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• W1569690763 abstract "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 (Mg2+ 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 (IC50, 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. 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 (Mg2+ 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 (IC50, 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. IntroductionA novel aspect of mitochondrial physiology has been disclosed by the application of electrophysiological techniques to the inner (IM) 1The abbreviations used are:IMinner membraneIMMinner mitochondrial membraneOMouter membranepSpicosiemensnSnanosiemensmCS channelmitochondrial centum picosiemens channelVDACvoltage-dependent anion channelPoopen probability. and outer (OM) membranes of mitochondria. In particular, the direct analysis of the native membranes with a patch clamp electrode has provided strong evidence of the presence of high conductance ion channels (reviewed in (1Sorgato M.C. Moran O. CRC Crit. Rev. Biochem. Mol. Biol. 1993; 18: 127-171Google Scholar)). By applying this technique to the IM of mitoplasts (obtained from mitochondria by removal of the OM), at least three types of channels have been found. One has a conductance of around 10 pS (in 100 mM salt) and is distinguishable for the selective permeability toward potassium ions. It is not regulated by voltage but is sensitive to a number of drugs and physiological effectors, including matrix ATP, which acts as inhibitor in the mM range(2Inoue I. Nagase H. Kishi K. Higuti T. Nature. 1991; 352: 244-247Google Scholar). Conversely, the main feature of the other channel, slightly anionic and with a conductance of around 100 pS (in 150 mM KCl) (mitochondrial centum picosiemens (mCS) channel), is the voltage dependence(3Sorgato M.C. Keller B.U. Stühmer W. Nature. 1987; 330: 498-500Google Scholar). At positive (unphysiological) matrix potentials the channel is mainly open, whereas the open probability decreases as the voltage is made negative. Interestingly, the current-voltage relationship of the entire IM shows an identical behavior, thus suggesting a major role of mCS channels in the electric activity of this membrane(1Sorgato M.C. Moran O. CRC Crit. Rev. Biochem. Mol. Biol. 1993; 18: 127-171Google Scholar). The third type of channel, frequently referred to as mitochondrial megachannel, is notable for the peak conductance of 1-1.3 nS (in 150 mM KCl) and the multiple substates(4Kinnally K.W. Campo M.L. Tedeschi H.T. J. Bioenerg. Biomembr. 1989; 21: 497-506Google Scholar, 5Petronilli V. Szabò I. Zoratti M. FEBS Lett. 1989; 259: 137-143Google Scholar). For the similar response to a variety of negative and positive effectors, the megachannel has been tentatively identified with the mitochondrial permeability transition pore and/or with the mitochondrial benzodiazepine receptor (for this topic, see (6Zoratti M. Szabò I. J. Bioenerg. Biomembr. 1994; 26: 543-553Google Scholar)). On the other hand, the electric features displayed in planar bilayers by voltage-dependent anion channel (VDAC), the most abundant OM protein, have not been detected in integral mitochondria analyzed with a patch clamp electrode, by which at least three other channels were identified(7Moran O. Sciancalepore M. Sandri G. Panfili E. Bassi R. Ballarin C. Sorgato M.C. Eur. Biophys. J. 1992; 20: 311-319Google Scholar).Undoubtedly, much has been learned by tackling mitochondria with electrophysiological tools. A number of unknowns, however, remain (for example, the physiological significance of the presence of high conductive pathways in the mitochondrial membranes, in particular of those in the IM, or their structural identity). Until now, almost all electrophysiological studies concerning the native inner mitochondrial membrane (IMM) have been carried out using mammalian tissues. In the light of the very similar physiology shared by this membrane in yeast and mammalian cells, it seemed of value to compare their electric properties. Indeed, were mammalian channels absent in yeast, this would imply that they perform a function specific to higher eukaryotes. Conversely, should they be present, then this result would indicate that channels are a fundamental property of all mitochondria. A long term aim of this study relates to the molecular understanding of IM channels, given that use of yeast mutants is the faster route to unraveling the structure and function of proteins.We report here the patch clamping of yeast mitoplasts isolated from two wild type strains of Saccharomyces cerevisiae and from a mutant lacking the VDAC gene. In all cases, channel activity was detected, but the overall electric behavior of the IM diverged from that observed in mammals. The electrophysiological analysis of proteoliposomes containing both mitochondrial membranes is also presented. IntroductionA novel aspect of mitochondrial physiology has been disclosed by the application of electrophysiological techniques to the inner (IM) 1The abbreviations used are:IMinner membraneIMMinner mitochondrial membraneOMouter membranepSpicosiemensnSnanosiemensmCS channelmitochondrial centum picosiemens channelVDACvoltage-dependent anion channelPoopen probability. and outer (OM) membranes of mitochondria. In particular, the direct analysis of the native membranes with a patch clamp electrode has provided strong evidence of the presence of high conductance ion channels (reviewed in (1Sorgato M.C. Moran O. CRC Crit. Rev. Biochem. Mol. Biol. 1993; 18: 127-171Google Scholar)). By applying this technique to the IM of mitoplasts (obtained from mitochondria by removal of the OM), at least three types of channels have been found. One has a conductance of around 10 pS (in 100 mM salt) and is distinguishable for the selective permeability toward potassium ions. It is not regulated by voltage but is sensitive to a number of drugs and physiological effectors, including matrix ATP, which acts as inhibitor in the mM range(2Inoue I. Nagase H. Kishi K. Higuti T. Nature. 1991; 352: 244-247Google Scholar). Conversely, the main feature of the other channel, slightly anionic and with a conductance of around 100 pS (in 150 mM KCl) (mitochondrial centum picosiemens (mCS) channel), is the voltage dependence(3Sorgato M.C. Keller B.U. Stühmer W. Nature. 1987; 330: 498-500Google Scholar). At positive (unphysiological) matrix potentials the channel is mainly open, whereas the open probability decreases as the voltage is made negative. Interestingly, the current-voltage relationship of the entire IM shows an identical behavior, thus suggesting a major role of mCS channels in the electric activity of this membrane(1Sorgato M.C. Moran O. CRC Crit. Rev. Biochem. Mol. Biol. 1993; 18: 127-171Google Scholar). The third type of channel, frequently referred to as mitochondrial megachannel, is notable for the peak conductance of 1-1.3 nS (in 150 mM KCl) and the multiple substates(4Kinnally K.W. Campo M.L. Tedeschi H.T. J. Bioenerg. Biomembr. 1989; 21: 497-506Google Scholar, 5Petronilli V. Szabò I. Zoratti M. FEBS Lett. 1989; 259: 137-143Google Scholar). For the similar response to a variety of negative and positive effectors, the megachannel has been tentatively identified with the mitochondrial permeability transition pore and/or with the mitochondrial benzodiazepine receptor (for this topic, see (6Zoratti M. Szabò I. J. Bioenerg. Biomembr. 1994; 26: 543-553Google Scholar)). On the other hand, the electric features displayed in planar bilayers by voltage-dependent anion channel (VDAC), the most abundant OM protein, have not been detected in integral mitochondria analyzed with a patch clamp electrode, by which at least three other channels were identified(7Moran O. Sciancalepore M. Sandri G. Panfili E. Bassi R. Ballarin C. Sorgato M.C. Eur. Biophys. J. 1992; 20: 311-319Google Scholar).Undoubtedly, much has been learned by tackling mitochondria with electrophysiological tools. A number of unknowns, however, remain (for example, the physiological significance of the presence of high conductive pathways in the mitochondrial membranes, in particular of those in the IM, or their structural identity). Until now, almost all electrophysiological studies concerning the native inner mitochondrial membrane (IMM) have been carried out using mammalian tissues. In the light of the very similar physiology shared by this membrane in yeast and mammalian cells, it seemed of value to compare their electric properties. Indeed, were mammalian channels absent in yeast, this would imply that they perform a function specific to higher eukaryotes. Conversely, should they be present, then this result would indicate that channels are a fundamental property of all mitochondria. A long term aim of this study relates to the molecular understanding of IM channels, given that use of yeast mutants is the faster route to unraveling the structure and function of proteins.We report here the patch clamping of yeast mitoplasts isolated from two wild type strains of Saccharomyces cerevisiae and from a mutant lacking the VDAC gene. In all cases, channel activity was detected, but the overall electric behavior of the IM diverged from that observed in mammals. The electrophysiological analysis of proteoliposomes containing both mitochondrial membranes is also presented." @default.
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• W1569690763 title "An Electrophysiological Study of Yeast Mitochondria" @default.
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