EGTA inhibits reverse uniport-dependent Ca2+ release from uncoupled mitochondria. Possible regulation of the Ca2+ uniporter by a Ca2+ binding site on the cytoplasmic side of the inner membrane.
When rat liver mitochondria are allowed to accumulate Ca2+, treated with ruthenium red to inhibit reverse activity of the Ca2+ uniporter, and then treated with an uncoupler, they release Ca2+ and endogenous Mg2+ and undergo large amplitude swelling with ultrastructural expansion of the matrix space. These effects are not produced by Ca2+ plus uncoupler alone. Like other "Ca2+-releasing agents" (i.e. N-ethylmaleimide, t-butylhydroperoxide, oxalacetate, etc.), the development of nonspecific permeability produced by ruthenium red plus uncoupler requires accumulated Ca2+ specifically and is antagonized by inhibitors of phospholipase A2. The permeability responses are also antagonized by ionophore A23187, indicating that a rapid pathway for Ca2+ efflux from deenergized mitochondria is necessary to prevent the development of nonspecific permeability. EGTA can be substituted for ruthenium red to produce the nonspecific permeability change in Ca2+-loaded, uncoupler-treated mitochondria. The permeability responses to EGTA plus uncoupler again require accumulated Ca2+ specifically and are antagonized by inhibitors of phospholipase A2 and by ionophore A23187. The equivalent effects of ruthenium red and EGTA on uncoupled, Ca2+-containing mitochondria indicate that reducing the extramitochondrial Ca2+ concentration to the subnanomolar range produces inhibition of reverse uniport activity. It is proposed that inhibition reflect regulation of the uniporter by a Ca2+ binding site which is available from the cytoplasmic side of the inner membrane. EDTA cannot substitute for EGTA to induce nonspecific permeability in Ca2+-loaded, uncoupled mitochondria. Furthermore, EDTA inhibits the response to EGTA with an I50 value of approximately 10 microM. These data suggest that the uniporter regulatory site also binds Mg2+. The data suggest further that Mg2+ binding to the regulatory site is necessary to inhibit reverse uniport activity, even when the site is not occupied by Ca2+.
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008854:Microscopy, Electron; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012430:Ruthenium Red / Q000494:pharmacology; D014475:Uncoupling Agents / Q000494:pharmacology*
D015220:Calcium Channels; D002135:Calcium-Binding Proteins; D014475:Uncoupling Agents; C034703:mitochondrial calcium uniporter; D012430:Ruthenium Red; D004533:Egtazic Acid; D004492:Edetic Acid; D002118:Calcium