Figure 2.

Ca²⁺-dependent/CAT-insensitive adenine nucleotide uptake through SCaMC-1. (a–c) 143B cells transiently expressing mitochondrial luciferase¹⁷ were permeabilized and 100 μM luciferin was added to report mitochondrial ATP levels. The black traces indicate control cells, whereas the red traces indicate SCaMC-1-KD cells. (a) ANT activity in the absence of Ca²⁺: The medium contained 100 μM EGTA and 1 mM ATP was added when indicated. The traces marked by arrows were obtained from cells treated with 10 μM CAT. (b and c) SCaMC-1 activity: The medium contained 100 μM Ca²⁺ and 10 μM CAT, and 1 mM ATP (b) or 2 mM ADP (c) was added when indicated. The bar charts indicate the transport rate of SCaMC-1-KD (KD) cells as compared with controls (C) (mean±S.E.M., n=6). (d–f) To determine the Ca²⁺ sensitivity of SCaMC-1-mediated ATP/ADP transport, mitochondrial ATP changes were measured in isolated mitochondria from COS-7 cells stably expressing mitochondrial luciferase after addition of 100 μM luciferin. (d) The medium contained 10 μM CAT and free Ca²⁺ at the indicated concentrations, and 1 mM ATP was added when indicated. (e) Efflux of ATP from mitochondria was triggered by addition of 10 mM Pi followed by different Ca²⁺ concentrations. The efflux was reverted at the end of the experiment by addition of 1 mM ADP. (f) Ca²⁺ activation of CAT-insensitive ATP transport. Transport rates were calculated from the slopes in panel d after ATP addition and fit to the following equation: V=V₀+[(V_max−V₀) × [Ca²⁺]^N)/(S_0.5^N+[Ca²⁺]^N)] (where V is transport activity at each [Ca²⁺] V₀ is the basal transport rate at [Ca²⁺]≈0; V_max is the maximal activity; N is the hill coefficient; and S_0.5 is the Ca²⁺ concentration, which generates half-maximal transport activity). Pooled data from six independent experiments are shown