ATP synthase complex from beef heart mitochondria. Role of the thiol group of the 25-kDa subunit of Fo in the coupling mechanism between Fo and F1.

In order to assess the role of thiol groups in the Fo part of the ATP synthase in the coupling mechanism of ATP synthase, we have treated isolated Fo, extracted from beef heart Complex V with urea, with thiol reagents, primarily with diazenedicarboxylic acid bis-(dimethylamide) (diamide) but also with Cd2+ and N-ethylmaleimide. FoF1 ATP synthase was reconstituted by adding isolated F1 and the oligomycin-sensitivity-conferring-protein (OSCP) to Fo. The efficiency of reconstitution was assessed by determining the sensitivity to oligomycin of the ATP hydrolytic activity of the reconstituted enzyme. Contrary to Cd2+, incubation of diamide with Fo, before the addition of F1 and OSCP, induced a severe loss of oligomycin sensitivity, due to an inhibited binding of F1 to Fo. This effect was reversed by dithiothreitol. Conversely, if F1 and OSCP were added to Fo before diamide, no effect could be detected. These results show that F1 (and/or OSCP) protects Fo thiols from diamide and are substantiated by the finding that the oligomycin sensitivity of ATP hydrolysis activity of isolated Complex V was also unaltered by diamide. Gel electrophoresis of FoF1 ATP synthase, reconstituted with diamide-treated Fo, revealed that the loss of oligomycin sensitivity was directly correlated with diminution of band Fo 1 (or subunit b). Concomitantly a band appeared of approximately twice the molecular weight of subunit Fo 1. As this protein contains only 1 cysteine residue (Walker, J. E., Runswick, M. J., and Poulter, L. (1987) J. Mol. Biol. 197, 89-100), the effect of diamide is attributed to the formation of a disulfide bridge between two of these subunits. These results offer further evidence for the proposal, based on aminoacid sequence and structural analysis, that subunit Fo 1 of mammalian Fo is involved in the binding with F1 (Walker et al. (1987]. N-Ethylmaleimide affects oligomycin sensitivity to a lesser extent than diamide, suggesting that the mode of action of these reagents (and the structural changes induced in Fo) is different

The prion protein regulates glutamate-mediated Ca2+ entry and mitochondrial Ca2+ accumulation in neurons

The cellular prion protein (PrPC) whose conformational misfolding leads to the production of deadly prions, has a still-unclarified cellular function despite decades of intensive research. Following our recent finding that PrPC limits Ca2+ entry via store-operated Ca2+ channels in neurons, we investigated whether the protein could also control the activity of ionotropic glutamate receptors (iGluRs). To this end, we compared local Ca2+ movements in primary cerebellar granule neurons and cortical neurons transduced with genetically encoded Ca2+ probes and expressing, or not expressing, PrPC. Our investigation demonstrated that PrPC downregulates Ca2+ entry through each specific agonist-stimulated iGluR and after stimulation by glutamate. We found that, although PrP-knockout (KO) mitochondria were displaced from the plasma membrane, glutamate addition resulted in a higher mitochondrial Ca2+ uptake in PrP-KO neurons than in their PrPC-expressing counterpart. This was because the increased Ca2+ entry through iGluRs in PrP-KO neurons led to a parallel increase in Ca2+-induced Ca2+ release via ryanodine receptor channels. These data thus suggest that PrPC takes part in the cell apparatus controlling Ca2+ homeostasis, and that PrPC is involved in protecting neurons from toxic Ca2+ overloads

Selective and Efficient Immunoprecipitation of the Disease-associated Form of the Prion Protein Can Be Mediated by Nonspecific Interactions between Monoclonal Antibodies and Scrapie-associated Fibrils

Transmissible spongiform encephalopathies are characterized by the accumulation in brain tissues of an abnormal isoform of the prion protein named PrPsc, which is the only direct marker known for transmissible spongiform encephalopathies. Here we show that PrPsc can be specifically immunoprecipitated by using several monoclonal antibodies (mAbs) of various specificities independently of the properties of their binding site (paratope). These results strongly suggest that a significant proportion of mAbs can interact with PrPsc aggregates through nonspecific paratope-independent interactions allowing selective immunoprecipitation of PrPsc when these mAbs are immobilized on a polydisperse solid phase like microbeads

High conductance pathways in mitochondrial membranes

The outer and inner membranes of mitochondria have recently been studied with the patch clamp technique. What has emerged is still an ill-defined picture for either membrane, primarily for the wide range of conductances found. Interestingly, however, a few conductances (in the range of 10-80 pS) seem to be ubiquitously distributed. Parallel studies in situ and in reconstituted systems have allowed the assignment to distinct membrane locations of some conductances, whose physiological role is, however, not yet elucidated

From cell protection to death: may Ca2+ signals explain the chameleonic attributes of the mammalian prion protein?

It is now accepted that a conformational change of the cellular prion protein (PrP(C)) generates the prion, the infectious agent responsible for lethal neurodegenerative disorders, named transmissible spongiform encephalopathies, or prion diseases. The mechanisms of prion-associated neurodegeneration are still obscure, as is the cell role of PrP(C), although increasing evidence attributes to PrP(C) important functions in cell survival. Such a behavioral dichotomy thus enables the prion protein to switch from a benign role under normal conditions, to the execution of neurons during disease. By reviewing data from models of prion disease and PrP(C)-null paradigms, which suggest a relation between the prion protein and Ca(2+) homeostasis, here we discuss the possibility that Ca(2+) is the factor behind the enigma of the pathophysiology of PrP(C). Ca(2+) features in almost all processes of cell signaling, and may thus tell us much about a protein that pivots between health and disease

Variable proton conductance of submitochondrial particles.

The relationship between the rate of substrate oxidation and the protonmotive force (electrochemical proton gradient) generated by bovine heart submitochondrial particles has been examined. Unexpectedly, oxidation of succinate generated a higher protonmotive force than the oxidation of NADH, although the rate of proton translocation across the membrane was inferred to be considerably lower with succinate as substrate. The data suggest that the flow of electrons through site 1 of the respiratory chain may increase the conductance of the mitochondrial membrane for protons. Upon reduction of the rate of succinate oxidation by titration with malonate, the protonmotive force remained essentially constant until the extent of inhibition was greater than 75%. The general conclusion from this work is that a constant passive membrane conductance for protons cannot be assumed

Anion channels of the inner membrane of mammalian and yeast mitochondria

The inner membrane of yeast and mammalian mitochondria has been studied in situ with a patch clamp electrode. Anion channels were found in both cases, although their behavior and regulation are different. In mammalian mitochondria, the principal channel is of around 100 pS conductance and opens mainly under depolarized membrane potentials. As no physiological compound able to alter its peculiar voltage dependence has yet been found, it is proposed that this channel may serve as a safeguard mechanism for recharging the mitochondrial membrane potential. Two other anion channels, each with a distinct conductance (one of approx. 45 pS, the second of at least a tenfold higher value) and kinetics are harbored in the yeast inner membrane. Matrix ATP was found to interact with both, but with a different mechanism. It is proposed that the 45 pS channel may be involved in the homeostatic mechanism of mitochondrial volume

An Electrophysiological study of yeast mitochondria. Evidence for two inner membrane anion channels sensitive to ATP

The inner membrane of mitochondria from various strains of Saccharomyces cerevisiae has been analyzed with the patch clamp technique for comparison with the better known homologous membrane in mammals (Sorgato, M. C., and Moran, O.(1993) CRC Crit. Rev. Biochem. Mol. Biol. 18, 127-171). Differently than in mammals, the yeast inner membrane was found to harbor essentially two channels with similar anionic selectivity but otherwise different functional behavior. One had a conductance of around 45 picosiemens (in symmetrical 150 mM KCl) and an activity only marginally sensitive to voltage. The other channel was prominent for the higher outwardly rectifying current and for the dependence upon voltage of the open probability that induced rapid closure at physiological (negative) membrane potentials. Particularly interesting was the effect of ATP (Mg free) added on the matrix side of the membrane. In the case of the lower conducting channel, the nucleotide caused an immediate block of activity (IC, 0.240 mM), whereas it locked the larger conductance in the open state at both positive and negative potentials. In proteoliposomes containing both mitochondrial membranes, the small conductance was clearly evident, whereas a larger channel, cationic and without the voltage dependence typical of that in the native inner membrane, was found

Mitochondrial Channels

Eukaryotic cells are living organisms surrounded by a surface membrane, which also house other membranes that define intracellular organelles. Membranes are lipidic structures impermeable to hydrophilic molecules (polar or charged); this is why they harbour proteins, called ion channels and carriers, catalysing the life-requiring exchange of material between a cell and the external space, and between organelles and the cytoplasm. At variance from carriers, ion channels form aqueous pores crossing the lipid bilayer that allow the highly selective transmembrane passage of charged species, namely inorganic ions (e.g. Na+, K+, Ca2+, Cl-), with high potency (105-108 ions per second are transported by a single molecule); they also possess regulatory domains that open and close the pore upon specific stimuli (electric or chemical). Mitochondria are organelles composed of two membranes, in either of which channels are present. However, while channels in the outer membrane (OM) are justified by its the overall high permeability, the finding of these entities in the inner membrane (IM) was unexpected in view of its implication in the process of oxidative phosphorylation that imposes an extremely controlled permeability to ions. After the initial phenomenological description, substantial advances in the functional - if not molecular - identification of several mitochondrial channels, disclose the possibility that they take part in crucial schemes of mitochondrial functionality, as well as in dramatic cell events