Peculiarities of mitochondrial chloride channel kinetics.

Cardiac mitochondrial chloride channels are involved in the regulation of mitochondrial membrane potential, with impact on the sarcolemma action potential. Despite their importance, they still lack molecular identity. So far, the most probable hypothesis is that they are part of the CLIC channel family. Here, we report a detailed profile of these channels under different conditions. We find this characterization essential for their identification and comparison with other chloride channels. The presence of many unresolved closed events at higher acquisition rate and extremely long closings were detected, which was consistent with the power-law distribution. On the other hand, the channel openings were described by a single-exponential function. We compare the results with ion channels of similar dwell time distribution and discuss the possible connections to other chloride channels and channel families, including the CLIC family. Moreover, the described kinetic features call for theoretical interpretation and proper single-channel analysis.

The effect of all-trans retinoic acid on the mitochondrial function and survival of cardiomyoblasts exposed to local photodamage.

Using H9C2 cardiomyoblasts, we have shown that all-trans retinoic acid (ATRA), the biologically active metabolite of vitamin A, affects mitochondrial dynamics and functions. The low dose (10 nM) ATRA stimulates the expression of nuclear retinoid receptors and induces mechanisms that are protective against severe local damage caused by laser irradiation at the mitochondrial level. These changes include increased density of the mitochondrial network, higher number of mitochondrial junctions, and enhanced mitochondrial velocity. Moreover, the treated cells had lower basal level of reactive oxygen species (ROS) and could maintain mitochondrial potential (ΔΨm ) after photodamage. Cells treated with 10 nM ATRA had significantly better survival rate after photodamage in comparison to control cells. Cells treated with pharmacological concentration of ATRA (1 µM) expressed higher mitochondrial connectivity without increased motility, which did not lead to better survival or decreased ROS level as was in the case of low-dose ATRA. The proteomics analysis showed changes in proteins related to cellular metabolism (glycolysis) and respiration in ATRA-treated cells. The l-lactate assay confirmed the shift to anaerobic glycolysis in cells treated with 1 µm ATRA, whereas the 10 nM ATRA decreased the level of lactate in medium. The increased levels of cytochrome c or peroxiredoxins 5 level and also lower expression of retinoid and rexinoid receptors were observed in cells treated with 1 µM ATRA. The effect of ATRA is concentration-dependent; the increased mitochondrial dynamics and slower metabolism at 10 nM ATRA contributed significantly to the chance of survival of the cells after photodamage whereas the higher concentration of ATRA overrode the protective effect and led to the unfavorable ones.

Subconductance states of mitochondrial chloride channels: implication for functionally-coupled tetramers.

Recently, it has been discovered that isoforms of intracellular chloride channels (CLIC) are present in cardiac mitochondria. By reconstituting rat cardiac mitochondrial chloride channels into bilayer lipid membranes, we detected three equally separated subconductance states with conductance increment of 45 pS and < 2% occupancy. The observed rare events of channel decomposition into substates, accompanied by disrupted gating, provide an insight into channel quaternary structure. Our findings suggest that the observed channels work as four functionally coupled subunits with synchronized gating. We discuss the putative connection of channel activity from native mitochondria with the recombinant CLIC channels. However, conclusive evidence is needed to prove this connection.

Effects of AP39, a novel triphenylphosphonium derivatised anethole dithiolethione hydrogen sulfide donor, on rat haemodynamic parameters and chloride and calcium Cav3 and RyR2 channels.

H2S donor molecules have the potential to be viable therapeutic agents. The aim of this current study was (i) to investigate the effects of a novel triphenylphosphonium derivatised dithiolethione (AP39), in the presence and absence of reduced nitric oxide bioavailability and (ii) to determine the effects of AP39 on myocardial membrane channels; CaV3, RyR2 and Cl(-). Normotensive, L-NAME- or phenylephrine-treated rats were administered Na2S, AP39 or control compounds (AP219 and ADT-OH) (0.25-1 µmol kg(-1)i.v.) and haemodynamic parameters measured. The involvement of membrane channels T-type Ca(2+) channels CaV3.1, CaV3.2 and CaV3.3 as well as Ca(2+) ryanodine (RyR2) and Cl(-) single channels derived from rat heart sarcoplasmic reticulum were also investigated. In anaesthetised Wistar rats, AP39 (0.25-1 µmol kg(-1) i.v) transiently decreased blood pressure, heart rate and pulse wave velocity, whereas AP219 and ADT-OH and Na2S had no significant effect. In L-NAME treated rats, AP39 significantly lowered systolic blood pressure for a prolonged period, decreased heart rate and arterial stiffness. In electrophysiological studies, AP39 significantly inhibited Ca(2+) current through all three CaV3 channels. AP39 decreased RyR2 channels activity and increased conductance and mean open time of Cl(-) channels. This study suggests that AP39 may offer a novel therapeutic opportunity in conditions whereby (•)NO and H2S bioavailability are deficient such as hypertension, and that CaV3, RyR2 and Cl(-) cardiac membrane channels might be involved in its biological actions.

Mitochondrial chloride channels: electrophysiological characterization and pH induction of channel pore dilation.

Physiological and pathological functions of mitochondria are highly dependent on the properties and regulation of mitochondrial ion channels. There is still no clear understanding of the molecular identity, regulation, and properties of anion mitochondrial channels. The inner membrane anion channel (IMAC) was assumed to be equivalent to mitochondrial centum picosiemens (mCS). However, the different properties of IMAC and mCS channels challenges this opinion. In our study, we characterized the single-channel anion selectivity and pH regulation of chloride channels from purified cardiac mitochondria. We observed that channel conductance decreased in the order: Cl⁻ > Br⁻ > I⁻ > chlorate ≈ formate > acetate, and that gluconate did not permeate under control conditions. The selectivity sequence was Br⁻ ≥ chlorate ≥ I⁻ ≥ Cl⁻ ≥ formate ≈ acetate. Measurement of the concentration dependence of chloride conductance revealed altered channel gating kinetics, which was demonstrated by prolonged mean open time value with increasing chloride concentration. The observed mitochondrial chloride channels were in many respects similar to those of mCS, but not those of IMAC. Surprisingly, we observed that acidic pH increased channel conductance and that an increase of pH from 7.4 to 8.5 reduced it. The gluconate current appeared and gradually increased when pH decreased from pH 7.0 to 5.6. Our results indicate that pH regulates the channel pore diameter in such a way that dilation increases with more acidic pH. We assume this newly observed pH-dependent anion channel property may be involved in pH regulation of anion distribution in different mitochondrial compartments.

Low molecular thiols, pH and O2 modulate H2S-induced S-nitrosoglutathione decomposition - •NO release.

We studied the involvement of O2, pH and low molecular thiols in H2S-induced decomposition of S-nitrosoglutathione (GSNO). The GSNO decomposition - •NO release was evaluated by UV-VIS spectroscopy and Griess assay. The H2S donor Na2S was used. O2 slightly increased, but was not necessary for the H2S-induced GSNO decomposition. The rate of GSNO decomposition depended on pH; the maximum rate was observed at pH 7.4-8.0, and this decreased with lowering pH (6.4-4.5) as well as with increasing pH at 9.0-12.0. H2S-induced GSNO decomposition was slowed by the presence of other thiols, such as L-cysteine (Cys), N-acetyl-L-cysteine (NAC) and L-glutathione (GSH), but not in the presence of L-methionine (Met) or oxidized glutathione (GSSG). In sharp contrast, at pH 6.0, H2S-induced GSNO decomposition was negligible, yet the presence of Cys, NAC and GSH induced the H2S-driven GSNO decomposition (whilst Met and GSSG were inactive). In conclusion we postulate an involvement of low molecular thiols and pH in •NO signaling, by modulating the interactions of H2S with nitroso compounds, and hence in part they also appear to control H2S-triggered •NO release. The interaction of H2S and/or its derivatives with the thiol group may be responsible for the observed effects.

Inhibitory effect of glybenclamide on mitochondrial chloride channels from rat heart.

Glybenclamide is used as a pharmacological tool in studies of mitochondrial functions supposing its main role to block ATP-dependent potassium (KATP) channel. The aim of this study was to test whether glybenclamide might interact with the mitochondrial chloride channels. Mitochondrial membranes, isolated from rat heart muscle, were incorporated into lipid bilayer membrane and single chloride channel currents were measured in 250/50 mM KCl cis/trans solutions. The observed chloride channels (N=11) with mean conductance 120±14 pS were sensitive to glybenclamide, which decreased the open probability (IC50=129 µM) and affected the channel gating kinetics (IC50=12 µM) by perturbing its open state. It did not influence the channel conductance or reversal potential. These results indicate that glybenclamide interacts with chloride channels what should be taken into consideration, when glybenclamide is used as a specific inhibitor of KATP channels.

The potassium channel opener CGS7184 activates Ca²âº release from the endoplasmic reticulum.

CGS7184 (ethyl 1-[[(4-chlorophenyl)amino]oxo]-2-hydroxy-6-trifluoromethyl-1H-indole-3-carboxylate) is a synthetic large-conductance Ca(2+)-activated potassium (BK(Ca)) channel opener. The existing literature suggests that potassium channels are involved in cardioprotection, particularly during ischemia-reperfusion events. However, the cellular mechanisms mediating the effects of CGS7184 remain unclear. In the present study, we investigated the effect of the BK(Ca) channel opener CGS7184 on Ca(2+) homeostasis in H9C2 and C2C12 cell lines, Ca(2+) uptake by isolated sarcoplasmic reticulum (SR) vesicles, SR Ca(2+)-ATPase (SERCA) activity, and single-channel properties of the ryanodine receptor calcium release channel (RYR2) when incorporated into a planar lipid bilayer. The effects of CGS7184 on calcium homeostasis in C2C12 and H9C2 cell lines were measured with a Fura-2 fluorescent indicator. The BK(Ca) channel opener CGS7184, when added to the H9C2 and C2C12 cells, caused a concentration-dependent release of calcium from internal stores. Calcium accumulation by the SR vesicles isolated from cardiac and skeletal muscle was inhibited by CGS7184 with a half-maximal inhibition value of 0.45 ± 0.04 µM and 0.37 ± 0.03 µM, respectively. The results of the present study indicate that the BK(Ca) channel opener CGS7184 modulates cytosolic Ca(2+) concentration in H9C2 and C1C12 cells due to its interaction with the endoplasmic reticulum (ER). CGS7184 approximately doubled the opening probability of RYR2 channels; however, the compound seemed to most strongly affect channels with a higher control activity. These results strongly suggest that the BK(Ca) channel opener CGS7184 affects intracellular calcium homeostasis by interacting with the sarcoplasmic reticulum RYR2 channels.

The aqueous garlic, onion and leek extracts release nitric oxide from S-nitrosoglutathione and prolong relaxation of aortic rings.

Garlic, onion and leek have beneficial effects in treatment of numerous health disorders. The aim of the present study was to investigate underlying molecular mechanisms. To test the potency of the aqueous garlic, onion and leek extracts to release NO from GSNO we have measured NO oxidation product, NO(2)-, by the Griess reagent method. Further, we studied the ability of garlic extract to relax noradrenaline-precontracted rat aortic rings in the presence of GSNO and effects of garlic extract on electrical properties of rat heart intracellular chloride channels. We have observed that: i) garlic, onion and leek extracts released NO from GSNO in the order: garlic > onion > leek; ii) the ability of garlic extract to release NO was pH-dependent (8.0 > 7.4 > 6.0) and potentiated by thiols (Cys >> GSH = N-acetyl-cysteine > oxidized glutathione) at concentration 100 µmol/l; iii) the garlic extract (0.045 mg/ml) prolonged relaxation time of aortic rings induced by GSNO (50 nmol/l) and inhibited intracellular chloride channels. We suggest that NO-releasing properties of the garlic, onion and leek extracts and their interaction with Cys and GSH are involved in NO-signalling pathway which contributes to some of its numerous beneficial biological effects.

On the involvement of H2S in nitroso signaling and other mechanisms of H2S action.

Both endogenously produced and exogenously administered H2S exert numerous biological effects. However, the molecular mechanisms underlying these effects are not fully understood. This review surveys the biological effects of H2S and summarizes the molecular mechanisms of H2S action. It focuses on the role of H2S/HS--induced NO release from nitroso compounds, modulation of ion channels and the antioxidant and radical properties of H2S in the molecular mechanism of its effects. The potential involvement of H2S in nitroso signaling underlying its diverse biological effects is also discussed.

The hypothesis of the main role of H2S in coupled sulphide-nitroso signalling pathway.

As a part of the nitroso signalling pathway, nitroso-compounds serve as stores and carriers of NO; as part of the sulphide signalling pathway, bound sulfane-sulphur compounds serve as stores and carriers of H2S. Here we hypothesise a coupled sulphide-nitroso signalling pathway, in which H2S plays a main role. H2S releases NO from the endogenous S-nitroso-compounds nitroso-cysteine, nitroso-acetylcysteine and nitroso-albumin. Relaxation of noradrenaline-precontracted aortic rings by H2S is also enhanced in the presence of nitroso-albumin, which may implicate the involvement of the nitroso signalling pathway. Pretreatment of albumin, cysteine, N-acetylcysteine and lipids with H2S results in binding of sulphur to these compounds creating thus new-modified sulphur compounds that release NO from nitroso-compounds directly and/or through released H2S, which suggests sulphide-nitroso signalling pathway participation. This hypothesis is supported by the observation that the pretreatment of noradrenaline-precontracted aortic rings with H2S significantly enhanced relaxation induced by nitroso-glutathione in the absence of H2S. We assume that the NO release from nitroso-compounds directly by H2S or indirectly by the H2S-induced sulphur-bound compounds represents coupled sulphide-nitroso signalling, which may explain some of the numerous biological effects of H2S that are shared with NO.

Modulation of intracellular chloride channels by ATP and Mg2+.

We report the effects of ATP and Mg2+ on the activity of intracellular chloride channels. Mitochondrial and lysosomal membrane vesicles isolated from rat hearts were incorporated into bilayer lipid membranes, and single chloride channel currents were measured. The observed chloride channels (n=112) possessed a wide variation in single channel parameters and sensitivities to ATP. ATP (0.5-2 mmol/l) modulated and/or inhibited the chloride channel activities (n=38/112) in a concentration-dependent manner. The inhibition effect was irreversible (n=5/93) or reversible (n=15/93). The non-hydrolysable ATP analogue AMP-PNP had a similar inhibition effect as ATP, indicating that phosphorylation did not play a role in the ATP inhibition effect. ATP modulated the gating properties of the channels (n=6/93), decreased the channels' open dwell times and increased the gating transition rates. ATP (0.5-2 mmol/l) without the presence of Mg2+ decreased the chloride channel current (n=12/14), whereas Mg2+ significantly reversed the effect (n=4/4). We suggest that ATP-intracellular chloride channel interactions and Mg2+ modulation of these interactions may regulate different physiological and pathological processes.

Mitochondrial chloride channels--What are they for?

This minireview focuses on observation of the properties, functional significance, and modulation of single chloride channels in the mitochondrial inner membrane using two electrophysiological methods--the patch-clamp and bilayer lipid membrane methods. Measurements of parameters such as conductance, Cl(-)/K(+) selectivity, voltage or pH dependence as well as their modulation by endogenous and exogenous compounds using individual mitochondrial chloride channels result in an unexpectedly wide range of values. This paper discusses the origin of this wide variety of channel parameters and the possible involvement of these channels in mitochondrial membrane potential oscillations, apoptosis, carrier function, and mitochondrial fusion and fission.

Lipids modulate H(2)S/HS(-) induced NO release from S-nitrosoglutathione.

Recently we observed that a gas messenger H(2)S/HS(-) released NO from S-nitrosoglutathione (Ondrias et al., Pflugers Arch. 457 (2008) 271-279). However, the effect of biological compounds on the release is not known. Measuring the NO oxidation product, which is nitrite, by the Griess reaction, we report that unsaturated fatty acid-linoleic acid and lipids having unsaturated fatty acids: asolectin, dioleoylphosphocholine and dioleoylphosphoserine depressed the H(2)S/HS(-) induced NO release from S-nitrosoglutathione. On the other hand, a depression effect of the saturated fatty acid-myristic acid and lipids having saturated fatty acids, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine was less pronounced. The inhibition effect increased with the decreasing gel-to-liquid phase transitions temperature of the fatty acids and lipids. We suggest that lipid composition of biological membranes modulates NO release from nitrosoglutathione induced by H(2)S/HS(-), assuming that a reaction of H(2)S/HS(-) with unsaturated bonds of fatty acids may be partially responsible for the effect.

The cardiac ryanodine receptor: looking for anomalies in permeation properties.

Anomalies in the permeation properties of the cardiac RyR channel reconstituted into bilayer lipid membranes were investigated systematically. We tested the presence of the anomalous mole fraction effect (AMFE) for the ion conductance and the reversal potential with varying mole fractions of two permeant ions, while the total ion concentration was lower, as in previous studies, to avoid the masking effect of the channel pore saturation with ions. Mixtures of Ba(2+) with other divalents (Ca(2+), Sr(2+)), of Ca(2+) with monovalents (Li(+), Cs(+)), and of Na(+) with other monovalents (Cs(+), Li(+)) were used. We revealed a clear anomaly only for the ion conductance measured in the Na(+)-Cs(+) and Ca(2+)-Li(+) mixtures as computed by a Poisson-Nernst-Planck/density functional theory (PNP/DFT) model. Furthermore, we found a significant minimum in the concentration dependence of the reversal potential determined under Li(+)/Ca(2+) bi-ionic conditions. Our study led to new observations that may have important implications for understanding the mechanisms involved in ion handling in the RyR channel pore; furthermore our results could be useful for further validation of ion permeation models developed for the RyR channel.

Inhibition of anion channels derived from mitochondrial membranes of the rat heart by stilbene disulfonate--DIDS.

The objective of this work was to characterize in more detail the inhibition effect of diisothiocyanatostilbene-2',2-disulfonic acid (DIDS) on anion channels isolated from the rat heart mitochondria. The channels reconstituted into a planar lipid membrane displayed limited powers of discrimination between anions and cations and the ion conductance measured under asymmetric (250/50 mM KCl, cis/trans) and symmetric (150 mM KCl) conditions was approximately 100 pS. DIDS caused a dramatic decrease in the channel activity (IC(50) = 11.7 +/- 3.1 microM) only when it was added to the cis side of a planar lipid membrane. The inhibition was accompanied by the significant prolongation of closings and the shortening of openings within the burst as well as gaps between bursts were prolonged and durations of bursts were reduced. The blockade was complete and irreversible when concentration of DIDS was increased up to 200 microM. Our data indicate that DIDS is an allosteric blocker of mitochondrial anion channels and this specific effect could be used as a tool for reliable identification of anion channels on the functional level.