Functional activity of permeability transition pore in energized and deenergized rat liver mitochondria

Abstract

permeability transition pore (mpTp) opening was studied under energized and deenergized conditions in rat liver mitochondria, and the effect of membrane depolarization on mPTP activity was evaluated. To assess mpTp activity, cyclosporine-sensitive swelling and cyclosporine sensitive ca 2+ efflux from mitochondria was studied using light absorbance techniques. In energized mitochondria, mpTp opening in subconductance states, at [ca 2+ ] ≤ K a , contributed positively to the rate of respiration, without affecting ΔΨ m . Threshold ca 2+ concentrations were found, which excess resulted in fast mitochondrial depolarization upon mpTp opening. An estimate of mpTp activity by cyclosporine-sensitive ca 2+ transport under energized and deenergized conditions showed that membrane depolarization by protonophore cccp essentially increased initial rate (V 0 ), at simultaneous decrease of the half-time (t 1/2 ) of ca 2+ efflux, which indicated mPTP activation, as compared to energized mitochondria. however, only partial release of ca 2+ via mpTp upon membrane depolarization was observed. With the use of selective blockers of ca 2+ uniporter and mpTp, ruthenium red (rr) and cyclosporine A (csA), partial contribution of ca 2+ uniporter and mpTp in ca 2+ transport was found. "Titration" of ca 2+ transport by adding RR at different times from the onset of depolarization showed that depolarization dramatically reduced "life span" of mpTp as compared to energized mitochondria, which agreed with the kinetic characteristics of csA-sensitive ca 2+ transport after the abolition of ΔΨ m . ca 2+ added from the outer side of mitochondrial membrane produced dual effect on mPTP activity: activation at the onset of depolarization, but consequent promotion of mPTP closure. Based on the experiments, it was concluded that mitochondrial energization was required for prolonged mpTp functioning in sub-conductance states, whereas membrane depolarization promoted the transition of mpTp to inactive state during calcium release from mitochondria. k e y w o r ds: rat liver mitochondria, calcium, permeability transition pore, ca 2+ transport, membrane potential.