(A) A HEK-293T cell mitoplast under differential interference contrast (far left), showing a typical figure-eight shape. The lobe bounded only by the mitochondrial inner membrane appears less dense (white arrow) than the lobe also bounded by the outer membrane (black arrow). Matrix-targeted mCherry demonstrates that the inner membrane forms a surface on both lobes (middle left). GFP-tagged mitofusin-1 (an outer membrane GTPase, middle right) reveals the outer membrane partially encapsulating only one lobe. Far right: merged image. (B) Western blots demonstrate reduced MCU expression following short-hairpin RNA-mediated knockdown of MCU but not GFP (control). ATP5B is a loading control. (C) Exemplar current traces demonstrate endogenous IMiCa in control knockdown (black trace), which is largely blocked by 100 nM RuR (red trace). Voltage ramps from −160 to +80 mV for 750 ms were delivered every 6 s from a holding potential of 0 mV. (D) Significantly reduced IMiCa after MCU knockdown. (E) Summary data, n = 6–10 per condition, error bars report SEM.