Peculiarities of functioning of wheat cut-off root cells at inhibition of complexes I and II of mitochondrial respiratory chain

Combined action of rotenone and malonate, inhibitors of complexes I and II of the mitochondrial electron transport chain (ETC), on wheat cut-off root seedlings was studied after 6 h of incubation. Intensity of oxygen consumption and release of potassium ions into incubation medium were determined simultaneously with the study of changes in cell ultrastructure. Malonic acid was added 1 h after the root incubation in the rotenone solution and produced inhibition of respiration, as well as a greater release of K⁺ into the incubation solution as compared with effect of rotenone alone. After 2 h of the combined action of these inhibitors, many mitochondria acquired a toroidal shape, thereby increasing the outer surface. For the ensuing hours, stimulation of oxygen consumption by the roots and a decrease of K⁺ content in the incubation medium were observed. Mitochondria once again acquired a round or oval shape and compensation-reparation processes took place. Contacts of endoplasmic reticulum channels with mitochondria were observed, which seems to be due to the synthesis of the enzyme splitting malonate to acetyl-CoA, which in turn can be included both into the Krebs cycle and into lipogenesis. It is suggested that the toroidal form of mitochondria is associated with the activation of the external NAD(P)H-dehydrogenase of the inner mitochondrial membrane, as under these conditions, at the inhibition of the ETC complexes I and II, the activity of other dehydrogenises is blocked. Thus, the use of the external NAD(P)H allows the activity of the ETC mitochondria to be restored, which facilitates the course of the reparation processes and allows cells to be adapted to this action.     

Changes in the Energy State of Wheat Root Cells Induced by the Protonophore CCCP

The effects of carbonyl cyanide 3-chlorophenylhydrazone (CCCP) on the rate of the oxygen uptake by excised wheat roots and their heat generation and K⁺ion content in the incubation medium were followed for 6 h. When the incubation medium contained 0.5 M CCCP, the roots were found to exhibit a reversible release of K⁺ions and the stimulation of the oxygen uptake. These responses were found to correlate with considerably enhanced heat generation by the plant tissues. It is proposed that these changes were due to the activation of both the energy system of the root cells and the H⁺-ATPase in the plasmalemma. The roots treated with 5 M CCCP exhibited an inhibition of the oxygen uptake and heat generation (1–3 h) followed by the stimulation of these processes by the 5th or 6th hour of the experiment; however, the potassium ion release by the roots was not reversed under these conditions. Uncoupling the processes of oxidation and phosphorylation in mitochondria of the root cells (the 4th–6th h) seems to underlie the observed responses. In the roots treated with 50 M CCCP, we observed the irreversible release of K⁺ions from the root cells, the considerable inhibition of the oxygen uptake by the latter, and the initial burst and then decline in heat generation. These effects suggest that, under the experimental conditions, a disturbance in cellular homeostasis and energy supply occurred and eventually resulted in cell death.     

Mitochondria from rat uterine smooth muscle possess ATP-sensitive potassium channel

The objective of this study was to detect ATP-sensitive K⁺ uptake in rat uterine smooth muscle mitochondria and to determine possible effects of its activation on mitochondrial physiology. By means of fluorescent technique with usage of K⁺-sensitive fluorescent probe PBFI (potassium-binding benzofuran isophthalate) we showed that accumulation of K ions in isolated mitochondria from rat myometrium is sensitive to effectors of K_ATP-channel (ATP-sensitive K⁺-channel) – ATP, diazoxide, glibenclamide and 5HD (5-hydroxydecanoate). Our data demonstrates that K⁺ uptake in isolated myometrium mitochondria results in a slight decrease in membrane potential, enhancement of generation of ROS (reactive oxygen species) and mitochondrial swelling. Particularly, the addition of ATP into incubation medium led to a decrease in mitochondrial swelling and ROS production, and an increase in membrane potential. These effects were eliminated by diazoxide. If blockers of K_ATP-channel were added along with diazoxide, the effects of diazoxide were removed. So, we postulate the existence of K_ATP-channels in rat uterus mitochondria and assume that their functioning may regulate physiological conditions of mitochondria, such as matrix volume, ROS generation and polarization of mitochondrial membrane.     

Dependency of the ATPase and 32 P--ATP exchange reaction of mitochondria on K + and electron transport

The 2,4-dinitrophenol-stimulated ATPase activity and the ³²P-ATP exchange reaction has been studied in rat liver mitochondria having less than 15 nmoles of K⁺ per milligram of protein. With 200 mm sucrose in the incubation media, the permeation of K⁺ and an oxidizable substrate is required for maximal stimulation of ATPase activity by 2,4-dinitrophenol. In these conditions, the 2,4-dinitrophenol-stimulated ATPase is inhibited by antimycin, acetate and mersalyl and depends to a certain extent on the rate of electron transport. The ³²P-ATP exchange reaction of mitochondria with a low content of K⁺ also requires K⁺ permeation and is inhibited by antimycin, cyanide, 2,4-dinitrophenol, and acetate. The results suggest that the entrance of ATP into the mitochondria is compulsory linked to K⁺ uptake in a process that depends on a negative internal potential.     

Adenosine Triphosphatases Bound to Plasmalemma, Mitochondria, and Microsomes or Present in the Cytoplasmic Phase from Potato Tubers

Between pH 4–10, basal ATPase activity, measured in the absence of mineral ions, was 10 to 100 times higher in the final cytoplasmic supernatant from potato tuber homogenates than in the membraneous fractions (purified plasmalemma, purified mitochondria and microsomes). The soluble ATPase was slightly inhibited, whereas the membrane-bound ATPases were all stimulated by Mg²⁺ ions. A further stimulation by Na⁺ or K⁺ ions was only observed in purified plasmalemma or mitochondria, at alkaline pH (7.5–9.5). At a fixed (Na⁺+ K⁺) concentrations (80 mM), this last stimulation was much greater in purified mitochondria (350%) than in plasmalemma (33%); it also increased with (Na⁺+ K⁺) concentrations up to 200 mM in mitochondria whereas, in plasmalemma, it was roughly constant for monovalent ion concentrations between 20 and 200 mM. General properties of the plasma membrane-bound ATPase have been determined, i.e. substrate specificity, activity variations with quantity of substrate, temperature, pH, etc. Divalent cations stimulated strongly the ATPase in the following order: Mn²⁺ > Mg²⁺ > Ca²⁺. The maximum ATP hydrolysis velocity for that part of ATPase activity which is strictly dependent on Mg²⁺ ions was 3.85 μmol × mg^?1 protein × h^?1. This plasma membrane ATPase was not sensitive to ouabaïn or to oligomycin.     

Ion transport processes in corn mitochondria : I. Effect of the local anesthetic dibucaine

The local anesthetic dibucaine inhibited respiration-dependent contraction mediated by the K⁺/H⁺ antiport system of isolated corn mitochondria. Respiration declined concurrently. Nigericin, an exogenous K⁺/H⁺ exchanger, restored ion efflux in dibucaine-blocked corn mitochondria. It was concluded that dibucaine inhibited ion efflux via blockage of the K⁺/H⁺ antiport. Further experiments determined that dibucaine also inhibited proton influx facilitated by protonophores and by the ATPase complex during state III respiration. These results are discussed in relation to the mechanism by which dibucaine inhibits proton translocation across the inner mitochondrial membrane.     

Effect of potential-dependent potassium uptake on production of reactive oxygen species in rat brain mitochondria

The effect of potential-dependent potassium uptake on reactive oxygen species (ROS) generation in mitochondria of rat brain was studied. It was found that the effect of K⁺ uptake on ROS production in the brain mitochondria under steady-state conditions (state 4) was determined by potassium-dependent changes in the membrane potential of the mitochondria (ΔΨ_m). At K⁺ concentrations within the range of 0–120 mM, an increase in the initial rate of K⁺-uptake into the matrix resulted in a decrease in the steady-state rate of ROS generation due to the K⁺-induced depolarization of the mitochondrial membrane. The selective blockage of the ATP-dependent potassium channel (K _ATP ⁺ -channel) by glibenclamide and 5-hydroxydecanoate resulted in an increase in ROS production due to the membrane repolarization caused by partial inhibition of the potential-dependent K⁺ uptake. The ATP-dependent transport of K⁺ was shown to be ～40% of the potential-dependent K⁺ uptake in the brain mitochondria. Based on the findings of the experiments, the potential-dependent transport of K⁺ was concluded to be a physiologically important regulator of ROS generation in the brain mitochondria and that the functional activity of the native K _ATP ⁺ -channel in these organelles under physiological conditions can be an effective tool for preventing ROS overproduction in brain neurons.     

The membrane potential of intact Rhodospirillum rubrum cells in the absence of light-dependent and oxygen-linked electron transfer

In the absence of oxygen-linked and light-dependent electron transfer, the steady-state membrane potential of intact Rhodospirillum rubrum cells was usually between 65 and 75% of that of dark aerated cells, as indicated by the relative extent of the bacteriochlorophyll electrochromic changes that were induced by oxygen and by uncouplers. That potential was not due to residual levels of oxygen or light, because its value was not significantly altered by the presence of oxygen-trapping systems or by exhaustive gassing with Ar, and because it was also exhibited by a reaction-center-less mutant. The dark anaerobic potential was unaffected by 0.11 M K⁺; that seemed to exclude a diffusion potential generated by dissipation of a previously built K⁺ gradient. In contrast, it was largely abolished by 0.5 mM N,N′-dicyclohexylcarbodiimide, suggesting its dependence on ATP hydrolysis by the proton-translocating ATPase of the bacterial membrane. That was not expected because R. rubrum did not grow fermentatively under the conditions used. Low concentrations of protonophores were more effective in dissipating the anaerobic than the aerobic membrane potential. That observation indicated a lower activity of the electrogenic system responsible for the anaerobic potential. In consequence, the addition of uncouplers at low levels resulted in a marked enhancement of the membrane potential decrease which followed the transition between the aerobic and the anaerobic steady states.     

Penetrating Cations Enhance Uncoupling Activity of Anionic Protonophores in Mitochondria

Protonophorous uncouplers causing a partial decrease in mitochondrial membrane potential are promising candidates for therapeutic applications. Here we showed that hydrophobic penetrating cations specifically targeted to mitochondria in a membrane potential-driven fashion increased proton-translocating activity of the anionic uncouplers 2,4-dinitrophenol (DNP) and carbonylcyanide-p-trifluorophenylhydrazone (FCCP). In planar bilayer lipid membranes (BLM) separating two compartments with different pH values, DNP-mediated diffusion potential of H⁺ ions was enhanced in the presence of dodecyltriphenylphosphonium cation (C₁₂TPP). The mitochondria-targeted penetrating cations strongly increased DNP- and carbonylcyanide m-chlorophenylhydrazone (CCCP)-mediated steady-state current through BLM when a transmembrane electrical potential difference was applied. Carboxyfluorescein efflux from liposomes initiated by the plastoquinone-containing penetrating cation SkQ1 was inhibited by both DNP and FCCP. Formation of complexes between the cation and CCCP was observed spectophotometrically. In contrast to the less hydrophobic tetraphenylphosphonium cation (TPP), SkQ1 and C₁₂TPP promoted the uncoupling action of DNP and FCCP on isolated mitochondria. C₁₂TPP and FCCP exhibited a synergistic effect decreasing the membrane potential of mitochondria in yeast cells. The stimulating action of penetrating cations on the protonophore-mediated uncoupling is assumed to be useful for medical applications of low (non-toxic) concentrations of protonophores.     

Carrier-mediated Potassium Efflux Across the Cell Membrane of Red Beet

The flux ratio (influx/efflux) of K⁺ across the plasmalemma of beet cells at an external potassium concentration of 0.6 mm does not respond to changes of membrane potential in the manner expected for the free diffusion of ions. The K⁺ efflux is affected by the presence of adsorbed Ca²⁺, but is apparently unrelated to the electrical potential or to the net uptake of potassium. The K⁺ efflux is greater than the efflux of the sulfate and organic anions which are accumulated with potassium, and is partially dependent on the presence of external potassium. Thus the loss of ⁴²K from the cell does not appear to be a leakage of freely diffusing K⁺ ions, nor a leakage of ion pairs, but a carrier-mediated transport or exchange of potassium across the cell membrane.     

Functional identification of ATP-sensitive K<Superscript>+</Superscript> uniporter in mitochondria from sugar beet taproot

Mitochondria isolated from sugar beet (Beta vulgaris L.) taproot were shown to swell spontaneously after the transfer from a sucrose-containing isolation medium to isoosmotic potassium chloride solutions. The kinetics of this process was strongly retarded after the replacement of potassium with sodium in the incubation medium and was substantially stimulated by the electron-transport chain activity and valinomycin. At neutral pH of the incubation medium, the rate of K⁺-dependent swelling of mitochondria decreased by 30–50% after adding 1 mM ATP but was insensitive to other nucleotides (GTP, UTP, and CTP). In the medium acidified to pH 6.0, the addition of ATP caused shrinkage of mitochondria that had been swollen in the KCl medium. In the absence of this nucleotide, the kinetics of K⁺-dependent swelling of mitochondria was considerably decelerated upon the acidification of the incubation medium. The effects of ATP were independent of the presence or absence of oligomycin and atractyloside. However, the ATP-dependent shrinkage of mitochondria was inhibited in the presence of quinine, and this agent also inhibited K⁺-dependent swelling of organelles in potassium acetate solutions. The presence of K⁺ ions in the incubation medium caused a rapid dissipation of the mitochondrial membrane potential () that was generated during succinate oxidation. The addition of ATP to the reaction medium resulted in the oligomycin-insensitive restoration of . The results are regarded as evidence that the membrane of taproot mitochondria is endowed with functionally active ATP-sensitive K⁺ uniporter. This system is likely to represent a K⁺ channel that catalyzes the electrogenic transfer of potassium ions to the mitochondrial matrix. It is supposed that the membrane of taproot mitochondria also contains a quinine-sensitive K⁺/H⁺ antiporter that catalyzes the efflux of potassium from the matrix or, on the contrary, the accumulation of K⁺ in the presence of potassium acetate.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 209–215.Original Russian Text Copyright © 2005 by Shugaev, Andreev, Vyskrebentseva.This revised version was published online in April 2005 with a corrected cover date.     

Effect of avermectin on the ultrastructural characteristics of loach embryos

The ultrastructure of loach (Misgurnus fossilis L.) embryo cells at the stages of the first and tenth blastomere divisions in the presence of avermectin B (a compound that belongs to the class of macrocyclic lactones, avermectins) in an incubation medium at concentrations of 0.01, 0.1, and 1.0 μg/ml was studied. It was found that the effect of this compound led to ultrastructural changes in cell organelles, such as hypertrophy of the granular and agranular endoplasmic reticulum and disorganization of the mitochondria and the embryo plasma membrane. Avermectin activity causes dose-dependent destructive changes in organelles; such changes are the consequences of the destruction of metabolic and regulatory processes, caused by the inhibitory influence of avermectin on the processes of active transport of Na⁺, K⁺ and Ca²⁺ ions. The data obtained in this study show that avermectin is characterized by high embryotoxicity.     

Ultrastructural localization of carbonic anhydrase activity in the rat choroid plexus epithelial cell

Summary Localization of carbonic anhydrase activity was studied electron microscopically on cells of the rat choroid plexus epithelium. For the ultracytochemical detection of these activities, Yokota's technique (1969), which is the modification of Hansson's method (1967) was employed. Numerous electron dense reaction products were observed in the microvilli of the choroidal epithelial cell. The reaction deposits were also remarkably present in the infoldings of the basal plasmalemma but to a lesser extent than in the microvilli. The localization sites were mainly on the plasma membrane, but some reaction products were also observed in the cytoplasm near the plasma membrane. Hardly any reaction product was found in the intracellular organelles except for the mitochondria in which reaction products were occasionally observed on the cristae. These activities were completely inhibited by acetazolamide. As the carbonic anhydrase activity was histochemically seen in the microvilli and the basal infoldings, it is likely that carbonic anhydrase is related to an active transport process in the secretion of cerebrospinal fluid as is Na⁺, K⁺-ATPase (Masuzawa et al. 1980).     

Ultracytochemical study of Ca++-ATPase and K+-NPPase activities in retinal photoreceptors of the guinea pig

Summary Ca⁺⁺-ATPase activity was demonstrated histochemically at light- and electron-microscopic levels in inner and outer segments of retinal photoreceptor cells of the guinea pig with the use of a newly developed one-step lead-citrate method (Ando et al. 1981). The localization of ouabain-sensitive, K⁺-dependent p-nitrophenylphosphatase (K⁺-NPPase) activity, which represents the second dephosphorylative step of the Na⁺-K⁺-ATPase system, was studied by use of the one-step method newly adapted for ultracytochemistry (Mayahara et al. 1980). In retinal photoreceptor cells fixed for 15 min in 2% paraformaldehyde the electron-dense Ca⁺⁺-ATPase reaction product accumulated significantly on the inner membranes of the mitochondria but not on the plasmalemma or other cytoplasmic elements of the inner segments. The membranes of the outer segments remained unstained except the membrane arrays in close apposition to the retinal pigment epithelium. The cytochemical reaction was Ca⁺⁺- and substrate-dependent and showed sensitivity to oligomycin. When Mg⁺⁺-ions were used instead of Ca⁺⁺-ions, a distinct reaction was also found on mitochondrial inner membranes.In contrast to the localization of the Ca⁺⁺ -ATPase activity, the K⁺-NPPase activity was demonstrated only on the plasmalemma of the inner segments, but not on the mitochondria, other cytoplasmic elements or the outer segment membranes. This reaction was almost completely abolished by ouabain or by elimination of K⁺ from the incubation medium.Fellow of the Alexander von Humboldt Foundation, Bonn, Federal Republic of Germany     

Mitochondrial membrane potential regulates matrix configuration and cytochrome c release during apoptosis

During apoptosis, the mitochondrial membrane potential (MMP) decreases, but it is not known how this relates to the apoptotic process. It was recently suggested that cytochrome c is compartmentalized in closed cristal regions and therefore, matrix remodeling is required to attain complete cytochrome c release from the mitochondria. In this work we show that, at the onset of apoptosis, changes in MMP control matrix remodeling prior to cytochrome c release. Early after growth factor withdrawal the MMP declines and the matrix condenses. Both phenomena are reversed by adding oxidizable substrates. In mitochondria isolated from healthy cells, matrix condensation can be induced by either denying oxidizable substrates or by protonophores that dissipate the membrane potential. Matrix remodeling to the condensed state results in cristal unfolding and exposes cytochrome c to the intermembrane space facilitating its release from the mitochondria during apoptosis. In contrast, when a transmembrane potential is generated due to either electron transport or a pH gradient formed by acidifying the medium, mitochondria maintain an orthodox configuration in which most cytochrome c is sequestered in the cristae and is resistant to release by agents that disrupt the mitochondrial outer membrane.     

Characterization of potassium-dependent currents in protoplasts of corn suspension cells

Protoplasts obtained from corn (Zea mays) suspension cells were studied using the whole cell patch-clamp technique. One time-independent current, as well as two time-dependent currents were identified. All three currents were reduced by tetraethylammonium (9 millimolar), a K⁺ channel blocker. The time-independent current had a nearly linear current-voltage relationship and its reversal potential, defined as the voltage at which there is zero current, was highly dependent on the extracellular potassium concentration. One of the two time-dependent currents was activated, with rapid kinetics, by membrane hyperpolarization to potentials more negative than −100 millivolts. The second time-dependent current was activated with a sigmoidal time course by membrane depolarization to potentials more positive than −60 millivolts. It exhibited no inactivation and was carried primarily by potassium ions. These characteristics suggest that this latter current is caused by the voltage-dependent opening of delayed-rectifier K⁺ channels. These three currents, which are not generated by the plasmalemma H⁺-ATPase, are likely to assist in the regulation of the cellular K⁺ fluxes and membrane potential.     

Über hydrophobe Acridinfarbstoffe zur Fluorochromierung von Mitochondrien in lebenden Zellen

Summary The hydrophobic fluorescence dye 10-n-nonyl-acridinium-orange-chloride, NAO, stains specifically the mitochondria of living HeLa-cells. A dye concentration of 1·10^–8 M is sufficient for vital staining and at 5·10^–7 M an incubation time less than 1 min is enough to generate the bright green fluorescence of the mitochondria. The retention of NAO by the mitochondria is longer than 7 days.The dye accumulation is not affected by the ionophores valinomycin, nigericin, gramicidin, the uncoupling agents DNP, CCCP or by ouabain. In contrast to Rh 123 the trans-membrane potential is not the driving force of the NAO accumulation. We assume that NAO is bound to the hydrophobic lipids and proteins in the mitochondrial membranes by hydrophobic interaction.With valinomycin, 500 ng/ml, 10 min, the mitochondria in HeLa-cells swell. Now it is possible to observe some details in the enlarged mitochondria by light microscopy. After vital staining with NAO, 5·10^–7 M, 10 min, the periphery of the swollen mitochondria shows an intense green fluorescence, the inner part is dark. Obviously the dye is bound to the membranes. By electron microscopy it can be shown that the valinomycin treated and NAO stained mitochondria have outer and inner membranes and cristae. They differ from untreated mitochondria mainly in the size.After incubation of the HeLa-cells with relatively high NAO concentrations, 5·10^–6 M, 10 min, the mitochondria show a weak orange fluorescence. It is generated by the dimers D of NAO. Therefore the dye concentration in the mitochondrial membranes is locally very high and causes dye dimerisation. The weak orange fluorescence is instable and disappeares within a few seconds. Instead we observe a green fluorescence with growing intensity that is generated by the monomers M of NAO. The intensity has its maximum value after a few seconds. Using low NAO concentrations for incubation, 1·10^–7 M, 10 min, we observe only the green fluorescence with increasing intensity. In this case the orange fluorescence is too weak for observation (concentration quenching). It can be shown by experiments and quantum mechanics that the orange fluorescence is assigned to an optical forbidden, the green fluorescence to an allowed electronic transition of D or M respectively. Our results indicate a dissoziation of D in 2 M by irradiation of the mitochondria under the fluorescence microscope.The intensity changes of the orange and the green fluorescence of bound D and M by irradiation has been measured in living cells with a microspectrophotometer. The experimental data agree quantitatively with a first-order reaction mechanism for the dissoziation of D in 2 M by irradiation. There is some evidence for energy transfer between dimers at higher NAO concentration.The oxygen consumption of HeLa-cell suspensions has been measured electrochemically at various NAO concentrations and incubation times with an oxygen electrode. Up to 5·10^–7 M NAO, 10 min, the respiratory activity is not affected. After that we observe an increasing inhibition of the oxygen consumption with growing NAO concentration and incubation time. At 5·10^–6 M, 30 min, the inhibition is 40% relative to the untreated cells.The ultrastructure of the mitochondria in incubated HeLa-cells has been investigated by electron microscopy and compared with untreated cells. Similar to the resiratory experiments there is no difference in ultrastructure up to 5·10^–7 M NAO, 10 min. Then the ultrastructure changes rapidly with increasing NAO concentration and incubation time. At the final stage, 5·10^–6 M, 1 h, the cristae totally or partially disappeared. The outer and inner membranes are still visible. Obviously the mitochondria without cristae are instable and collapse. They change into liposomes with stacks of four, eight and more membranes on the periphery. They enclose cytoplasm. The genesis of the liposomes is discussed in some detail.These experiments show that the dye NAO is accumulated at the inner mitochondrial membrane and the cristae. It blocks the enzymes of the oxydative phosphorylation in the inner membranes and affects the self-organization of the cristae. NAO is specifically bound to the membranes of the mitochondria. Neither by fluorescence microscopy nor by electron microscopy we observe binding of NAO to the membranes of the nuclei.     

Is the ATP — dependent protection of lysosomes against osmotic lysis a function of the lysosomal proton pump

Summary Rat liver lysosomes have been used to characterize further the effects of ATP on lysosomal stability during incubation at 37°C at hypo-osmolarity. As previously reported, when the osmotically-supporting solute is the salt of a strong base (K⁺), ATP protects against lysis during incubation. However, if the osmotically-supporting solute is the salt of a weak base, e.g. Tris HCl or NH₄Cl, ATP actually promotes lysis during incubation. Thus, ATP can exert destabilizing as well as protective effects on lysosomes. The destabilizing effect is eliminated by protonophores. The protective effect in the presence of potassium salts is not eliminated by protonophores. Moreover, when incubation is in the presence of a salt of a weak base, protonophores actually cause an ATP-dependent protective effect to be established. The destabilizing effect occurs at 37°C, but not at 0°C. The Mg^–+-dependence of the destabilizing effect was found to be similar to that found earlier for the ATP-dependent protective effect, insofar as only 1 mM MgCl₂ in the presence of 1 mM EDTA is sufficient for nearly maximal stimulation of both effects. The destabilizing effect may result from a H^– ion gradient across the lysosomal membrane which is maintained by the lysosomal ATP-dependent proton pump. The protective effect, on the other hand, does not depend on such a gradient being maintained; on the contrary, protonophores appear to act as enablers of the protective effect. The question that remains to be answered is: does the protective effect derive in some way from the same ATP-driven mechanism which constitutes the proton pump? Some possible answers to this question are considered.Abbreviations Mops 2-(N-morpholine)-propanesulfonic acid - CCCP Carbonyl cyanide m-chlorophenylhydrazone - DNP 2,4-Dinitrophenol - EDTA Ethylenediaminetetracetic acid     

Effects of human T-cell leukemia virus type 1 (HTLV-1) p13 on mitochondrial K permeability: A new member of the viroporin family?

Human T-cell leukemia virus type-1 (HTLV-1) encodes a mitochondrial protein named p13. p13 mediates an inward K⁺ current in isolated mitochondria that leads to mitochondrial swelling, depolarization, increased respiratory chain activity and reactive oxygen species (ROS) production. These effects trigger the opening of the permeability transition pore and are dependent on the presence of K⁺ and on the amphipathic alpha helical domain of p13. In the context of cells, p13 acts as a sensitizer to selected apoptotic stimuli. Although it is not known whether p13 influences the activity of endogenous K⁺ channels or forms a channel itself, it shares some structural and functional analogies with viroporins, a class of small integral membrane proteins that form pores and alter membrane permeability.     

The effect of acetylcholine on Characeae K+ channels at rest and during action potential generation

The role of acetylcholine (ACh) as a signalling molecule in plants was investigated using a model system of Characeae cells. The effect of ACh on conductance of K⁺ channels in Nitella flexilis cells and on the action potential generation in Nitellopsis obtusa cells after H⁺-ATPase inhibition, where repolarization occurs after the opening of outward rectifying K⁺ channels, was investigated. Voltage-clamp method based on only one electrode impalement was used to evaluate the activity of separate potassium ion transport system at rest. We found that ACh at high concentrations (1 mM and 5 mM) activates K⁺ channels as the main membrane transport system at the resting state involved in electrogenesis of Characeaen membrane potential. We observed that ACh caused an increase in duration of AP repolarization of cells in K⁺ state when plasmalemma electrical characteristics are determined by large conductance K⁺ channels irrespective of whether AP were spontaneous or electrically evoked. These results indicate interference of ACh with electrical cellular signalling pathway in plants.