Bcl-2 prevents apoptotic mitochondrial dysfunction by regulating proton flux

Abstract

We and others have recently shown that loss of the mitochondrial membrane potential (Δψ) precedes apoptosis and chemical-hypoxia-induced necrosis and is prevented by Bcl-2. In this report, we examine the biochemical mechanism used by Bcl-2 to prevent Δψ loss, as determined with mitochondria isolated from a cell line overexpressing human Bcl-2 or from livers of Bcl-2 transgenic mice. Although Bcl-2 had no effect on the respiration rate of isolated mitochondria, it prevented both Δψ loss and the permeability transition (PT) induced by various reagents, including Ca²⁺, H₂O₂, and tert-butyl hydroperoxide. Even under conditions that did not allow PT, Bcl-2 maintained Δψ, suggesting that the functional target of Bcl-2 is regulation of Δψ but not PT. Bcl-2 also maintained Δψ in the presence of the protonophore SF6847, which induces proton influx, suggesting that Bcl-2 regulates ion transport to maintain Δψ. Although treatment with SF6847 in the absence of Ca²⁺ caused massive H⁺ influx in control mitochondria, the presence of Bcl-2 induced H⁺ efflux after transient H⁺ influx. In this case, Bcl-2 did not enhance K⁺ efflux. Furthermore, Bcl-2 enhanced H⁺ efflux but not K⁺ flux after treatment of mitochondria with Ca²⁺ or tert-butyl hydroperoxide. These results suggest that Bcl-2 maintains Δψ by enhancing H⁺ efflux in the presence of Δψ-loss-inducing stimuli.