Potassium affects MMP and OCR in isolated mitochondria by altering the ionic strength of the mitochondrial environment. (A) Effect of potassium ion (as KCl) on the MMP of isolated mitochondria, as measured by the TMRM ratio (Materials and methods). Conditions were applied sequentially to the same wells, such that the same samples of mitochondria were exposed sequentially to substrates (green), ADP (orange), and FCCP (purple). Slopes of “-adp, -fccp” (green) and “+adp, -fccp” (orange) conditions are significantly different from zero (P < 0.001 in each case). Differences between -adp, -fccp (green) and +adp, -fccp (orange) at every KCl concentration have a P-value ≤0.001. Data are from three experiments with three replicates per condition. (B) Isolated mitochondria were incubated in 250 mM KCl for 15 min and then switched to the indicated buffer by dilution. Data are from two experiments with three replicates per condition. (C) MMP of isolated mitochondria incubated in the indicated buffers. Data are from two experiments, with three replicates per condition. (D) Isolated mitochondria were incubated with the indicated substrate(s) in 50 or 250 mM KCl. Decline in MMP with increased KCl was equivalent for all substrates (P > 0.08 across comparisons of substrate by KCl interaction terms). Data are from three experiments, with three replicates per condition. In A–D, to facilitate comparisons across experiments, TMRM ratios were normalized to the condition containing all substrates, 250 mM KCl, and no FCCP or ADP. (E) OCR of isolated mitochondria incubated in the indicated amount of potassium. Data are from two experiments, with 15 replicates per condition. (F) Cytochrome c oxidase activity as a function of ionic strength in milliequivalents (mEq.) Ionic strength was altered using KCl (green) or 40 mM KCl plus different amounts of NaCl (orange) to obtain the indicated ionic strength. Data are from four experiments, with 3–4 replicates per experiment. All error bars show 95% CIs.