GRAMICIDIN AND ION TRANSPORT IN ISOLATED LIVER MITOCHONDRIA.
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ABSTRACT:
SUBMITTER: CHAPPELL JB
PROVIDER: S-EPMC1214336 | biostudies-other | 1965 May
REPOSITORIES: biostudies-other
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Project description:Glutathione (GSH) is transported into renal mitochondria by the dicarboxylate (DIC; Slc25a10) and 2-oxoglutarate carriers (OGC; Slc25a11). To determine whether these carriers function similarly in liver mitochondria, we assessed the effect of competition with specific substrates or inhibitors on GSH uptake in isolated rat liver mitochondria. GSH uptake was uniphasic, independent of ATP hydrolysis, and exhibited K(m) and V(max) values of 4.08 mM and 3.06 nmol/min per mg protein, respectively. Incubation with butylmalonate and phenylsuccinate inhibited GSH uptake by 45-50%, although the individual inhibitors had no effect, suggesting in rat liver mitochondria, the DIC and OGC are only partially responsible for GSH uptake. H4IIE cells, a rat hepatoma cell line, were stably transfected with the cDNA for the OGC, and exhibited increased uptake of GSH and 2-oxoglutarate and were protected from cytotoxicity induced by H(2)O(2), methyl vinyl ketone, or cisplatin, demonstrating the protective function of increased mitochondrial GSH transport in the liver.
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Project description:1. Liver mitochondria suspended in an iso-osmotic buffered potassium chloride medium containing an oxidizable substrate and phosphate accumulated added Ca(2+). During this process H(+) appeared in the medium and the mitochondrial suspension showed increased light-scattering. Respiration was markedly stimulated. 2. The addition of excess of Ca(2+), respiratory inhibitors or uncoupling agents caused extensive mitochondrial swelling associated with release of Ca(2+) into the suspending medium. When the suspension became anaerobic extensive swelling also occurred. Only under conditions when the addition of uncoupling agents would have produced high rates of electron transport, e.g. in the presence of succinate, was the structural integrity of the mitochondrion maintained after Ca(2+) accumulation. 3. Conditions that prevented respiration-dependent Ca(2+) accumulation also prevented Ca(2+)-induced swelling. Bovine plasma albumin was without effect, indicating that U-factor was not involved. Oligomycin together with ADP or ATP partially stabilized the mitochondria against Ca(2+)-induced swelling. 4. It is suggested that a ;high-energy' intermediate generated by coupled electron transport is required to prevent the mitochondrial swelling that results as a consequence of Ca(2+) accumulation.
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Project description:Ion channels are key proteins in mammalian cell membranes. They have a central role in the physiology of excitable cells such as neurons, muscle, and heart cells. They also play a crucial role in kidney physiology. The gramicidin ion channel is one of the most studied ion channels, in particular it was intensively employed to investigate the lipid–protein interactions in model cell membranes. For example, even though the sequence of gramicidin is extremely hydrophobic, its motion is impaired in membrane bilayer, i.e., it does not rapidly flip to the other membrane leaflet, and low channel activity were observed when gramicidin is added asymmetrically to only one leaflet of a model cell membrane. In this article, we study the transport properties of gramicidin channel in a heterogeneous model membrane. Using microfluidics, we are forming freestanding bilayers as model cell membranes including heterogeneous domains that are created by oil inclusions. The presence of oil inclusions is then demonstrated by measuring the bilayer capacity via a patch-clamp amplifier and fluorescent confocal inspection. Based on electrophysiological and optical measurements Gramicidin A (gA) ion channels are dispersed into the buffer phases on both side of the formed lipid bilayer and insert spontaneously into the bilayer upon formation. The presence of functional Gramicidin A is then demonstrated by measuring conductivity signals. Based on electrophysiological and optical measurements, we explore the consequence of the presence of these oil inclusions on the functionality of incorporated gA ion channels. For low oil concentration, we measure a decrease of gA transport properties due to the reduction of the bilayer tension. For large oil concentration, we measure a saturation of gA transport properties due to an increase of the bilayer thickness.
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Project description:1. The excessive accumulation of Ca(2+) by mitochondria suspended in an iso-osmotic buffered potassium chloride medium containing oxidizable substrate and phosphate led to extensive swelling and release of accumulated Ca(2+) from the mitochondria. When the Ca(2+) was removed from the medium by chelation with ethylene glycol bis(aminoethyl)tetra-acetate, the swelling was reversed in a respiration-dependent contraction. The contracted mitochondria were shown to have regained some degree of respiratory control. 2. The respiration-dependent contraction could be supported by electron transport through a restricted portion of the respiratory chain, and by substrates donating electrons at different levels in the respiratory chain. 3. Respiratory inhibitors appropriate to the substrate present completely inhibited the contraction. Uncoupling agents, and the inhibitors oligomycin and atractyloside, were without effect. 4. When the reversal of swelling had been prevented by respiratory inhibitors, the addition of ATP induced a contraction of the mitochondria. In the absence of added chelating agent the contraction was very slow. The ATP-induced contraction was completely inhibited by oligomycin and atractyloside, was incomplete in the presence of uncoupling agents and was unaffected by respiratory inhibitors. 5. The relationship between the energy requirements of respiration-dependent contraction and the requirements of ion transport and other contractile systems are discussed.
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Project description:The free energy governing K(+) conduction through gramicidin A channels is characterized by using over 0.1 micros of all-atom molecular dynamics simulations with explicit solvent and membrane. The results provide encouraging agreement with experiments and insights into the permeation mechanism. The free energy surface of K(+), as a function of both axial and radial coordinates, is calculated. Correcting for simulation artifacts due to periodicity and the lack of hydrocarbon polarizability, the calculated single-channel conductance for K(+) ions is 0.8 pS, closer to experiment than any previous calculation. In addition, the estimated single ion dissociation constants are within the range of experimental determinations. The relatively small free energy barrier to ion translocation arises from a balance of large opposing contributions from protein, single-file water, bulk electrolyte, and membrane. Mean force decomposition reveals a remarkable ability of the single-file water molecules to stabilize K(+) by -40 kcal/mol, roughly half the bulk solvation free energy. The importance of the single-file water confirms the conjecture of Mackay et al. [Mackay, D. H. J., Berens, P. H., Wilson, K. R. & Hagler, A. T. (1984) Biophys. J. 46, 229-248]. Ion association with the channel involves gradual dehydration from approximately six to seven water molecules in the first shell, to just two inside the narrow pore. Ion permeation is influenced by the orientation of the single-file water column, which can present a barrier to conduction and give rise to long-range coupling of ions on either side of the pore. Small changes in the potential function, including contributions from electronic polarization, are likely to be sufficient to obtain quantitative agreement with experiments.
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Project description:1. Addition of N-ethylmaleimide to rat liver mitochondria respiring with succinate as substrate decreases both the initial rate of Ca(2+) transport and the ability of mitochondria to retain Ca(2+). As a result, Ca(2+) begins to leave the mitochondria soon after it has entered. Half-maximal effects occur at an N-ethylmaleimide concentration of about 100nmol/mg of protein. 2. The efflux of Ca(2+) induced by N-ethylmaleimide is not prevented by Mg(2+) or by Ruthenium Red at concentrations known to prevent Ca(2+) efflux when exogenous phosphate also is present. Swelling of mitochondria does not accompany N-ethylmaleimide-induced Ca(2+) efflux. 3. Addition of Ca(2+) to rat liver mitochondria in the presence of N-ethylmaleimide produces an immediate decrease in DeltaE (membrane potential), which decreases further to only a slight extent over the next 8min. Concomitant with this is an immediate increase and then levelling off of the -59DeltapH (transmembrane pH gradient). 4. Preincubation of rat liver mitochondria with p-chloromercuribenzenesulphonate, which by contrast with N-ethylmaleimide is unable to penetrate the inner mitochondrial membrane, also prevents Ca(2+) retention. The DeltaE and -59DeltapH respond to Ca(2+) addition in a manner similar to that which occurs when N-ethylmaleimide is present. Subsequent addition of mercaptoethanol produces an immediate increase in both DeltaE and -59DeltapH. At the same time Ca(2+) is rapidly accumulated by the organelles. 5. The above data are interpreted as indicating that under the conditions of Ca(2+) efflux seen here, the mitochondria retain their functional integrity. This contrasts with the uncoupling effect of Ca(2+) seen in the presence of P(i), which generally leads to a loss of mitochondrial integrity. We suggest that a unique mechanism of Ca(2+) cycling is able to take place when mitochondria have been treated with N-ethylmaleimide.
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Project description:The EGTA (ethanedioxybis(ethylamine)tetra-acetic acid)-Ruthenium Red-quench technique (Reed & Bygrave, 1974a) was used to measure initial rates of Ca-2+ transport in mitochondria from flight muscle of the blowfly Lucilia cuprina. Evidence is provided for the existence in these mitochondria of a Ca-2+-transport system that has many features in common with that known to exist in rat liver mitochondria. These include requirement for energy, saturation at high concentrations of Ca-2+, a sigmoidal relation between initial rates of Ca-2+ transport and Ca-2+ concentration, a high affinity for free Ca-2+ (Km approx. 5 muM) and high affinity for the Ca-2+-transport inhibitoy, Ruthenium Red (approx. 0.03 nmol of carrier-specific binding-sites/mg of protein; Ki approx. 1.6 x 10- minus 8 M). Controlled respiration can be stimulated by Ca-2+ after a short lag-period provided the incubation medium contains KCl and not sucrose. The ability of Lucilia mitochondria to transport Ca-2+ critically depends on the stage of mitochondrial development; Ca-2+ transport is minimal in mitochondria from pharate adults, is maximal between 0 and 2h post-emergence and thereafter rapidly declines to reach less than 20% of the maximum value by about 2-3 days post-emergence. Respiration in mitochondria from newly emerged flies does not respond to added Ca-2+; that from 3-5-day-old flies is stimulated approx. 50%. Whereas very low concentrations of Ca-2+ inhibit ADP-stimulated respiration and oxidative phosphorylation in mitochondria from newly emerged flies (Ki approx. 60 ng-ions of Ca-2+/mg of protein); much higher concentrations (approx. 200 ng-ion/mg of protein) are needed to inhibit these processes in those from older flies. The potential of this system for studying the function and development of metabolite transport systems in mitochondria is discussed.
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Project description:All-atom molecular dynamics simulations have been applied in the recent past to explore the free energetics underlying ion transport processes in biological ion channels. Roux and co-workers, Kuyucak and co-workers, Busath and co-workers, and others have performed rather elegant and extended time scale molecular dynamics simulations using current state-of-the-art fixed-charge (nonpolarizable) force fields to calculate the potential of mean force defining the equilibrium flux of ions through prototypical channels such as gramicidin A. An inescapable conclusion of such studies has been the gross overestimation of the equilibrium free energy barrier, generally predicted to be from 10 to 20 kcal/mol depending on the force field and simulation protocol used in the calculation; this translates to an underestimation of experimentally measurable single channel conductances by several orders of magnitude. Next-generation polarizable force fields have been suggested as possible alternatives for more quantitative predictions of the underlying free energy surface in such systems. (1) Presently, we consider ion permeation energetics in the gramicidin A channel using a novel polarizable force field. Our results predict a peak barrier height of 6 kcal/mol relative to the channel entrance; this is significantly lower than the uncorrected value of 12 kcal/mol for nonpolarizable force fields such as GROMOS and CHARMM27 which do not account for electronic polarization. These results provide promising initial indications substantiating the long-conjectured importance of polarization effects in describing ion-protein interactions in narrow biological channels.
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