MODULATION OF THE MITOCHONDRIAL CYCLOSPORINE A-SENSITIVE PERMEABILITYTRANSITION PORE BY MATRIX PH - EVIDENCE THAT THE PORE OPEN CLOSED PROBABILITY IS REGULATED BY REVERSIBLE HISTIDINE PROTONATION

Energized mitochondria in sucrose medium take up a Ca2+ pulse but do n ot show opening of the permeability transition pore (MTP) upon membran e depolarization by uncoupler. This is due to locking of the pore in t he closed conformation by matrix acidification and fast Ca2+ efflux fo llowing membrane depolarization (Petronilli, V., Cola, C., & Bernardi P. (1993) J. Biol. Chem. 268, 1011-1016). Here we show that addition o f diethyl pyrocarbonate (DPC) prior to membrane depolarization restore s the ability of uncoupler to induce MTP opening. Since DPC does not m odify the rate and extent of matrix acidification and the rate and ext ent of Ca2+ release following addition of uncoupler, its effects on po re opening appear to be due to modification of histidyl residues regul ating the pore open-closed probability. This hypothesis was confirmed in studies with deenergized mitochondria incubated in potassium thiocy anate medium. While at acidic pH values pore opening is otherwise prev ented, DPC allows Ca2+-dependent pore opening at pH 6.5 in a process t hat maintains full sensitivity to cyclosporin A. Pore induction by DPC can be completely prevented and partially reversed by hydroxylamine, indicating that the effect of DPC can be specifically traced to carbet hoxylation of histidyl residue(s) rather than to reaction with tyrosyl or sulfhydryl groups, while the possible involvement of lysyl residue s cannot be excluded. Since DPC increases the pore open probability ev en at matrix pH values between 7.0 and 7.7, we propose that reversible protonation of one or more histidyl residues on the matrix side of th e MTP plays a role in the physiological modulation of pore opening.