Antagonistic Regulation of Native Ca2+- and ATP-sensitive Cation Channels in Brain Capillaries by Nucleotides and Decavanadate

Regulation by cytosolic nucleotides of Ca2+- and ATP-sensitive nonselective cation channels (CA-NSCs) in rat brain capillary endothelial cells was studied in excised inside-out patches. Open probability (Po) was suppressed by cytosolic nucleotides with apparent KI values of 17, 9, and 2 μM for ATP, ADP, and AMP, as a consequence of high-affinity inhibition of channel opening rate and low-affinity stimulation of closing rate. Cytosolic [Ca2+] and voltage affected inhibition of Po, but not of opening rate, by ATP, suggesting that the conformation of the nucleotide binding site is influenced only by the state of the channel gate, not by that of the Ca2+ and voltage sensors. ATP inhibition was unaltered by channel rundown. Nucleotide structure affected inhibitory potency that was little sensitive to base substitutions, but was greatly diminished by 3′-5′ cyclization, removal of all phosphates, or complete omission of the base. In contrast, decavanadate potently (K1/2 = 90 nM) and robustly stimulated Po, and functionally competed with inhibitory nucleotides. From kinetic analyses we conclude that (a) ATP, ADP, and AMP bind to a common site; (b) inhibition by nucleotides occurs through simple reversible binding, as a consequence of tighter binding to the closed-channel relative to the open-channel conformation; (c) the conformation of the nucleotide binding site is not directly modulated by Ca2+ and voltage; (d) the differences in inhibitory potency of ATP, ADP, and AMP reflect their different affinities for the closed channel; and (e) though decavanadate is the only example found to date of a compound that stimulates Po with high affinity even in the presence of millimolar nucleotides, apparently by competing for the nucleotide binding site, a comparable mechanism might allow CA-NSC channels to open in living cells despite physiological levels of nucleotides. Decavanadate now provides a valuable tool for studying native CA-NSC channels and for screening cloned channels

Structural alterations by five disease-causing mutations in the low-pH conformation of human dihydrolipoamide dehydrogenase (hLADH) analyzed by molecular dynamics – implications in functional loss and modulation of reactive oxygen species generation by pathogenic hLADH forms

AbstractHuman dihydrolipoamide dehydrogenase (hLADH) is a flavoenzyme component (E3) of the human alpha-ketoglutarate dehydrogenase complex (α-KGDHc) and few other dehydrogenase complexes. Pathogenic mutations of hLADH cause severe metabolic diseases (atypical forms of E3 deficiency) that often escalate to cardiological or neurological presentations and even premature death; the pathologies are generally accompanied by lactic acidosis. hLADH presents a distinct conformation under acidosis (pH 5.5–6.8) with lower physiological activity and the capacity of generating reactive oxygen species (ROS). It has been shown by our laboratory that selected pathogenic mutations, besides lowering the physiological activity of hLADH, significantly stimulate ROS generation by hLADH, especially at lower pH, which might play a role in the pathogenesis of E3-deficiency in respective cases. Previously, we generated by molecular dynamics (MD) simulation the low-pH hLADH structure and analyzed the structural changes induced in this structure by eight of the pathogenic mutations of hLADH. In the absence of high resolution mutant structures these pieces of information are crucial for the mechanistic investigation of the molecular pathogeneses of the hLADH protein. In the present work we analyzed by molecular dynamics simulation the structural changes induced in the low-pH conformation of hLADH by five pathogenic mutations of hLADH; the structures of these disease-causing mutants of hLADH have never been examined before

A Novel Kinetic Assay of Mitochondrial ATP-ADP Exchange Rate Mediated by the ANT

AbstractA novel method exploiting the differential affinity of ADP and ATP to Mg2+ was developed to measure mitochondrial ADP-ATP exchange rate. The rate of ATP appearing in the medium after addition of ADP to energized mitochondria, is calculated from the measured rate of change in free extramitochondrial [Mg2+] reported by the membrane-impermeable 5K+ salt of the Mg2+-sensitive fluorescent indicator, Magnesium Green, using standard binding equations. The assay is designed such that the adenine nucleotide translocase (ANT) is the sole mediator of changes in [Mg2+] in the extramitochondrial volume, as a result of ADP-ATP exchange. We also provide data on the dependence of ATP efflux rate within the 6.8–7.8 matrix pH range as a function of membrane potential. Finally, by comparing the ATP-ADP steady-state exchange rate to the amount of the ANT in rat brain synaptic, brain nonsynaptic, heart and liver mitochondria, we provide molecular turnover numbers for the known ANT isotypes

Human 2-Oxoglutarate Dehydrogenase Complex E1 Component Forms a Thiamin-derived Radical by Aerobic Oxidation of the Enamine Intermediate.

Herein are reported unique properties of the human 2-oxoglutarate dehydrogenase multienzyme complex (OGDHc), a rate-limiting enzyme in the Krebs (citric acid) cycle. (a) Functionally competent 2-oxoglutarate dehydrogenase (E1o-h) and dihydrolipoyl succinyltransferase components have been expressed according to kinetic and spectroscopic evidence. (b) A stable free radical, consistent with the C2-(C2alpha-hydroxy)-gamma-carboxypropylidene thiamin diphosphate (ThDP) cation radical was detected by electron spin resonance upon reaction of the E1o-h with 2-oxoglutarate (OG) by itself or when assembled from individual components into OGDHc. (c) An unusual stability of the E1o-h-bound C2-(2alpha-hydroxy)-gamma-carboxypropylidene thiamin diphosphate (the "ThDP-enamine"/C2alpha-carbanion, the first postdecarboxylation intermediate) was observed, probably stabilized by the 5-carboxyl group of OG, not reported before. (d) The reaction of OG with the E1o-h gave rise to superoxide anion and hydrogen peroxide (reactive oxygen species (ROS)). (e) The relatively stable enzyme-bound enamine is the likely substrate for oxidation by O2, leading to the superoxide anion radical (in d) and the radical (in b). (f) The specific activity assessed for ROS formation compared with the NADH (overall complex) activity, as well as the fraction of radical intermediate occupying active centers of E1o-h are consistent with each other and indicate that radical/ROS formation is an "off-pathway" side reaction comprising less than 1% of the "on-pathway" reactivity. However, the nearly ubiquitous presence of OGDHc in human tissues, including the brain, makes these findings of considerable importance in human metabolism and perhaps disease

Formation of reactive oxygen species by human and bacterial pyruvate and 2- oxoglutarate dehydrogenase multienzyme complexes reconstituted from recombinant components

Individual recombinant components of pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes (PDHc, OGDHc) of human and Escherichia coli (E. coli) origin were expressed and purified from E. coli with optimized protocols. The four multienzyme complexes were each reconstituted under optimal conditions at different stoichiometric ratios. Binding stoichiometries for the highest catalytic efficiency were determined from the rate of NADH generation by the complexes at physiological pH. Since some of these complexes were shown to possess ‘moonlighting’ activities under pathological conditions often accompanied by acidosis, activities were also determined at pH 6.3. As reactive oxygen species (ROS) generation by the E3 component of hOGDHc is a pathologically relevant feature, superoxide generation by the complexes with optimal stoichiometry was measured by the acetylated cytochrome c reduction method in both the forward and the reverse catalytic directions. Various known affectors of physiological activity and ROS production, including Ca(2+), ADP, lipoylation status or pH, were investigated. The human complexes were also reconstituted with the most prevalent human pathological mutant of the E3 component, G194C and characterized; isolated human E3 with the G194C substitution was previously reported to have an enhanced ROS generating capacity. It is demonstrated that: i. PDHc, similarly to OGDHc, is able to generate ROS and this feature is displayed by both the E. coli and human complexes, ii. Reconstituted hPDHc generates ROS at a significantly higher rate as compared to hOGDHc in both the forward and the reverse reactions when ROS generation is calculated for unit mass of their common E3 component, iii. The E1 component or E1-E2 subcomplex generates significant amount of ROS only in hOGDHc; iv. Incorporation of the G194C variant of hE3, the result of a disease-causing mutation, into reconstituted hOGDHc and hPDHc indeed leads to a decreased activity of both complexes and higher ROS generation by only hOGDHc and only in its reverse reaction

Enhanced hydrogen peroxide generation accompanies the beneficial bioenergetic effects of methylene blue in isolated brain mitochondria

The redox dye methylene blue (MB) is proven to have beneficial effects in various models of neurodegenerative diseases. Here we investigated the effects of MB (100 nM, 300 nM, and 1 μM) on key bioenergetic parameters and on H2O2 production/elimination in isolated guinea pig brain mitochondria under normal as well as respiration-impaired conditions. As measured by high-resolution Oxygraph the rate of resting oxygen consumption was increased, but the ADP-stimulated respiration was unaffected by MB with any of the substrates (glutamate malate, succinate, or α-glycerophosphate) used for supporting mitochondrial respiration. In mitochondria treated with inhibitors of complex I or complex III MB moderately but significantly increased the rate of ATP production, restored ΔΨm, and increased the rate of Ca2+ uptake. The effects of MB are consistent with transferring electrons from upstream components of the electron transport chain to cytochrome c, which is energetically favorable when the flow of electrons in the respiratory chain is compromised. On the other hand, MB significantly increased the production of H2O2 measured by Amplex UltraRed fluorimetry under all conditions, in resting, ATP-synthesizing, and respiration-impaired mitochondria, with each substrate combination supporting respiration. Furthermore, it also decreased the elimination of H2O2. Generation of H2O2 without superoxide formation, observed in the presence of MB, is interpreted as a result of reduction of molecular oxygen to H2O2 by the reduced MB. The elevated generation and impaired elimination of H2O2 should be considered for the overall oxidative state of mitochondria treated with MB

Molecular dynamics study of the structural basis of dysfunction and the modulation of reactive oxygen species generation by pathogenic mutants of human dihydrolipoamide dehydrogenase

Human dihydrolipoamide dehydrogenase (LADH, E3) is a component in the pyruvate-, alpha-ketoglutarate- and branched-chain ketoacid dehydrogenase complexes and in the glycine cleavage system. The pathogenic mutations of LADH cause severe metabolic disturbances, called E3 deficiency that often involve cardiological and neurological symptoms and premature death. Our laboratory has recently shown that some of the known pathogenic mutations augment the reactive oxygen species (ROS) generation capacity of LADH, which may contribute to the clinical presentations. A recent report concluded that elevated oxidative stress generated by the above mutants turns the lipoic acid cofactor on the E2 subunits dysfunctional. In the present contribution we generated by molecular dynamics (MD) simulation the conformation of LADH that is proposed to be compatible with ROS generation. We propose here for the first time the structural changes, which are likely to turn the physiological LADH conformation to its ROS-generating conformation. We also created nine of the pathogenic mutants of the ROS-generating conformation and again used MD simulation to detect structural changes that the mutations induced in this LADH conformation. We propose the structural changes that may lead to the modulation in ROS generation of LADH by the pathogenic mutations. © 2013 Elsevier Inc. All rights reserved

Distinct Mechanisms of Pathogenic DJ-1 Mutations in Mitochondrial Quality Control

The deglycase and chaperone protein DJ-1 is pivotal for cellular oxidative stress responses and mitochondrial quality control. Mutations in PARK7, encoding DJ-1, are associated with early-onset familial Parkinson’s disease and lead to pathological oxidative stress and/or disrupted protein degradation by the proteasome. The aim of this study was to gain insights into the pathogenic mechanisms of selected DJ-1 missense mutations, by characterizing protein–protein interactions, core parameters of mitochondrial function, quality control regulation via autophagy, and cellular death following dopamine accumulation. We report that the DJ-1M26I mutant influences DJ-1 interactions with SUMO-1, in turn enhancing removal of mitochondria and conferring increased cellular susceptibility to dopamine toxicity. By contrast, the DJ-1D149A mutant does not influence mitophagy, but instead impairs Ca2+ dynamics and free radical homeostasis by disrupting DJ-1 interactions with a mitochondrial accessory protein known as DJ-1-binding protein (DJBP/EFCAB6). Thus, individual DJ-1 mutations have different effects on mitochondrial function and quality control, implying mutation-specific pathomechanisms converging on impaired mitochondrial homeostasis

Neuroprotective coordination of cell mitophagy by the ATPase Inhibitory Factor 1

The mitochondrial ATPase Inhibitory Factor 1 (hereafter referred to as IF1) blocks the reversal of the F1Fo-ATPsynthase to prevent detrimental consumption of cellular ATP and associated demise. Herein, we infer further its molecular physiology by assessing its protective function in neurons during conditions of challenged homeostatic respiration. By adopting in vitro and in vivo protocols of hypoxia/ischemia and re-oxygenation, we show that a shift in the IF1:F1Fo-ATPsynthase expression ratio occurs in neurons. This increased IF1 level is essential to induce accumulation of the PTEN-induced putative kinase 1 (PINK-1) and recruitment of the mitophagic ubiquitin ligase PARK-2 to promote autophagic “control” of the mitochondrial population. In IF1 overexpressing neurons ATP depletion is reduced during hypoxia/ischemia and the mitochondrial membrane potential (ΔYm) resilient to re-oxygenation as well as resistant to electrogenic, Ca2+ dependent depolarization. These data suggest that in mammalian neurons mitochondria adapt to respiratory stress by upregulating IF1, which exerts a protective role by coordinating pro-survival cell mitophagy and bioenergetics resilience