Effects of magnesium and nucleotides on the proton conductance of rat skeletal-muscle mitochondria

During oxidative phosphorylation most of the protons pumped out to the cyto sol across the mitochondrial inner membrane return to the matrix through th e ATP synthase, driving ATP synthesis. However, some of them leak back to t he matrix through a proton-conductance pathway in the membrane. When the AT P synthase is inhibited with oligomycin and ATP is not being synthesized, a ll of the respiration is used to drive the proton leak. We report here that Mg2+ inhibits the proton conductance in rat skeletal-muscle mitochondria. Addition of Mg2+ inhibited both oligomycin-inhibited respiration and the pr oton conductance, while removal of Mg2+ using EDTA activated these processe s. The proton conductance was inhibited by more than 80 % as free Mg2+ was raised from 25 nM to 220 mu M. HalF-maximal inhibition occurred at about 1 mu M free Mg2+, which is close to the contaminating free Mg2+ concentration in our incubations in the absence of added magnesium chelators. ATP, GTP, CTP, TTP or UTP at a concentration of 1mM increased the oligomycin-inhibite d respiration rate by about 50%. However, these NTP effects were abolished by addition of 2 mM Mg2+ and any NTP-stimulated proton conductance was expl ained completely by chelation of endogenous free Mg2+. The corresponding nu cleoside diphosphates (ADP, GDP, CDP, TDP or UDP) at 1 mM had no effect on oligomycin-inhibited respiration. We conclude that proton conductance in ra t skeletal-muscle mitochondria is very sensitive to free Mg2+ concentration but is insensitive to NTPs or NDPs at 1 mM.