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Modal gating of human Ca(v)2.1 (P/Q-type) calcium channels: II. The b mode and reversible uncoupling of inactivation
No full texthe single channel gating properties of human Ca(V)2.1 (P/Q-type) calcium channels were investigated with cell-attached patch-clamp recordings on HEK293 cells stably expressing these calcium channels. Human Ca(V)2.1 channels showed a complex modal gating, which is described in this and the preceding paper (Luvisetto, S., T. Fellin, M. Spagnolo, B. Hivert, RE Brust, M.M. Harpold, K.A. Stauderman, M.E. Williams, and D. Pietrobon. 2004. J Gev. Physiol. 124:445-461). Here, we report the characterization of the so-called b gating mode. A Ca(V)2.1 channel in the b gating mode shows a bell-shaped voltage dependence of the open probability and a characteristic low open probability at high positive voltages, that decreases with increasing voltage, as a consequence of both shorter mean open time and longer mean closed time. Reversible transitions of single human Ca(V)2.1 channels between the b gating mode and the mode of gating in which the channel shows the usual voltage dependence of the open probability (nb gating mode) were much more frequent (time scale of seconds) than those between the slow and fast gating modes (time scale of minutes; Luvisetto et al., 2004), and Occurred independently of whether the channel was in the fast or slow mode. We show that the b gating mode produces reversible uncoupling of inactivation in human Ca(V)2.1 channels. In fact, a Ca(V)2.1 channel in the b gating mode does not inactivate during long pulses at high positive voltages, where the same channel in both fast-nb and slow-jib gating modes inactivates relatively rapidly Moreover, a Ca(V)2.1 channel in the b gating mode shows a larger availability to open than in the rib gating modes. Regulation of the complex modal gating of human Ca(V)2.1 channels could be a potent and verb satile mechanism for the modulation of synaptic strength and plasticity as well as of neuronal excitability and other postsynaptic Ca2+-dependent processes
Uncoupling of oxidative phosphorylation. 2. Alternative mechanisms: intrinsic uncoupling or decoupling?
No full textThe mechanism of uncoupling of oxidative phosphorylation by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), oleic acid, and chloroform is further investigated by measuring in the presence of a certain concentration of each type of uncoupler (i) the mitochondrial P/O and respiratory control ratios upon progressive inhibition of the redox pumps and (ii) delta mu H and the rate of either electron transfer or adenosine 5'-triphosphate (ATP) hydrolysis in static head upon progressive inhibition of either the redox or the adenosine triphosphatase (ATPase) proton pumps. Chloroform exhibits in all the experiments a behavior very different from that of FCCP and oleic acid. For example, upon addition of antimycin to chloroform-supplemented mitochondria, the respiratory control ratio remains unchanged and the P/O ratio slightly increases (in a certain range of inhibition) instead of decreasing as expected for an increased membrane conductance (and as indeed measured in the presence of either FCCP or oleic acid). From the kinetic model of chemiosmotic free energy coupling described by Pietrobon and Caplan [Pietrobon, D., & Caplan, S.R. (1986) Biochemistry 25, 7690-7696] all the results can be simulated by making the assumptions that (i) chloroform acts specifically at the level of the proton pumps and intrinsically uncouples electron transfer and ATP hydrolysis/synthesis from proton translocation and (ii) FCCP and oleic acid have a mixed behavior and act both as protonophores and as intrinsic uncouplers of the redox pumps (but not of the ATPases). The consistency of the results with the alternative hypothesis that the three agents interfere either with localized energy coupling sites or with a direct interaction between proton pumps is discussed
Uncoupling of oxidative phosphorylation. 1. Protonophoric effects account only partially for uncoupling.
No full textThe mechanism of uncoupling of oxidative phosphorylation by carbonyl cyanide p-trifluoromethoxy)phenylhydrazone (FCCP), a typical weak acid protonophore, oleic acid, a fatty acid, and chloroform, a general anesthetic, has been investigated by measuring in mitochondria their effect on (i) the transmembrane proton electrochemical potential gradient (delta mu H) and the rates of electron transfer and adenosine 5'-triphosphate (ATP) hydrolysis in static head, (ii) delta mu H and the rates of electron transfer and ATP synthesis in state 3, and (iii) the membrane proton conductance. Both FCCP and oleic acid increase the membrane proton conductance, and accordingly, they cause a depression of delta mu H [generated by either the redox proton pumps or the adenosinetriphosphatase (ATPase) proton pumps]. Although their effects on ATP synthesis/hydrolysis, respiration, and delta mu H are qualitatively consistent with a pure protonophoric uncoupling mechanism and an additional inhibitory action of oleic acid on both the ATPases and the electron-transfer enzymes, a quantitative comparison between the dissipative proton influx and the rate of either electron transfer or ATP hydrolysis (multiplied by either the H+/e- or the H+/ATP stoichiometry, respectively) at the same delta mu H shows that the increase in membrane conductance induced by FCCP and oleic acid accounts for the stimulation of the rate of ATP hydrolysis but not for that of the rate of electron transfer. Chloroform (at concentrations that fully inhibit ATP synthesis) only very slightly increases the proton conductance of the mitochondrial membrane and causes only a little depression of delta mu H.(ABSTRACT TRUNCATED AT 250 WORDS
NATURE OF RESPIRATORY STIMULATION IN HYPERTHYROIDISM - THE REDOX BEHAVIOR OF CYTOCHROME-C
No full textCentral injection of botulinum neurotoxins: behavioural effects in mice
No full textStrains of Clostridium botulinum produce seven antigenically distinct botulinum neurotoxins (BoNTs) designated as serotypes A-G. All serotypes interfere with neural transmission by blocking the release of acetylcholine in cholinergic neurons. They cleave specific sites on proteins of the SNARE [soluble n-ethylmaleimide-sensitive factor (NSF) attachment protein receptor] complex, which play a key role in neuroexocytosis. This study assessed the behavioural effects due to central administration of BoNTs in mice. CD1 mice were injected intracerebroventricularly (icv) with sub-lethal doses of BoNT/A or /B and their behavioural responses in conditioning of active avoidance, object recognition test and pharmacologically induced locomotor activity were tested. Compared to control mice, BoNT-treated mice showed: (1) a reduced capacity to discriminate a novel object within a familiar environment; (2) an enhanced stimulant effect by scopolamine and a depressant effect by oxotremorine on locomotor activity. In contrast, central injection of BoNTs did not alter active avoidance acquisition. These results suggest an in vivo functional alteration due to the action of BoNTs directly administered into the central nervous system. The present data demonstrate that BoNTs may represent an analytical tool for studying the functional role of cholinergic neurons
ON THE NATURE OF THE UNCOUPLING EFFECT OF FATTY-ACIDS
No full textThe effect of palmitic acid on the electrical potential differences delta psi across the inner mitochondrial membrane appears to depend on the medium in which mitochondria are incubated. In medium A (cf. Luvisetto et al. (1987), Biochemistry, 26, 7332-7338) delta psi decreases much more than in medium B (cf. Rottenberg and Hashimoto (1986), Biochemistry, 25, 1747-1755) at concentrations of fatty acid which equally stimulate the rate of respiration in state 4. Valinomycin and NaCl were both present in medium B and absent in medium A. However, in both media the pattern of the P/O ratio as a function of antimycin in the presence of a constant amount of palmitic acid or of FCCP shows similar behaviour. We conclude that in both media palmitic acid increases the membrane conductance to protons, but for unclear reasons the delta psi assay fails to measure the decline of delta psi in medium B. However, the increase in membrane conductance induced by palmitic acid does not quantitatively account for the stimulation of the rate of respiration
THE NATURE OF MITOCHONDRIAL RESPIRATION AND DISCRIMINATION BETWEEN MEMBRANE AND PUMP PROPERTIES
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Enhancement of anxiety, facilitation of avoidance behavior and occurrence of adult-onset obesity in mice lacking mitochondrial cyclophilin D
No full textIn this report, we have assessed the behavioral responses of mice missing the Ppif gene (CyPD-KO), encoding mitochondrial cyclophilin D (CyPD). Mitochondrial CyPD is a key modulator of the mitochondrial permeability transition which is involved in the regulation of calcium- and oxidative damage–induced cell death. Behavioral screening of CyPD-KO mice (ranging between 4 and 15 months of age) was accomplished using a battery of behavioral paradigms which included testing of motor functions, exploratory activity, and anxiety/emotionality, as well as learning and memory skills. We found that, compared with wild-type mice, CyPD-KO mice were (i) more anxious and less explorative in open field and elevated plus maze and (ii) performed better in learning and memory of avoidance tasks, such as active and passive avoidance. However, the absence of CyPD did not alter the nociceptive threshold for thermal stimuli. Finally, deletion of CyPD caused also an abnormal accumulation of white adipose tissue resulting in adult-onset obesity, which was not dependent on increased food and/or water intake. Taken together, our results suggest a new fundamental role of mitochondrial CyPD in basal brain functions and body weight homeostasis
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