Abstract
Mitochondria from transgenic mice, expressing enzymatically active mitochondrial creatine kinase in liver, were analyzed for opening of the permeability transition pore in the absence and presence of creatine kinase substrates but with no external adenine nucleotides added. In mitochondria from these transgenic mice, cyclosporin A-inhibited pore opening was delayed by creatine or cyclocreatine but not by beta-guanidinopropionic acid. This observation correlated with the ability of these substrates to stimulate state 3 respiration in the presence of extramitochondrial ATP. The dependence of transition pore opening on calcium and magnesium concentration was studied in the presence and absence of creatine. If mitochondrial creatine kinase activity decreased (i.e. by omitting magnesium from the medium), protection of permeability transition pore opening by creatine or cyclocreatine was no longer seen. Likewise, when creatine kinase was added externally to liver mitochondria from wild-type mice that do not express mitochondrial creatine kinase in liver, no protective effect on pore opening by creatine and its analog was observed. All these findings indicate that mitochondrial creatine kinase activity located within the intermembrane and intercristae space, in conjunction with its tight functional coupling to oxidative phosphorylation, via the adenine nucleotide translocase, can modulate mitochondrial permeability transition in the presence of creatine. These results are of relevance for the design of creatine analogs for cell protection as potential adjuvant therapeutic tools against neurodegenerative diseases.
Highlights
Most vertebrate cell types express cytosolic as well as mitochondrial isoforms of the enzyme creatine kinase (CK).1 CK
In an attempt to define the role of mitochondrial creatine kinase on MPT, we have shown in an earlier study that isolated liver mitochondria from transgenic mice containing mitochondrial CK (mtCK) did not respond by MPT pore opening upon treatment with Ca2ϩ plus atractyloside if Cr or CyCr were present in the medium carbonyl cyanide p-trifluoromethoxyphenylhydrazone; GPA, -guanidinopropionic acid; IMS, intermembrane space; MPT, mitochondrial permeability transition; mtCK, mitochondrial creatine kinase; PCr, phosphocreatine; Mops, 4-morpholinepropanesulfonic acid
In the present study we investigated these effects in more detail, asking the question of whether inhibition of MPT pore opening by CK substrates depends on mtCK activity and whether the tight functional coupling between the CK reaction and oxidative phosphorylation, demonstrated recently to take place in situ [25], would lead to cycling of mitochondrial adenine nucleotides, resulting in net production of phosphorylated CK substrates
Summary
Most vertebrate cell types express cytosolic as well as mitochondrial isoforms of the enzyme creatine kinase (CK). CK. In liver mitochondria from control mice without mtCK, pore opening induced by Ca2ϩ plus atractyloside was independent on the presence or absence of Cr or CyCr. In the present study we investigated these effects in more detail, asking the question of whether inhibition of MPT pore opening by CK substrates depends on mtCK activity and whether the tight functional coupling between the CK reaction and oxidative phosphorylation, demonstrated recently to take place in situ [25], would lead to cycling of mitochondrial adenine nucleotides, resulting in net production of phosphorylated CK substrates. Our results indicate that substrate channeling between mtCK and adenine nucleotide translocase (ANT) takes place in a tight functionally coupled microcompartment that seems absolutely required for protection of MPT pore opening by creatine
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