The effect of antimycin A on mouse liver inner mitochondrial membrane channel activity.

In the present study, we describe the existence of a large-conductance Ca2+-activated potassium (BKCa) channel in the mitochondria of the human endothelial cell line EA.hy926. A single-channel current was recorded from endothelial mitoplasts (i.e., inner mitochondrial membrane) using the patch-clamp technique in the mitoplast-attached mode. A potassium-selective current was recorded with a mean conductance equal to 270 ± 10 pS in a symmetrical 150/150 mM KCl isotonic solution. The channel activity, which was determined as the open probability, increased with the addition of calcium ions and the potassium channel opener NS1619. Conversely, the activity of the channel was irreversibly blocked by paxilline and iberiotoxin, BKCa channel inhibitors. The open-state probability was found to be voltage dependent. The substances known to modulate BKCa channel activity influenced the bioenergetics of mitochondria isolated from human endothelial EA.hy926 cells. In isolated mitochondria, 100 μM Ca2+, 10 μM NS1619, and 0.5 μM NS11021 depolarized the mitochondrial membrane potential and stimulated nonphosphorylating respiration. These effects were blocked by iberiotoxin and paxilline in a potassium-dependent manner. Under phosphorylating conditions, NS1619-induced, iberiotoxin-sensitive uncoupling diverted energy from ATP synthesis during the phosphorylating respiration of the endothelial mitochondria. Immunological analysis with antibodies raised against proteins of the plasma membrane BKCa channel identified a pore-forming α-subunit and an auxiliary β2-subunit of the channel in the endothelial mitochondrial inner membrane. In conclusion, we show for the first time that the inner mitochondrial membrane in human endothelial EA.hy926 cells contains a large-conductance calcium-dependent potassium channel with properties similar to those of the surface membrane BKCa channel.

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Hypoxia increases BK channel activity in the inner mitochondrial membrane

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2007.04.110 ◽

2007 ◽

Vol 358 (1) ◽

pp. 311-316 ◽

Cited By ~ 27

Author(s):

Xiang Q. Gu ◽

Detlef Siemen ◽

Suhel Parvez ◽

Yu Cheng ◽

Jin Xue ◽

...

Keyword(s):

Mitochondrial Membrane ◽

Bk Channel ◽

Channel Activity ◽

Inner Mitochondrial Membrane

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The F1FO-ATP Synthase in Yeast Also Functions as an Inner Mitochondrial Membrane Channel♦

Journal of Biological Chemistry ◽

10.1074/jbc.p114.559633 ◽

2014 ◽

Vol 289 (23) ◽

pp. 15986-15986

Keyword(s):

Atp Synthase ◽

Mitochondrial Membrane ◽

Inner Mitochondrial Membrane ◽

Membrane Channel ◽

F1fo Atp Synthase

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Identification of anion and cation pathways in the inner mitochondrial membrane by patch clamping of mouse liver mitoplasts

The Journal of Membrane Biology ◽

10.1007/bf01870459 ◽

1991 ◽

Vol 124 (2) ◽

pp. 151-158 ◽

Cited By ~ 25

Author(s):

Yuri N. Antonenko ◽

Kathleen W. Kinnally ◽

Henry Tedeschi

Keyword(s):

Mitochondrial Membrane ◽

Mouse Liver ◽

Patch Clamping ◽

Inner Mitochondrial Membrane

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Coupled transmembrane mechanisms control MCU-mediated mitochondrial Ca2+uptake

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2005976117 ◽

2020 ◽

Vol 117 (35) ◽

pp. 21731-21739 ◽

Cited By ~ 5

Author(s):

Horia Vais ◽

Riley Payne ◽

Usha Paudel ◽

Carmen Li ◽

J. Kevin Foskett

Keyword(s):

Mitochondrial Membrane ◽

Permeability Transition Pore ◽

Permeability Transition ◽

Regulatory Mechanisms ◽

Intermembrane Space ◽

Channel Activity ◽

Inner Mitochondrial Membrane ◽

Energy Demands ◽

Transport Chain ◽

The Matrix

Ca2+uptake by mitochondria regulates bioenergetics, apoptosis, and Ca2+signaling. The primary pathway for mitochondrial Ca2+uptake is the mitochondrial calcium uniporter (MCU), a Ca2+-selective ion channel in the inner mitochondrial membrane. MCU-mediated Ca2+uptake is driven by the sizable inner-membrane potential generated by the electron-transport chain. Despite the large thermodynamic driving force, mitochondrial Ca2+uptake is tightly regulated to maintain low matrix [Ca2+] and prevent opening of the permeability transition pore and cell death, while meeting dynamic cellular energy demands. How this is accomplished is controversial. Here we define a regulatory mechanism of MCU-channel activity in which cytoplasmic Ca2+regulation of intermembrane space-localized MICU1/2 is controlled by Ca2+-regulatory mechanisms localized across the membrane in the mitochondrial matrix. Ca2+that permeates through the channel pore regulates Ca2+affinities of coupled inhibitory and activating sensors in the matrix. Ca2+binding to the inhibitory sensor within the MCU amino terminus closes the channel despite Ca2+binding to MICU1/2. Conversely, disruption of the interaction of MICU1/2 with the MCU complex disables matrix Ca2+regulation of channel activity. Our results demonstrate how Ca2+influx into mitochondria is tuned by coupled Ca2+-regulatory mechanisms on both sides of the inner mitochondrial membrane.

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The utilization of iron and its complexes by mammalian mitochondria

Biochemical Journal ◽

10.1042/bj1281043 ◽

1972 ◽

Vol 128 (5) ◽

pp. 1043-1055 ◽

Cited By ~ 44

Author(s):

R. Barnes ◽

J. L. Connelly ◽

O. T. G. Jones

Keyword(s):

Mitochondrial Membrane ◽

Nadh Dehydrogenase ◽

Oxygen Electrode ◽

Permeability Barrier ◽

Antimycin A ◽

Inner Mitochondrial Membrane ◽

Mammalian Mitochondria ◽

Ferrioxamine B ◽

Lag Period ◽

Subsequent Incorporation

Sonicated mitochondria catalyse the reduction of ferric salts, and the subsequent incorporation of Fe2+ into haem, when provided with a reducing substrate such as succinate or NADH. The rate of haem synthesis was low under aerobic conditions and, after a short lag period, accelerated once anaerobic conditions were achieved; it was insensitive to antimycin A. The lag period was decreased by preincubating the mitochondria with NADH and Fe3+. Newly formed Fe2+ was autoxidized rapidly and the consequent O2 uptake was measured with an oxygen electrode to determine the rate of enzymic formation of Fe2+ from FeCl3; this reaction was rapid in sonicated mitochondria provided with NADH or succinate and was insensitive to antimycin A. The reaction was very slow in intact mitochondria, suggesting a permeability barrier to Fe3+ ions. This system was used to test the permeability of the mitochondrial membrane to various iron complexes of biological importance. Of the compounds tested only ferrioxamine G appeared to penetrate readily and the iron of this complex was reduced when intact mitochondria were supplied with succinate or NADH-linked substrates. The reduction was insensitive to rotenone or antimycin A. Both ferrioxamine G and ferrioxamine B were, however, reduced by particles. The membrane fraction of sonicated mitochondria was necessary for the reduction. The rate of ferrioxamine B reduction by sonicated mitochondria was measured by a dual-wavelength spectrophotometric assay and was found to be stimulated in conditions where the Fe2+ produced was utilized for haem synthesis. The addition of FeCl3 to anaerobic particles caused an oxidation of cytochrome b when this region of the respiratory chain was isolated by treatment with rotenone and antimycin A. These results suggest that the reduction of ferric iron and its complexes occurs inside the inner mitochondrial membrane in proximity to ferrochelatase. Possible sites for this reduction are the flavoproteins, succinate and NADH dehydrogenase.

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Strongly coupled transmembrane mechanisms control MCU-mediated mitochondrial Ca2+ uptake

10.1101/2020.04.07.029637 ◽

2020 ◽

Author(s):

Horia Vais ◽

Riley Payne ◽

Carmen Li ◽

J. Kevin Foskett

Keyword(s):

Mitochondrial Membrane ◽

Permeability Transition Pore ◽

Permeability Transition ◽

Regulatory Mechanisms ◽

Intermembrane Space ◽

Channel Activity ◽

Inner Mitochondrial Membrane ◽

Strongly Coupled ◽

Energy Demands ◽

The Matrix

Ca2+ uptake by mitochondria regulates bioenergetics, apoptosis, and Ca2+ signaling. The primary pathway for mitochondrial Ca2+ uptake is the mitochondrial calcium uniporter (MCU), a Ca2+-selective ion channel in the inner mitochondrial membrane. MCU-mediated Ca2+ uptake is driven by the sizable inner-membrane potential generated by the electron-transport chain. Despite the large thermodynamic driving force, mitochondrial Ca2+ uptake is tightly regulated to maintain low matrix [Ca2+] and prevent opening of the permeability transition pore and cell death, while meeting dynamic cellular energy demands. How this is accomplished is controversial. Here we define a regulatory mechanism of MCU-channel activity in which cytoplasmic Ca2+ regulation of intermembrane space-localized MICU1/2 is controlled by strongly-coupled Ca2+-regulatory mechanisms localized across the membrane in the mitochondrial matrix. Ca2+ that permeates through the channel pore regulates Ca2+ affinities of coupled inhibitory and activating sensors in the matrix. Ca2+ binding to the inhibitory sensor within the MCU amino-terminus closes the channel despite Ca2+ binding to MICU1/2. Conversely, disruption of the interaction of MICU1/2 with the MCU complex abolishes matrix Ca2+ regulation of channel activity. Our results demonstrate how Ca2+ influx into mitochondria is tuned by coupled Ca2+-regulatory mechanisms on both sides of the inner mitochondrial membrane.

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