Alternative sulfonylurea receptor expression defines metabolic sensitivity of K-ATP channels in dopaminergic midbrain neurons.
(1/808)
ATP-sensitive potassium (K-ATP) channels couple the metabolic state to cellular excitability in various tissues. Several isoforms of the K-ATP channel subunits, the sulfonylurea receptor (SUR) and inwardly rectifying K channel (Kir6.X), have been cloned, but the molecular composition and functional diversity of native neuronal K-ATP channels remain unresolved. We combined functional analysis of K-ATP channels with expression profiling of K-ATP subunits at the level of single substantia nigra (SN) neurons in mouse brain slices using an RT-multiplex PCR protocol. In contrast to GABAergic neurons, single dopaminergic SN neurons displayed alternative co-expression of either SUR1, SUR2B or both SUR isoforms with Kir6.2. Dopaminergic SN neurons expressed alternative K-ATP channel species distinguished by significant differences in sulfonylurea affinity and metabolic sensitivity. In single dopaminergic SN neurons, co-expression of SUR1 + Kir6.2, but not of SUR2B + Kir6.2, correlated with functional K-ATP channels highly sensitive to metabolic inhibition. In contrast to wild-type, surviving dopaminergic SN neurons of homozygous weaver mouse exclusively expressed SUR1 + Kir6.2 during the active period of dopaminergic neurodegeneration. Therefore, alternative expression of K-ATP channel subunits defines the differential response to metabolic stress and constitutes a novel candidate mechanism for the differential vulnerability of dopaminergic neurons in response to respiratory chain dysfunction in Parkinson's disease. (+info)
Energy depletion differently affects membrane transport and intracellular metabolism of riboflavin taken up by isolated rat enterocytes.
(2/808)
Isolated rat enterocytes, both normal and those de-energized with rotenone, were used to study the energy dependence of membrane and intracellular intestinal riboflavin transport in vitro. Membrane and intracellular transport were investigated by using short (3 min) and long (20 min) incubation times, respectively. For both types of cells and incubation times, [3H]-riboflavin uptake presented a saturable component prevailing at physiologic intraluminal concentrations. At 3 min incubation, saturable [3H]-riboflavin transport was apparently an energy-independent process with high affinity and low capacity. Values of the saturable component and its apparent constants, Km and Jmax, did not differ in normal and de-energized enterocytes. At 20 min incubation, saturable [3H]-riboflavin transport was a strictly energy-dependent process in which values of the saturable component were significantly greater in normal than in de-energized enterocytes. Km values did not differ in the two types of cells and were unmodified over 3 min, whereas in normal enterocytes, Jmax at 20 min [6.25 +/- 0.2 pmol/(mg protein. 20 min)] was significantly greater than at 3 min [2.67 +/- 0.33 pmol/(mg protein. 3 min)] and compared with de-energized enterocytes at 20 min [2.54 +/- 0.16 pmol/(mg protein. 20 min)]. Both membrane and intracellular events were inhibited by unlabeled riboflavin and analogs, which are good substrates for flavokinase, thus demonstrating the paramount role of this enzyme in riboflavin intestinal transport. (+info)
Dual actions of the metabolic inhibitor, sodium azide on K(ATP) channel currents in the rat CRI-G1 insulinoma cell line.
(3/808)
1. The effects of various inhibitors of the mitochondrial electron transport chain on the activity of ATP-sensitive K+ channels were examined in the Cambridge rat insulinoma G1 (CRI-G1) cell line using a combination of whole cell and single channel recording techniques. 2. Whole cell current clamp recordings, with 5 mM ATP in the pipette, demonstrate that the mitochondrial uncoupler sodium azide (3 mM) rapidly hyperpolarizes CRI-G1 cells with a concomitant increase in K+ conductance. This is due to activation of K(ATP) channels as the sulphonylurea tolbutamide (100 microM) completely reversed the actions of azide. Other inhibitors of the mitochondrial electron transport chain, rotenone (10 microM) or oligomycin (2 microM) did not hyperpolarize CRI-G1 cells or increase K+ conductance. 3. In cell-attached recordings, bath application of 3 mM sodium azide (in the absence of glucose) resulted in a rapid increase in K(ATP) channel activity, an action readily reversible by tolbutamide (100 microM). Application of sodium azide (3 mM), in the presence of Mg-ATP, to the intracellular surface of excised inside-out patches also increased K(ATP) channel activity, in a reversible manner. 4. In contrast, rotenone (10 microM) or oligomycin (2 microM) did not increase K(ATP) channel activity in either cell-attached, in the absence of glucose, or inside-out membrane patch recordings. 5. Addition of sodium azide (3 mM) to the intracellular surface of inside-out membrane patches in the presence of Mg-free ATP or the non-hydrolysable analogue 5'-adenylylimidodiphosphate (AMP-PNP) inhibited, rather than increased, K(ATP) channel activity. 6. In conclusion, sodium azide, but not rotenone or oligomycin, directly activates K(ATP) channels in CRI-G1 insulin secreting cells. This action of azide is similar to that reported previously for diazoxide. (+info)
NADH-quinone oxidoreductase: PSST subunit couples electron transfer from iron-sulfur cluster N2 to quinone.
(4/808)
The proton-translocating NADH-quinone oxidoreductase (EC 1.6.99.3) is the largest and least understood enzyme complex of the respiratory chain. The mammalian mitochondrial enzyme (also called complex I) contains more than 40 subunits, whereas its structurally simpler bacterial counterpart (NDH-1) in Paracoccus denitrificans and Thermus thermophilus HB-8 consists of 14 subunits. A major unsolved question is the location and mechanism of the terminal electron transfer step from iron-sulfur cluster N2 to quinone. Potent inhibitors acting at this key region are candidate photoaffinity probes to dissect NADH-quinone oxidoreductases. Complex I and NDH-1 are very sensitive to inhibition by a variety of structurally diverse toxicants, including rotenone, piericidin A, bullatacin, and pyridaben. We designed (trifluoromethyl)diazirinyl[3H]pyridaben ([3H]TDP) as our photoaffinity ligand because it combines outstanding inhibitor potency, a suitable photoreactive group, and tritium at high specific activity. Photoaffinity labeling of mitochondrial electron transport particles was specific and saturable. Isolation, protein sequencing, and immunoprecipitation identified the high-affinity specifically labeled 23-kDa subunit as PSST of complex I. Immunoprecipitation of labeled membranes of P. denitrificans and T. thermophilus established photoaffinity labeling of the equivalent bacterial NQO6. Competitive binding and enzyme inhibition studies showed that photoaffinity labeling of the specific high-affinity binding site of PSST is exceptionally sensitive to each of the high-potency inhibitors mentioned above. These findings establish that the homologous PSST of mitochondria and NQO6 of bacteria have a conserved inhibitor-binding site and that this subunit plays a key role in electron transfer by functionally coupling iron-sulfur cluster N2 to quinone. (+info)
The NDUFA1 gene product (MWFE protein) is essential for activity of complex I in mammalian mitochondria.
(5/808)
The MWFE polypeptide of mammalian complex I (the proton-translocating NADH-quinone oxidoreductase) is 70 amino acids long, and it is predicted to be a membrane protein. The NDUFA1 gene encoding the MWFE polypeptide is located on the X chromosome. This polypeptide is 1 of approximately 28 "accessory proteins" identified in complex I, which is composed of 42 unlike subunits. It was considered accessory, because it is not one of the 14 polypeptides making up the core complex I; a homologous set of 14 polypeptides can make a fully functional proton-translocating NADH-quinone oxidoreductase in prokaryotes. One MWFE mutant has been identified and isolated from a collection of respiration-deficient Chinese hamster cell mutants. The CCL16-B2 mutant has suffered a deletion that would produce a truncated and abnormal MWFE protein. In these mutant cells, complex I activity is reduced severely (<10%). Complementation with hamster NDUFA1 cDNA restored the rotenone-sensitive complex I activity of these mutant cells to approximately 100% of the parent cell activity. Thus, it is established that the MWFE polypeptide is absolutely essential for an active complex I in mammals. (+info)
Selective induction of apoptosis by capsaicin in transformed cells: the role of reactive oxygen species and calcium.
(6/808)
Capsaicin is a vanilloid quinone analog that inhibits the plasma membrane electron transport (PMOR) system and induces apoptosis in transformed cells. Using a cytofluorimetric approach we have determined that capsaicin induces a rapid increase of reactive oxygen species (ROS) followed by a subsequent disruption of the transmembrane mitochondrial potential (DeltaPsim) and DNA nuclear loss in transformed cell lines and in mitogen activated human T cells. This apoptotic pathway is biochemically different from the typical one induced by either ceramide or edelfosine where, in our system, the DeltaPsim dissipation precedes the generation of reactive oxygen species. Neither production of ROS nor apoptosis was found in capsaicin-treated resting T cells where the activity of the PMOR system is minimal when compared with mitogen activated or transformed T cells. Capsaicin also induces Ca2+ mobilization in activated but not in resting T cells. However, preincubation of cells with BAPTA-AM, which chelate cytosolic free calcium, did not prevent ROS generation or apoptosis induced by capsaicin, suggesting that ROS generation in capsaicin treated cells is not a consequence of calcium signaling and that the apoptotic pathway may be separated from the one that mobilizes calcium. Moreover, we present data for the implication of a possible vanilloid receptor in calcium mobilization, but not in ROS generation. These results provide evidence that the PMOR system may be an interesting target to design antitumoral and anti-inflammatory drugs. (+info)
Titrating the effects of mitochondrial complex I impairment in the cell physiology.
(7/808)
The mitochondrial oxidative phosphorylation system consists of five multimeric enzymes (complexes I-V). NADH dehydrogenase or complex I (CI) is affected in most of the mitochondrial diseases and in some neurodegenerative disorders. We have studied the physiological consequences of a partial CI inhibition at the cellular level. We used a genetic model (40% CI-inhibited human-ape xenomitochondrial cybrids) and a drug-induced model (0-100% CI-inhibited cells using different concentrations of rotenone). We observed a quantitative correlation between the level of CI impairment and cell respiration, cell growth, free radical production, lipid peroxidation, mitochondrial membrane potential, and apoptosis. We showed that cell death was quantitatively associated with free radical production rather than with a decrease in respiratory chain function. The results obtained with human xenomitochondrial cybrid cells were compatible with those observed in rotenone-induced 40% CI-inhibited cells. At high concentrations (5-6-fold higher than the concentration necessary for 100% CI inhibition), rotenone showed a second toxic effect at the level of microtubule assembly, which also led to apoptosis. The correlation found among all the parameters studied helped clarify the physiological consequences of partial CI inhibitions at the cellular level. (+info)
Modulation of oxidative phosphorylation of human kidney 293 cells by transfection with the internal rotenone-insensitive NADH-quinone oxidoreductase (NDI1) gene of Saccharomyces cerevisiae.
(8/808)
In contrast to the mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I), which consists of at least 43 different subunits, the internal rotenone-insensitive NADH-quinone oxidoreductase (Ndi1) of Saccharomyces cerevisiae is a single polypeptide enzyme. The NDI1 gene was stably transfected into the human embryonal kidney 293 (HEK 293) cells. The transfected NDI1 gene was then transcribed and translated in the HEK 293 cells to produce the functional enzyme. The immunochemical and immunofluorescence analyses indicated that the expressed Ndi1 polypeptide was located to the inner mitochondrial membranes. The expression of Ndi1 did not alter the content of existing complex I in the HEK 293 mitochondria, suggesting that the expressed Ndi1 enzyme does not displace the endogenous complex I. The NADH oxidase activity of the NDI1-transfected HEK 293 cells was not affected by rotenone but was inhibited by flavone. The ADP/O ratios coupled to NADH oxidation were lowered from 2.4 to 1.8 by NDI1-transfection while the ADP/O ratios coupled to succinate oxidation (1.6) were not changed. The NDI1-transfected HEK 293 cells were able to grow in media containing a complex I inhibitor such as rotenone and 1-methyl-4-phenylpyridinium ion. The potential usefulness of incorporating the Ndi1 protein into mitochondria of human cells is discussed. (+info)