Abstract

Cell function depends on the distribution of cytosolic and mitochondrial factors across the outer mitochondrial membrane (OMM). Passage of metabolites through the OMM has been attributed to the voltage-dependent anion-selective channel (VDAC), which can form a large conductance and permanently open a channel in lipid bilayers. However, recent data indicate that the transport of metabolites through the OMM is controlled in the cells. Recognizing that the bilayer studies had been commonly conducted at supraphysiological [Ca2+] and [K+], we determined the effect of Ca2+ on VDAC activity. In liposomes, the purified VDAC displays Ca2+-dependent control of the molecular cut-off size and shows Ca2+-regulated Ca2+ permeability in the physiological [Ca2+] range. In bilayer experiments, at submicromolar [Ca2+], the purified VDAC or isolated OMM does not show sustained large conductance but rather exhibits gating between a nonconducting state and various subconductance states. Ca2+ addition causes a reversible increase in the conductance and may evoke channel opening to full conductance. Furthermore, single cell imaging data indicate that Ca2+ may facilitate the cation and ATP transport across the OMM. Thus, the VDAC gating is dependent on the physiological concentrations of cations, allowing the OMM to control the passage of ions and some small molecules. The OMM barrier is likely to decrease during the calcium signal.

Highlights

  • The voltage-dependent anion-selective channel (VDAC)/mitochondrial porin is the most abundant protein of the outer mitochondrial membrane (OMM)

  • Characterization of Isolated VDAC and OMM—To study VDAC activity, both VDAC-prep and OMM-prep were isolated from rat liver mitochondria (Fig. 1)

  • The enrichment of the 32-kDa band in the OMM-prep was apparent when compared with the mitochondrial homogenate (Mito), inner mitochondrial membrane (IMM), and CP fractions (Fig. 1A, top)

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Summary

Introduction

The voltage-dependent anion-selective channel (VDAC)/mitochondrial porin is the most abundant protein of the OMM. Selective permeabilization of the OMM by tBid, a proapoptotic protein [21], and by overexpression of VDAC [22] was reported to enhance the inositol 1,4,5-trisphosphate-induced mitochondrial matrix [Ca2ϩ] ([Ca2ϩ]m) signal. These results suggested that the OMM Ca2ϩ permeability could limit rapid Ca2ϩ uptake to the mitochondria during physiological calcium signals. To establish the VDAC behavior in the presence of physiological (submicromolar) and elevated [Ca2ϩ] as well, we set up both liposomal transport and BLM experiments with purified VDAC and OMM and fluorescence imaging of mitochondrial ion transport in single cells

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