Figure 2. Increasing the propensity of the MPTP to open with either KSCN buffer or PAO increases the number of GNX-4975-binding sites.

Opening of the MPTP was measured as described in Figure 1 but for each separate mitochondrial preparation a single protein concentration (1 mg/ml) was employed. In (A), parallel measurements were made using either the standard 150 mM KSCN medium or one in which the KSCN was replaced with 125 mM KCl+2.5 mM potassium phosphate. For both media, MPTP opening was initiated by addition of 150 μM Ca²⁺ and rates of swelling were expressed as a percentage of the zero inhibitor rate in the KCl medium. Mean data ± S.E.M. (error bars) for five separate mitochondrial preparations are given. Data were fitted as described in Figure 1 and generated mean derived values (±S.E.M., n=5) for E_t of 9.43±1.4 and 19.2±2.2 pmol/mg in KCl and KSCN medium respectively (P ≤ 0.01 calculated using the unpaired Student's t test). The corresponding calculated K_i values were 1.68±0.13 and 2.29±0.17 nM respectively. In (B), mitochondria were either untreated (control) or pre-incubated with 20 μM PAO for 3 min before addition of 50 μM Ca²⁺ in KSCN medium to initiate swelling. Mean data ± S.E.M. (error bars) for six separate mitochondrial preparations are given. The rate of swelling in the absence of both PAO and GNX-4975 was set at 100 and all other rates calculated relative to this. Data, fitted as in (A) gave mean values (±S.E.M.) for E_t in the absence and presence of PAO of 11.9±0.92 and 17.2±1.3 pmol/mg respectively (P<0.01) with corresponding K_i values of 1.93±0. 21 and 2.56±0.14 nM.