Km for Ca2+ without affecting the Hill slope. ADP in the presence of carboxyatractyloside closes the pore almost completely even in the presence of 40 microM Ca2+. ADP shows mixed type inhibition of the Ca(2+)-dependent pore, and cyclosporin A increases the affinity of the pore for ADP. It is concluded that cyclosporin A and ADP act synergistically to close the Ca(2+)-dependent pore of the mitochondrion and that the pore is probably not formed directly from the adenine nucleotide transporter.

Effects of the membrane potential upon the Ca(2+)- and cumene hydroperoxide-induced permeabilization of the inner mitochondrial membrane.

A protonophore-induced delta psi decrease in a 180-140 mV range causes an increase in the lag-period of Ca(2+)-induced mitochondrial permeabilization but has little effect on the cumene hydroperoxide-induced permeability transition of mitochondria. Suppression of the non-specific permeability induction seems to be mediated by an increase in [ADP] in the mitochondrial matrix. A further decrease in delta psi leads to additional suppression of the non-specific permeability as a result of a partial ruthenium red-sensitive efflux of the previously accumulated Ca2+. On the other hand, complete dissipation of delta psi causes immediate induction of the non-specific permeability. It is concluded that only complete dissipation of delta psi caused by H+ leakages may act as a trigger for non-specific permeability induction.

The nonspecific inner membrane pore of liver mitochondria: modulation of cyclosporin sensitivity by ADP at carboxyatractyloside-sensitive and insensitive sites.

Cyclosporin A prevents the opening of a nonspecific pore in the inner membrane of liver mitochondria when added prior to Ca2+. In the presence of 10 microM Ca2+ cyclosporin is unable to close the pore and restore the original permeability unless ADP is also added. ADP acts at a high-affinity site (Km 5 microM), corresponding to the adenine nucleotide transporter. This effect of ADP is prevented and reversed by carboxyatractyloside. In the presence of carboxyatractyloside, cyclosporin added with higher concentrations of ADP (Km 70 microM) also can close the pore. This suggests that a lower-affinity ADP-binding component as well as cyclophilin and the adenine nucleotide transporter can modulate the sensitivity of the pore to cyclosporin.

Mechanism accounting for the induction of nonspecific permeability of the inner mitochondrial membrane by hydroperoxides.

The effect of antioxidants on the nonspecific permeability of the inner mitochondrial membrane induced by cumene hydroperoxide or Ca(2+) has been studied. Butylated hydroxytoluene, butylated hydroxyanisole and 2,2,5,7,8-pentamethyl-6-chromanol, taken at a concentration up to 50 microM, suppress the cumene hydroperoxide-induced accumulation of lipid peroxidation products. In the same range of concentrations, these antioxidants inhibit the activation of nonspecific permeability by cumene hydroperoxide or Ca(2+). Propyl gallate, being less effective under such conditions, fails to affect the induction of nonspecific permeability. Additionally, 2,2,5,7,8-pentamethyl-6-chromanol at a concentration decreasing the accumulation of lipid peroxidation products by 70% has been shown not to increase the lag period of nonspecific permeability induction. Higher antioxidant concentrations, while leading to an increase in the lag period of nonspecific permeability induction, cause but minor suppression of lipid peroxidation. From the results obtained we can assume that free radicals formed in the course of hydroperoxide decomposition or on mitochondrial redox complex interact directly with a system responsible for nonspecific permeability or with regulating components of this system.

[The effect of cyclosporin A and oligomycin on the nonspecific permeability of the mitochondria inner membrane]. / Deistvie tsiklosporina A i oligomitsina na nespetsificheskuiu pronitsaemost' vnutrennei membrany mitokhondrii.

The effect of oligomycin and cyclosporine A on Ca(2+)-induced nonspecific permeability of the inner mitochondrial membrane was under study. Both oligomycin and cyclosporine A were able to prevent the activation of nonspecific permeability; however, but cyclosporine A was the only agent which could restore the initial permeability of the inner mitochondrial membrane. The effect of cyclosporine A was not shown to be mediated through redistribution of Ca2+ ions between different subpopulations of mitochondria.

[The role of the ADP/ATP-antiporter in the inhibition of nonspecific permeability of the inner mitochondrial membrane by cyclosporin A]. / Rol' ADP/ATP-antiportera v podavlenii tsiklosporinom A nespetsificheskoi pronitsaemosti vnutrennei membrany mitokhondrii.

The effect of the conformational state of the ADP/ATP-antiporter on the efficiency of inhibition by cyclosporine A of the Ca(2+)-induced increase of nonspecific permeability of the inner mitochondrial membrane was under study. It was found that the ADP/ATP-antiporter inhibitor carboxyatractyloside is able to reverse the cyclosporine A-induced inhibition of this nonspecific permeability. The effect of carbocyatractyloside is manifested only in mitochondria depleted of adenine nucleotides. The bifunctional SH-reagent phenylarsine oxide is also able to reverse the cyclosporine A effect. The data obtained testify to the fact that inhibition by cyclosporine A of nonspecific permeability is due to its effect on the conformational state of the ADP/ATP-antiporter.

Effect of ADP/ATP antiporter conformational state on the suppression of the nonspecific permeability of the inner mitochondrial membrane by cyclosporine A.

The influence of the conformational state of ADP/ATP antiporter on the efficiency of the inhibitory effect of cyclosporine A on the Ca2(+)-induced nonspecific permeability of the inner mitochondrial membrane has been studied. Carboxyatractiloside, the inhibitor of ADP/ATP-antiporter, was shown to prevent the cyclosporine A-induced suppression of the nonspecific permeability. The carboxyatractiloside effect was displayed only in mitochondria depleted of adenine nucleotides. Bifunctional SH reagent, phenylarsine oxide, was also able to reverse the effect of cyclosporine A. The data are consistent with the suggestion that cyclosporine A causes suppression of the nonspecific permeability due to its effect on the ADP/ATP antiporter conformation.

[The effect of ionizing radiation on the respiration of rat thymocytes]. / Vliianie ioniziruiushchego izlucheniia na dykhanie timotsitov krys.

The oligomycin-sensitive component of the rate of oxygen consumption by rat thymocytes increased by 22% after X-irradiation. The postirradiation increase in the thymocyte respiration rate was connected with the impairment of a balance between sodium, potassium and calcium and with the protein synthesis activation, and was independent of lymphokine production by lipoxygenase. Irradiation was ineffective with respect to electron transport in the respiratory chain and electrogenic leakages through the inner membrane of thymocyte mitochondria.

Effect of cyclosporine A and oligomycin on non-specific permeability of the inner mitochondrial membrane.

The effect of oligomycin and cyclosporine A on the induction of non-specific permeability of the inner mitochondrial membrane by Ca2+ was under study. Both oligomycin and cyclosporine A were able to prevent the activation of non-specific permeability, but cyclosporine A was the only agent which could restore initial permeability of the inner mitochondrial membrane. The effect of cyclosporine A was shown not to be mediated through redistribution of Ca2+ between different mitochondrial subpopulations.

ATP-synthase complex: the mechanism of control of ion fluxes induced by cumene hydroperoxide in mitochondria.

In state 4, the cumene hydroperoxide-induced decrease of mitochondrial transmembrane potential is prevented by the free radical scavenger, butylhydroxytoluene. This decrease is accompanied by accumulation of lipid hydroperoxides. Oligomycin suppresses the cumene hydroperoxide-induced uncoupling of mitochondria, but has no significant effect on lipid peroxidation. The transition of mitochondria from state 4 to state 3 by ADP addition leads to the suppression of both lipid peroxidation and mitochondrial uncoupling. This effect on lipid peroxidation is prevented by carboxyatractyloside and oligomycin.