Voltage-dependent anion channels (VDACs), also known as mitochondrial porins, are small channel proteins involved in the translocation of metabolites across the mitochondrial outer membrane. A single channelforming protein is found in yeast, whereas higher eukaryotes express multiple VDACs, with humans and mice each harboring three distinct channels (VDAC1-3) encoded by separate genes. To begin to assess the functions of each of the three isoforms, the VDAC3 gene was inactivated by targeted disruption in embryonic stem cells. Here we show that mice lacking VDAC3 are healthy, but males are infertile. Although there are normal sperm numbers, the sperm exhibit markedly reduced motility. Structural defects were found in twothirds of epididymal axonemes, with the most common abnormality being loss of a single microtubule doublet at a conserved position within the axoneme. In testicular sperm, the defect was only rarely observed, suggesting that instability of a normally formed axoneme occurs with sperm maturation. In contrast, tracheal epithelial cilia showed no structural abnormalities. In addition, skeletal muscle mitochondria were abnormally shaped, and activities of the respiratory chain complexes were reduced. These results demonstrate that axonemal defects may be caused by associated nonaxonemal components such as mitochondrial channels and illustrate that normal mitochondrial function is required for stability of the axoneme.

Voltage-dependent anion channels (VDACs) are poreforming proteins found in the outer mitochondrial membrane of all eucaryotes. VDACs are the binding sites for several cytosolic enzymes, including the isoforms of hexokinase and glycerol kinase. VDACs have recently been shown to conduct ATP when in the open state, allowing bound kinases preferential access to mitochondrial ATP and providing a possible mechanism for the regulation of adenine nucleotide flux. Two human VDAC cDNAs have been described previously, and we recently reported the isolation of mouse VDAC1 and VDAC2 cDNAs, as well as a third novel VDAC cDNA, designated VDAC3. In this report we describe the structural organization of each mouse VDAC gene and demonstrate that, based on conserved exon/intron boundaries, the three VDAC isoforms belong to a single gene family. Voltage-dependent anion channels (VDACs, 1 also known as mitochondrial porins) are 30 -35-kilodalton (kDa) pore-forming proteins found in the outer mitochondrial membrane of eucaryotes (reviewed in Ref. 1). VDACs play a role in the regulated flux of metabolites across the outer mitochondrial membrane, but their exact cellular role is not well understood. VDACs from a variety of organisms have remarkably similar electrophysiological properties (1). Gating of the channel depends upon the transmembrane potential, while its voltage sensitivity is modulated by an intermembrane protein (2). VDACs are "open" at low transmembrane potentials, with a preference for anions such as phosphate, chloride, and adenine nucleotides. At higher transmembrane potentials, VDACs are in a "closed" configuration and more selective for cations (2, 3). VDACs have been shown to reversibly bind several cytosolic kinases, including glycerol kinase and the hexokinase isoforms I-IV (reviewed in Ref. 4). This interaction is believed to allow bound kinases preferential access to mitochondrial ATP derived from oxidative phosphorylation (5, 6). VDACs have also been associated with the adenine nucleotide translocator of the inner mitochondrial membrane and octomeric creatine kinase of the intermembrane space (6 -8). It has been suggested that this complex has properties resembling the permeability transition pore (9).A direct demonstration of voltage-gated ATP flux through VDAC was recently reported (10). Physiological concentrations of NADH also affect VDAC permeability, suggesting one possible mechanism for the observed ability of glycolysis to suppress oxidative phosphorylation (the Crabtree effect; Refs. 11 and 12). VDACs have been identified as a component of the peripheral benzodiazepine receptor complex (13), which is linked to steroid biosynthesis (14). Finally, VDACs have been shown to co-purify with the brain ␥-aminobutyric acid subunit A receptor complex (15). cDNAs encoding two human VDAC isoforms were reported by . We have previously described the isolation of mouse orthologues for VDAC1 and VDAC2, as well as a novel mouse VDAC termed VDAC3 (17,18). Each isoform is 65-70% identical to the other isoforms. Phylogene...

Mitochondrial outer membrane permeability is conferred by a family of porin proteins. Mitochondrial porins conduct small molecules and constitute one component of the permeability transition pore that opens in response to apoptotic signals. Because mitochondrial porins have significant roles in diverse cellular processes including regulation of mitochondrial ATP and calcium flux, we sought to determine their importance in learning and synaptic plasticity in mice. We show that fear conditioning and spatial learning are disrupted in porin-deficient mice. Electrophysiological recordings of porin-deficient hippocampal slices reveal deficits in long and short term synaptic plasticity. Inhibition of the mitochondrial permeability transition pore by cyclosporin A in wild-type hippocampal slices reproduces the electrophysiological phenotype of porin-deficient mice. These results demonstrate a dynamic functional role for mitochondrial porins and the permeability transition pore in learning and synaptic plasticity.

The channel-forming protein called VDAC forms the major pathway in the mitochondrial outer membrane and controls metabolite flux across that membrane. The different VDAC isoforms of a species may play different roles in the regulation of mitochondrial functions. The mouse has three VDAC isoforms (VDAC1, VDAC2 and VDAC3). These proteins and different versions of VDAC3 were expressed in yeast cells (S. cerevisiae) missing the major yeast VDAC gene and studied using different approaches. When reconstituted into liposomes, each isoform induced a permeability in the liposomes with a similar molecular weight cutoff (between 3,400 and 6,800 daltons based on permeability to polyethylene glycol). In contrast, electrophysiological studies on purified proteins showed very different channel properties. VDAC1 is the prototypic version whose properties are highly conserved among other species. VDAC2 also has normal gating activity but may exist in 2 forms, one with a lower conductance and selectivity. VDAC3 can also form channels in planar phospholipid membranes. It does not insert readily into membranes and generally does not gate well even at high membrane potentials (up to 80 mV). Isolated mitochondria exhibit large differences in their outer membrane permeability to NADH depending on which of the mouse VDAC proteins was expressed. These differences in permeability could not simply be attributed to different amounts of each protein present in the isolated mitochondria. The roles of these different VDAC proteins are discussed.

Voltage-dependent anion channels (VDACs, also known as mitochondrial porins) are small pore-forming proteins of the mitochondrial outer membrane found in all eukaryotes. Mammals harbor three distinct VDAC isoforms, with each protein sharing 65-70% sequence identity. Deletion of the yeast VDAC1 gene leads to conditional lethality that can be partially or completely complemented by the mammalian VDAC genes. In vitro, VDACs conduct a variety of small metabolites and in vivo they serve as a binding site for several cytosolic kinases involved in intermediary metabolism, yet the specific physiologic role of each isoform is unknown. Here we show that mouse embryonic stem cells lacking each isoform are viable but exhibit a 30% reduction in oxygen consumption. VDAC1 and VDAC2 deficient cells exhibit reduced cytochrome c oxidase activity, whereas VDAC3 deficient cells have normal activity. These results indicate that VDACs are not essential for cell viability and we speculate that reduced respiration in part reflects decreased outer membrane permeability for small metabolites necessary for oxidative phosphorylation.