Key regions of VDAC1 functioning in apoptosis induction and regulation by hexokinase.

The voltage-dependent anion channel (VDAC), located in the mitochondrial outer membrane, functions as gatekeeper for the entry and exit of mitochondrial metabolites, and thus controls cross-talk between mitochondria and the cytosol. VDAC also serves as a site for the docking of cytosolic proteins, such as hexokinase, and is recognized as a key protein in mitochondria-mediated apoptosis. The role of VDAC in apoptosis has emerged from various studies showing its involvement in cytochrome c release and apoptotic cell death as well as its interaction with proteins regulating apoptosis, including the mitochondria-bound isoforms of hexokinase (HK-I, HK-II). Recently, the functional HK-VDAC association has shifted from being considered in a predominantly metabolic light to the recognition of its major impact on the regulation of apoptotic responsiveness of the cell. Here, we demonstrate that the HK-VDAC1 interaction can be disrupted by mutating VDAC1 and by VDAC1-based peptides, consequently leading to diminished HK anti-apoptotic activity, suggesting that disruption of HK binding to VDAC1 can decrease tumor cell survival. Indeed, understanding structure-function relationships of VDAC is critical for deciphering how this channel can perform such a variety of differing functions, all important for cell life and death. By expressing VDAC1 mutants and VDAC1-based peptides, we have identified VDAC1 amino acid residues and domains important for interaction with HK and protection against apoptosis. These include negatively- and positively-charged residues, some of which are located within beta-strands of the protein. The N-terminal region of VDAC1 binds HK-I and prevents HK-mediated protection against apoptosis induced by STS, while expression of a VDAC N-terminal peptide detaches HK-I-GFP from mitochondria. These findings indicate that the interaction of HK with VDAC1 involves charged residues in several beta-strands and in the N-terminal domain. Displacing HK, serving as the 'guardian of the mitochondrion', from its binding site on VDAC1 may thus be exploited as an approach to cancer therapy.

Retina expresses a novel variant of the ryanodine receptor.

Calcium released from intracellular stores via the ryanodine receptor (RyR) mediates a variety of signalling processes. We previously showed that retina expresses the three known types of RyR, but retinal membrane preparations exhibit unique characteristics such as Ca2+-independent [3H]ryanodine-binding and inhibition by caffeine. We have heretofore suggested that the major retinal RyR isoform is novel. The present study aimed to identify this receptor isoform and to localize RyR in mammalian retina. Immunoblotting with specific and pan-antibodies showed that the major retinal RyR has a mobility similar to that of RyR2 or RyR3. Real-time PCR revealed that the major type is RyR2, and RT-PCR followed by sequencing showed a transcript that encodes a protein with approximately 99% identity to RyR2, yet lacking two regions of seven and 12 amino acids and including an additional insertion of eight amino acids. An antibody against RyR2 localized this type to somas and primary dendrites of most retinal neurons. An antibody against RyR1 localized RyR to most somas but also revealed staining in photoreceptor outer segments, concentrated on the disk membranes at their rim. The ryanodine-binding properties and the electrophoretic mobility of RyR from the outer segments were similar to those of the whole retinal preparation. The results thus identify a novel variant of RyR2 which can contribute to regulating photoreceptor Ca2+ concentrations. The restricted localization of the outer segment RyR to the disk rim suggests that its activation mechanism involves a coupling between retinal RyR and the cGMP-gated channel.