Fig. 9. The PT pore increases diffusibility of intermembrane space proteins but is not involved in the action of tBid at low concentrations. CyP-D(+) mitochondria were used throughout. (A) Calcein-loaded mitochondria were incubated in SRM containing either 400 µM Ca²⁺ (400 Ca) with/without 1 µM CSA, or 1 µM buffered free Ca²⁺ (1 Ca) with/without 20 nM tBid. The Ca²⁺ buffer comprised 5 mM EGTA and 4 mM CaCl₂. After incubation for the time specified, mitochondria were sedimented and calcein in the supernatant was determined fluorometrically. (B) Calcein-loaded mitochondria were incubated in SRM containing 400 µM Ca²⁺, 1 µM free Ca²⁺ and 20 nM tBid, as indicated. Cytochrome c release was measured at the times specified. The data of A and B are representative of three such experiments. (C) The two-step procedure was used to determine the diffusibility rate constants for cytochrome c (white columns) and Smac (black columns) in the presence of 20 nM tBid. Other additions were 400 µM Ca²⁺, 1 µM buffered free Ca²⁺ and 1 µM CSA, as indicated. Means ± s.e.m. (four experiments).