Knockout of cyclophilin D in Ppif⁻/⁻ mice increases stability of brain mitochondria against Ca²âº stress.

The mitochondrial peptidyl prolyl isomerase cyclophilin D (CypD) activates permeability transition (PT). To study the role of CypD in this process we compared the functions of brain mitochondria isolated from wild type (BMWT) and CypD knockout (Ppif(-/-)) mice (BMKO) with and without CypD inhibitor Cyclosporin A (CsA) under normal and Ca(2+) stress conditions. Our data demonstrate that BMKO are characterized by higher rates of glutamate/malate-dependent oxidative phosphorylation, higher membrane potential and higher resistance to detrimental Ca(2+) effects than BMWT. Under the elevated Ca(2+) and correspondingly decreased membrane potential the dose response in BMKO shifts to higher Ca(2+) concentrations as compared to BMWT. However, significantly high Ca(2+) levels result in complete loss of membrane potential in BMKO, too. CsA diminishes the loss of membrane potential in BMWT but has no protecting effect in BMKO. The results are in line with the assumption that PT is regulated by CypD under the control of matrix Ca(2+). Due to missing of CypD the BMKO can favor PT only at high Ca(2+) concentrations. It is concluded that CypD sensitizes the brain mitochondria to PT, and its inhibition by CsA or CypD absence improves the complex I-related mitochondrial function and increases mitochondria stability against Ca(2+) stress.

Beta(3)-adrenergic stimulation and insulin inhibition of non-selective cation channels in white adipocytes of the rat.

Single-channel currents were recorded from the plasma membrane of white adipocytes of 6-8-week-old male Sprague-Dawley rats. In outside-out patches (high K(+), no Ca(2+) in pipette), a voltage-dependent K-channel (delayed rectifier) with a single-channel conductance (gamma) of 16 pS (24 degrees C) in modified Ringer's was active at a density of 0.5/microm(2). It was blocked by TEA (IC(50)=1.5 mM). A Ca(2+)-activated non-selective cation channel (NSC-channel) appeared at a mean density of 1/microm(2) in inside-out patches ([Ca(2+)](i)=1.2 mM). gamma was 28 pS (24 degrees C). The NSC showed weak voltage dependence and was blocked by mefenamic acid and by internal ATP. In the cell-attached mode spontaneous activity could be blocked reversibly by 100 nM insulin. Noradrenaline (NA, 100 nM) induced a flickering activity of the NSC-channels. Isoproterenol (100 nM) caused activity of the NSC-channel as well. After 1 microM propranolol even 1 microM NA did not induce any activity. The alpha-antagonist phentolamine had no effect on isoproterenol- or on NA-induced currents. The beta(3)-agonists BRL 37344 and BRL 35135A induced activity of the NSC-channel at 100 nM as well. We conclude that white adipocytes express ion channels which are comparable to those in brown adipocytes and that beta-receptor activation opens NSC-channels thus allowing for Na(+) entry into white adipocytes.

A voltage-dependent and a voltage-independent potassium channel in brown adipocytes of the rat.

Single-channel recordings of a voltage-dependent potassium channel in brown adipocytes of the rat confirm recordings of macroscopic currents. Single-channel conductance (gamma) is 8 pS at 20 degrees C in KF solution inside vs. a modified Ringer's solution outside. With KCl solution outside, gamma is 17 pS for outward currents and 21 pS for inward currents. The majority of the channels inactivate with a time constant around 200 ms; deactivation occurs within milliseconds. The channel is blocked by tetraethylammonium (TEA) with an inhibiting constant of 1.8 mM. The type of block is fast. Selectivity sequence for monovalent cations is K+ > Rb+ >> NH4+ >> Li+ > or = Na+ approximately Cs+. Cs+ at the outside causes a voltage-dependent block of inward currents. This channel is remarkably similar to the delayed rectifier of the F-type in the node of Ranvier. Occasionally, an additional K+ channel was found. This channel is voltage-insensitive, not blocked by 10 mM TEA, and has not been recorded in brown adipocytes before. Physiological relevance of this channel could be the steady-state membrane potential.

Slow permeation of organic cations in acetylcholine receptor channels.

Block, permeation, and agonist action of small organic amine compounds were studied in acetylcholine receptor (AChR) channels. Single channel conductances were calculated from fluctuation analysis at the frog neuromuscular junction and measured by patch clamp of cultured rat myotubes. The conductance was depressed by a few millimolar external dimethylammonium, arginine, dimethyldiethanolammonium, and Tris. Except with dimethylammonium, the block was intensified with hyperpolarization. A two-barrier Eyring model describes the slowed permeation and voltage dependence well for the three less permeant test cations. The cations were assumed to pause at a site halfway across the electric field of the channel while passing through it. For the voltage-independent action of highly permeant dimethylammonium, a more appropriate model might be a superficial binding site that did not prevent the flow of other ions, but depressed it. Solutions of several amine compounds were found to have agonist activity at millimolar concentrations, inducing brief openings of AChR channels on rat myotubes in the absence of ACh.

Large conductance channel in plasma membranes of astrocytic cells is functionally related to mitochondrial VDAC-channels.

Large conductance anion channels with similar electrophysiological characteristics were found in plasma membranes and in outer mitochondrial membranes of various cell types. Although their large conductance and their peculiar voltage dependence point to a close relation, it was questioned whether they belong to the same family. We therefore compared some biochemical features of a plasmalemmal channel with those known from the mitochondrial channel. Current events were recorded from excised patches of plasma membranes of a rat astrocytic cell line (RGCN). The underlying channels exhibited a conductance of 401 +/- 50 pS. Open probability was highest between +/- 10 mV and gradually approached zero beyond +/- 25 mV. Activity as induced by voltage ramps between +/- 40 mV appeared after a delay of up to several min. The delay could be reduced by bathing either side of the patch in an acidic Ringer solution (pH 6.2). 1 mM Al3+ increased the open time at potentials more positive than 20 mV. 10 mM dextran sulfate (MW 8000) caused reversible flickering, increasing the closed probability. 4,4'-diisothiocyanatostilbene-2,2' disulfonic acid (DIDS) also caused a reversible flickering into the closed state, reducing the apparent single channel amplitude by up to 70% at 0.5 mM DIDS. Application of 5 mM ATP resulted in reversible blockade; ATP was more effective from the outside than from the inside (blocking activity 65% vs. 16% of the patches). We conclude that the large conductance anion channel from astrocytic cells displays electrophysiological and pharmacological characteristics that resemble those of VDAC (Voltage Dependent Anion Channel) from the outer mitochondrial membrane.

Electrophysiological and neurobiochemical evidence for the blockade of a potassium channel by dendrotoxin.

The effects of dendrotoxin (DTX), a toxic peptide from Dendroaspis angusticeps venom, were studied electrophysiologically on peripheral frog nerve fibres, and biochemically on large synaptosomes from rat brain. On nerve fibres, DTX reduced the amplitude and prolonged the duration of the action potential; even at 0.1 nmol/l DTX produced significant effects. Maximum block of potassium currents occurred at about 30 nmol/l. Turning on of the remaining current was slowed. Reversibility was incomplete. The reduction of potassium currents was between 31% and 85% at 85 nmol/l DTX (n = 8). The remainder appeared to be resistant to DTX. Sodium channels were not affected. On large synaptosomes DTX (above 1 nmol/l) produced a slight depolarization, indicated by an outward shift of the lipophilic cation tetraphenylphosphonium, and promoted the release of radioactivity after preloading with [3H] GABA. DTX had similar potency but lower efficacy in this respect than sea anemone toxin II (ATX II). In contrast to the effects of ATX II, those due to DTX were only partially inhibited by tetrodotoxin. The actions of 4-aminopyridine resembled those of DTX, but the latter was about 500 times more potent. The electrophysiological data provide direct evidence for blockade of a potassium channel by DTX. This action is sufficient to explain the biochemical observations, although additional effects on synaptosomes cannot be excluded.

Modulation of the permeability transition pore by inhibition of the mitochondrial K(ATP) channel in liver vs. brain mitochondria.

Inhibition of the mitochondrial K(ATP) (mitoK(ATP)) channel abrogates the beneficial effects of preconditioning induced by a brief episode of sublethal ischemia. We studied the effect of 5-hydroxydecanoate, a well-known inhibitor of the mitoK(ATP) channel, on swelling of isolated liver and brain mitochondria. Volume changes were determined by measurement of light absorbance at 540 nm. Mitochondrial swelling induced by adding Ca(2+ )ions correlated with opening of the permeability transition pore as shown by modulation by 1 microM cyclosporin A. In brain mitochondria, 5-hydroxydecanoate did not significantly affect Ca(2+)-induced swelling. In contrast, 50 or 500 microM 5-hydroxydecanoate increased swelling of liver mitochondria by 9.7 +/- 5.1% (n = 6, P = 0.057) and 29.4 +/- 1.4% (n = 5, P < 0.0001), respectively. The effect of 5-hydroxydecanoate was blocked by cyclosporin A and was dependent on the presence of potassium in the medium. In medium containing 200 microM ATP to inhibit the mitoK(ATP )channel, 5-hydroxydecanoate did not further increase Ca(2+)-induced swelling. We conclude that inhibition of the mitoK(ATP) channel exerts its detrimental effect by facilitation of permeability transition pore opening.

Inner mitochondrial membrane anion channel is present in brown adipocytes but is not identical with the uncoupling protein.

Vesicles of inner mitochondrial membrane, mitoplasts, from rat brown adipose tissue were prepared by osmotic swelling and studied using the patch-clamp technique. Current events of a 107.8 +/- 8.7 pS (n = 16, 21 degrees C) channel were recorded in the mitoplast-attached mode. This channel was selective for anions and its kinetics resembled those of channels previously found in liver and heart mitochondria of mouse and ox. In whole-mitoplast mode each of five purine nucleotides (20 microM) blocked the channel. This is the first demonstration of pharmacological blockade of this type of channel. Although a similar anion channel in mouse and ox mitochondria was suggested to be the uncoupling protein (UCP) associated with nonshivering thermogenesis, we present several arguments against this possibility. Thus we describe a high-conductance, purine-nucleotide-binding, anion selective mitochondrial channel, that is not the UCP.

Kinetic parameters of the ionic currents in myelinated axons: characterization of temperature effects in a hibernator and a nonhibernator.

Na+ and K+ currents were measured by the patch-clamp method in the paranodal region of single sciatic nerve fibres of rats and of warm-adapted and cold-adapted golden hamsters. Kinetic parameters and temperature dependence of the Na+ currents were determined. The time constant for activation (about 0.2 ms for rats and hamsters) as well as the time constant for inactivation (about 1.6 ms for rats and hamsters) at 15 degrees C and at -35 mV compared well with single fibre voltage-clamp data from the rat. Differences amongst the three groups of animals were not significant. The temperature coefficient, Q10, for the activation and the inactivation time constant as well as for the time-to-peak of the Na+ current ranged between 2.3 and 3.1. No data have previously been published on the temperature dependence of the delayed-rectifier K channels of mammalian nerve fibres. Most of the K+ current was carried by intermediate (KI) and fast (KF) K channels. Dendrotoxin block indicated that "approximate"55% of the K+ current was due to KI channels, with no significant difference amongst the three groups of animals tested. The Arrhenius plot of the time constant of K+ current activation, "tau"n, yielded a mean Q10 of 3.3 at -40 mV (4. 0 at + 60 mV). No significant differences of the channel kinetics between rats, warm-adapted hamsters and cold-adapted hamsters were detected. We observed, however, a significant decrease of the Na channel density in the paranodal region of cold-adapted hamsters.

Permeability of the non-selective channel in brown adipocytes to small cations.

The non-selective channel for monovalent cations of cultured brown adipocytes was studied concerning its permeability to alkali metal ions, NH4+, Tris+, Ca2+, and Ba2+. Experiments were done by means of the patch clamp technique using inside-out patches. With symmetrically increasing sodium concentrations the ion fluxes saturated. They are described by a dissociation constant (KNa) of 155 mmol/l and a maximum single channel conductance of 50 pS. Permeabilities were determined in relation to those for sodium yielding values of 0.80 for potassium and 1.55 for ammonium. The complete permeability sequence for ammonium and the alkali metals is: NH4+ greater than Na+ greater than Li+ greater than K+ greater than or equal to Rb+ congruent to Cs+ . Ca2+ and Ba2+ as well as the buffer ion Tris+ are not able to pass the channel measurably. It is shown that the conductance behaviour of the non-selective channel is not sufficiently described by the Goldman-Hodgkin-Katz theory. Deviations from independence are saturation with increased activity of the permeant ion and non-linear current voltage relations in symmetrical solutions. A simple two barrier model with one binding site in the center of the electric field is shown to be more appropriate.

Non-selective cationic channel in primary cultured cells of brown adipose tissue.

Single channel current events were recorded from membranes of cultured brown adipose tissue cells with the patch-clamp technique. In excised inside-out patches the predominant type of events showed a slope conductance of about 30 pS (25 degrees C). The current was carried by both, Na+ ions and K+ ions. Discrimination between them was poor. The frequency of events increased with increasing temperature. Their amplitude was temperature dependent as well (Q10 approximately equal to 1.4). A single exponential was not sufficient for fitting the histograms of "on-time" or "off-time". We conclude that these events belong to a type of non-selective cation channel described previously for other tissues.

Norepinephrine-induced sustained inward current in brown fat cells: alpha(1)-mediated by nonselective cation channels.

The nature of the sustained norepinephrine-induced depolarization in brown fat cells was examined by patch-clamp techniques. Norepinephrine (NE) stimulation led to a whole cell current response consisting of two phases: a first inward current, lasting for only 1 min, and a sustained inward current, lasting as long as the adrenergic stimulation was maintained. The nature of the sustained current was here investigated. It could be induced by the alpha(1)-agonist cirazoline but not by the beta(3)-agonist CGP-12177A. Reduction of extracellular Cl(-) concentration had no effect, but omission of extracellular Ca(2+) or Na(+) totally eliminated it. When unstimulated cells were studied in the cell-attached mode, some activity of approximately 30 pS nonselective cation channels was observed. NE perfusion led to a 10-fold increase in their open probability (from approximately 0.002 to approximately 0.017), which persisted as long as the perfusion was maintained. The activation was much stronger with the alpha(1)-agonist phenylephrine than with the beta(3)-agonist CGP-12177A, and with the Ca(2+) ionophore A-23187 than with the adenylyl cyclase activator forskolin. We conclude that the sustained inward current was due to activation of approximately 30 pS nonselective cation channels via alpha(1)-adrenergic receptors and that the effect may be mediated via an increase in intracellular free Ca(2+) concentration.

108-pS channel in brown fat mitochondria might Be identical to the inner membrane anion channel.

Single-channel and whole-mitoplast patch-clamp recordings were employed to characterize the 108-pS (Cl-) channel in brown fat mitochondrial mitoplasts. We demonstrated the ability of this channel to conduct di- and trivalent anions, such as sulfate, phosphate, and benzenetricarboxylates, and its blockage by propranolol, 1,4-dihydropyridine-type Ca2+ antagonists, and Cibacron blue. Moreover, we have revealed its pH dependence for the first time. As a basis for the characteristic potential dependence of the whole-mitoplast current, we identified an open probability, increasing with depolarizing (positive) potentials, Eh, and being almost zero in the hyperpolarizing range. Events at negative Eh exhibit a short flickering behavior, whereas at positive Eh, they become much longer. This voltage dependence is influenced by pH in such a way that, at acidic pH, the 108-pS channel possesses a low open probability throughout the observed potential range, whereas at alkaline pH, the channel switches to long openings, even at a negative potential. All these properties lead us to conclude that the inner membrane anion channel, which has been characterized only by light scattering studies, and the 108-pS inner membrane channel, which has been characterized electrophysiologically, are one and the same process.

Reversible blockade of the calcium-activated nonselective cation channel in brown fat cells by the sulfhydryl reagents mercury and thimerosal.

We have used patch-clamp techniques to study the effect of the sulfhydryl group oxidizing agents mercury and thimerosal on calcium-activated nonselective cation channels from brown adipose tissue. 100 nmol/l mercury and 50 mumol/l thimerosal induced a complete block. Blockade could be reversed by reduction of the mercaptide by dithiotreitol (DTT). Mercury was found to be the most potent blocker (IC50-value 21 x 10(-9) mol/l), whereas thimerosal (IC50-value 1.5 x 10(-6) mol/l) was as effective as 3',5-dichlorodiphenylamine-2-carboxylic acid (DCDPC). The DCDPC effect, however, could not be reversed by DTT, indicating different blocking mechanisms. It is concluded that SH-groups are involved in gating of the calcium-activated nonselective channel.

Regulation of the activity of 27 pS nonselective cation channels in excised membrane patches from rat brown-fat cells.

The regulation of the activity of the approximately 30 pS nonselective cation channel (NSC channel) was studied by the patch-clamp technique in inside-out patches obtained from rat brown-fat cells. NSC channel activity was induced by excision; reduced redox state induced by dithiothreitol accelerated the kinetics in the excised state. The NSC channels were inhibited by the fenamates flufenamic acid and mefenamic acid but not by NS-1619 or SKF-96365. The channels were inhibited by purine nucleotides but not by polyamines. No evidence for protein kinase C, CaM kinase or protein kinase A activation of the NSC channel was obtained. NSC-channel activity was stimulated in a concentration-dependent manner by Ca2+ but the EC50 was very high (0.81 mM), in comparison to expected cytosolic Ca2+ levels. In the presence of ATP, even higher Ca2+ levels were necessary for comparable NSC-channel activation. The increase in Po was not associated with an increase in open-time constants. We conclude that although high Ca2+ levels can experimentally activate the NSC channel, a further mediatory step must probably be postulated in order to link alpha1-adrenergic stimulation to NSC-channel activation.

Development of sodium permeability inactivation in nodal membranes.

1. The time course of inactivation of the sodium permeability was studied in myelinated nerve fibres of Xenopus laevis using the voltage-clamp technique of Nonner (1969). The potassium currents were blocked by 10 mM-tetraethylammonium chloride (TEA). The remaining currents were corrected for leakage and capacity currents. 2. The decay of the sodium current was double-exponential confirming Chiu's (1977) findings The slower time constant was observed in TEA-free solutions by clamping to VK in high potassium concentrations. 3. The fast time constant was similar to tau h of Frankenhaeuser (1960), whereas the slower time constant was about four times larger and also decreased with increasing depolarization. 4. Arrhenius plots of both time constants can be well approximated by straight lines for temperatures between 0 and 25 degrees C. 5. The entire sodium current induced by single voltage-clamp pulses was fitted by a non-linear least-square routine to the Frankenhaeuser-Huxley equations extended by Chiu's three-state kinetics. With rare exceptions for potentials between 37 and 70 mV the fit was better without a delay in the onset of the inactivation of the sodium current. 6. The time course of inactivation was also studied in two-pulse experiments where a prepulse of varying duration was directly followed by a test pulse. The peak sodium currents were normalized by the associated peak currents without a prepulse. 7. The relative peak sodium current as a function of the prepulse duration had a sigmoid time course. The early deviation from an exponential decay is due to the activation arising during the prepulse and is implicit in the classical equations quoted above. It is therefore not necessarily a sign of a delay in the inactivation process. 8. To eliminate errors due to activation in two-pulse experiments, the decaying part of the test sodium currents was extrapolated back to the onset of the test pulse. These current values at t = 0 plotted as a function of the prepulse duration revealed no delay at the beginning of inactivation. Within the accuracy of our measurements a possible delay of more than 100 microseconds (at 5-10 degrees C) could be excluded.

The involvement of caspases in the CD95(Fas/Apo-1)- but not swelling-induced cellular taurine release from Jurkat T-lymphocytes.

Following a delay of 45 min, stimulation of the CD95 (Fas/Apo-1)-receptor in Jurkat T-lymphocytes leads to the release of the osmolyte taurine, an event coinciding with apoptotic cell shrinkage. The present study has been performed to elucidate the cellular mechanisms involved in CD95-induced taurine release as compared to swelling-induced taurine release, and to explore whether taurine modifies apoptotic DNA fragmentation and cell shrinkage. Taurine release stimulated by osmotic cell swelling is insensitive to the tyrosine kinase inhibitor herbimycin A and the caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD) but is blunted in the absence of extracellular Ca2+. Conversely, the Ca2+ ionophore ionomycin stimulates taurine release. However, the taurine release following CD95 stimulation is not paralleled by an increase of cytosolic Ca2+ and not inhibited by complexation of extracellular Ca2+. None of herbimycin A, the phosphatase inhibitor vanadate, spingomyelinase or Lck56 deficiency prevent CD95-induced taurine release. In contrast, the caspase inhibitor zVAD, but not the caspase inhibitor Ac-Tyr-Val-Ala-Asp-chloromethylketone (YVAD), almost abolishes CD95-induced taurine release. Both caspase inhibitors blunt CD95-induced cell shrinkage and DNA fragmentation, zVAD being more effective than YVAD. Preloading of the cells with 40 mM taurine but not with 40 mM mannitol significantly inhibits CD95-induced DNA fragmentation (by 28%) and apoptotic cell shrinkage (by 25%). In conclusion, CD95-receptor triggering leads to caspase-dependent stimulation of cellular taurine release, which facilitates, but is not sufficient for, the triggering of apoptotic DNA fragmentation and cell shrinkage.

Physiology of Receptor-Mediated Lymphocyte Apoptosis.

Apoptosis (programmed cell death), a physiological mechanism eliminating abundant and potentially harmful cells, is triggered by a variety of stimuli including activation of distinct receptors. The machinery mediating CD95 receptor-induced apoptosis includes caspases, ceramide, kinases, Ras and Rac, formation of O(2)(-), mitochondrial proteins, inhibition of K(+) channels, activation of Cl(-) channels, and osmolyte release.