O. V. Akopova*, L. I. Kolchinskaya, V. I. Nosar

Bogomoletz Institute of Physiology, National Academy of Sciences of Ukraine, Kyiv;
*e-mail: ov_akopova@ukr.net

Received: 15 June 2020; Accepted: 13 November 2020

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 Ca²⁺ efflux from mitochondria was studied using light absorbance techniques. In energized mitochondria, mPTP opening in sub-conductance states, at [Ca²⁺] ≤ K_a, contributed positively to the rate of respiration, without affecting ΔΨ_m. Threshold Ca²⁺ concentrations were found, which excess resulted in fast mitochondrial depolarization upon mPTP opening. An estimate of mPTP activity by cyclosporine-sensitive Ca²⁺ transport under energized and deenergized conditions have shown that membrane depolarization by protonophore CCCP essentially increased initial rate (V₀), at simultaneous decrease of the half-time (t_1/2) of Ca²⁺ efflux, which indicated mPTP activation, as compared to energized mitochondria. However, only partial release of Ca²⁺ via mPTP upon membrane depolarization was observed. With the use of selective blockers of Ca²⁺ uniporter and mPTP, ruthenium red (RR) and cyclosporine A (CsA), partial contribution of Ca²⁺ uniporter and mPTP in Ca²⁺ transport was found. “Titration” of Ca²⁺ 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 Ca²⁺ transport after the abolition of ΔΨ_m. Ca²⁺ 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.