Lack of voltage-dependent anion channel in human mitochondrial myopathies.

SIR—In several hundreds of patients with a mitochondrial myopathy an enzyme defect in mitochondrial energy metabolism is identified. However, in a substantial number of subjects no enzyme defect can be detected, although diminished substrate oxidation and ATP production rates are found in their muscle in vitro. The hypothesis, that in this group of patients proteins for transport of various ions and substrates across mitochondrial membranes might be affected, led us to study these transport proteins more systematically. Among 40 investigated patients, 1 was found with a diminished content of the voltage-dependent anion channel (VDAC or human porin). Figure: Immunoblots with VDAC and ANT antisera Samples of muscle 600 g supernatants from patient (lanes 4) and 3 controls (lanes 3, 5, 6) were dissolved in sodium dodecylsulphate and loaded on polyacrylamide gels (10-15% gradient), together with biotinylated molecular weight markers (lanes 1: 39-8, 29-0, 20.1 kDa) and purified ANT (lanes 2: 29 kDa). All lanes of controls and patient contained 100 mU cytochrome c oxidase (the patient's muscle had normal cytochrome c oxidase activity). After electrophoresis (Phast System, Pharmacia-LKB, Woerden, Netherlands) proteins were electroblotted to nitrocellulose. Immunolabelling of the proteins was done with 4 different antibodies: human B-lymphocyte VDAC monoclonal antibodies from 2 different cell lines (mAb-No 4 in panel I and mAb-No 6 in panel 11),' anti-bovine heart VDAC2 (panel III), and anti-C-terminal bovine heart ANT3 (panel IV). Immunodetection was done applying the Biotin-ECL method (Amersham International, UK). The patient was born at term from non-consanguineous parents. He had dysmorphism, hypotonia, respiration and feeding problems, and seizures. He was treated for hypothyroidism. Because of lactic acidosis a quadriceps muscle biopsy specimen was taken at the age of 2 years. Decreased rates of pyruvate and malate oxidation and of ATP production were found. The content of the membrane transport proteins VDAC and ATP/ADP translocator (ANT) were estimated immunochemically. The amount of VDAC protein (35 kDa, marked by-v-in figure) appeared to be clearly decreased, with use of monoclonal anti-N-terminal human VDAC (figure; panels I, II), and polyclonal anti-bovine-heart VDAC (panel III) antibodies. The ANT content (29 kDa, marked by-A-) appeared to be slightly reduced. The polyclonal antiserum also reacted with other supernatant proteins (panel III). In this panel the VDAC deficiency of the patient was additionally confirmed: apart from the VDAC protein the amounts of all proteins, including ANT, were similar to those in controls. To our knowledge, this case is the …