Mitochondria-derived and extra-mitochondrial human type-1 porin are identical as revealed by amino acid sequencing and electrophysiological characterisation.

In mammalian cells porin channels are localised in both mitochondrial outer membranes and extra-mitochondrial membranes. We isolated mitochondria-derived porin of a human lymphoblastoid B cell line, determined its amino acid sequence and characterised its channel properties. Interestingly, the amino acid sequence of this porin preparation and, correspondingly, its electrophysiological characteristics in a reconstituted system were identical to those of 'Porin 31HL', the human type-1 porin purified from a crude membrane preparation of the same cell line using a different purification protocol. The results raise questions about targeting, insertion and orientation of human type-1 porin in different membranes.

Studies on human porin. XII. Eight monoclonal mouse anti-"porin 31HL" antibodies discriminate type 1 and type 2 mammalian porin channels/VDACs in western blotting and enzyme-linked immunosorbent assays.

Eight mouse monoclonal antibodies directed against the acetylated N-terminal part of the type 1 human VDAC Porin 31HL clearly discriminate type 1 and type 2 mammalian porin channels. This is shown by comparing synthetic N-terminal peptides of either channel type in Western dot blots or by ELISA. The data support the specificity of the anti-Porin 31HL antibodies and thus give further support to our recent observations on extramitochondrial expression of VDAC. In the plasmalemma of different mammalian cells VDAC forms part of an ubiquitous chloride channel complex, which in patch clamp measurements may figure as the outwardly rectifying depolarization-induced chloride channel that is affected in cystic fibrosis.

Channel active mammalian porin, purified from crude membrane fractions of human B lymphocytes and bovine skeletal muscle, reversibly binds adenosine triphosphate (ATP).

A new aspect of mammalian porin (mammalian VDAC = mammalian voltage-dependent anion channel) is presented: channel active VDAC binds adenosine triphosphate (ATP) in the absence of Ca2+. Channel active "Porin 31HL" or "Porin 31BM", enriched from crude membranes of human B lymphocytes or whole cell lysates of bovine skeletal muscle, respectively, was bound to a nine atoms spacer ATP-agarose at pH 7.4 or 5.0 and reeluted from the resin by 10 mM ATP disodium salt. Furthermore, channel active "Porin 31BM" was labelled by [32P]ATP in a 1:1 stoichiometric relation. Binding of ATP to human porin was confirmed by studying the interaction of the synthetic porin fragment Type-1/Ac-35, comprising the putative nucleotide binding site G Y G F G, with trinitrophenyl-ATP (TNT-ATP) by scanning fluorometry. Peptide/TNP-ATP complexes clearly show enhancement of fluorescence intensity and a spectral shift of the fluorescence maximum. In a control experiment, using a porin fragment lacking the putative nucleotide binding site, no change of fluorescence emission was observed. Further confirmation for ATP binding by human VDAC arose from an autoradiographic experimental approach: the porin fragment Type-1/Ac-35 could be labelled by [32P]ATP, while a second porin fragment ending immediately before the putative nucleotide binding site could not; nor could a synthetic non porin peptide.

Channel active mammalian porin, purified from crude membrane fractions of human B lymphocytes or bovine skeletal muscle, reversibly binds the stilbene-disulfonate group of the chloride channel blocker DIDS.

Two new aspects of mammalian porin are presented. First, by affinity chromatography we show that channel active human or bovine porin reversibly bind the stilbene-disulfonate group of the chloride channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). The procedure is suitable for further purification of porin after enrichment by ion exchange chromatography and shows a yield of 24.3%. The data support our recent proposal that VDAC forms part of the ORDIC channel complex which is affected in cystic fibrosis. Second, a purification scheme for mammalian porin is given starting with direct solubilisation of ground bovine skeletal muscle to avoid breaking up tissue. About 130 mg of channel active "Porin 31BM" are enriched from 946 g muscle tissue. Concerning its apparent molecular mass, primary structure, channel activity, channel conductance and voltage dependence the molecule shows high similarity to human porin. "Porin 31BM" is furthermore labelled by antibodies raised against human B lymphocyte derived "Porin 31HL".