Molecular and functional characterization of VDAC2 purified from mammal spermatozoa

VDAC (voltage-dependent anion channel) is the pore-forming protein located in the outer mitochondrial membrane.In higher eukaryotes, three genes encode VDAC. Nevertheless, the knowledge of VDAC isoforms is mainly restrictedto VDAC1, the only isoform that has been characterized from living tissues to date. We have highly enriched theisoform VDAC2 using as starting material bovine spermatozoa. VDAC2 was obtained in the hydroxyapatite/celitepass-through of sperm proteins solubilized with Triton X-100. This fraction showed in SDS/PAGE two major bands andone faint band in the molecular mass range of 30–35 kDa. Two-dimensional electrophoresis resolved these bands inten spots with various Coomassie Blue staining intensities. Western-blot analysis with antibodies monospecific foreach isoform and MS peptide sequencing showed that the main protein resolved in electrophoresis was VDAC2 withminor contaminations of the other isoforms. Proteomic analysis of the higher molecular mass VDAC2 protein allowedthe coverage of the whole protein with the exception of the tripeptide A24AR26. In the same material, the presenceof two possible amino acid substitutions (T88 to L88 and A97 to Q97) was revealed. Reconstitution of VDAC2 pores inplanar lipid bilayers showed typical features of mitochondrial porins. Stepwise increases in membrane conductancewere observed with a predominant conductance of approx. 3.5 nS (nanoSiemens) in 1 M KCl. Very often, small shortlivedfluctuations were observed with single-channel conductance of approx. 1.5 nS. Bovine spermatozoa VDAC2 wasanion selective and showed voltage dependence. The present study is the first work to report the purification andcharacterization of VDAC2 from a mammalian tissue.