Susanne Reymann

New findings concerning vertebrate porin.

Eukaryotic porin can be considered to be a good candidate for forming the channel component of the protein complex which, depending on the approach used, may realize its expression either as the outwardly-rectifying depolarization-induced chloride channel or as the volume-sensitive organic osmolyte-anion channel. As a basis for this proposition, we point to a series of correspondences in properties between mammalian porin and the ORDIC channel complex. Specifically, mammalian porin is expressed in the plasmalemma of different cells and chloride channels can be blocked by anti-human porin antibodies in astrocytes and endothelial cells. There is an indication of colocalisation of human porin and the cystic fibrosis (CF) gene product, CFTR, in the apical region of epithelial cells. The primary structure of porin from a CF patient was found to be normal. Cytosol and amniotic fluid fractions influence the channel characteristics of mammalian porin. Channel-active mammalian porin binds ATP and the stilbene disulphonate grouping of the chloride channel inhibitor DIDS. Human porin in black membranes is a pathway for taurine, and biogenic polyamines reduce the voltage dependence of human porin. Assuming the relationship between human porin and the ORDIC channel/VSOAC complex, studies on plasmalemma-integrated human porin have a relevance for CF research. In addition, we refer to a case study on a child with encephalomyopathy in which porin could not be detected using monoclonal anti-human porin antibodies. Our studies were based on purified and sequenced human porin from different cells and from different cell compartments. In addition, we raised antibodies against mature human porin or synthetic parts of the molecule. This provided a firm foundation for our topochemical work with which we were able to establish the multi-topological expression of eukaryotic porin channels. The data are summarized and discussed.

Studies on human porin: XIII. The type‐1 VDAC ‘Porin 31HL’ biotinylated at the plasmalemma of Trypan blue excluding human B lymphocytes

In 1989, we demonstrated for the first time the expression of the VDAC ‘Porin 31HL’ in the plasmalemma of human B lymphocytes, then giving first evidence of a multi‐topological expression of VDAC. Meanwhile, data from this and other laboratories support our proposal of a multi‐compartment distribution of the channel in mammalian tissues. Here, by biotinylation of plasmalemma‐integrated proteins of proven living B lymphocytes, followed by two‐dimensional electrophoresis, immuno‐ and streptavidin affinity blotting, we show that part of the channel molecules can be labelled at the outer membrane of the cells. Thus, by a relevant approach our results invalidate objections concerning putative cross‐reactivity of anti‐human Type‐1 porin antibodies with non‐VDAC proteins at the outer cell membrane.

The plasma membrane of Xenopus laevis oocytes contains voltage-dependent anion-selective porin channels.

Recent patch-clamp studies have shown that anti-porin antibodies, applied to the external side of excised plasma membrane patches of mammalian astrocytes, close chloride channels that are thought to be engaged in cell volume regulation. Frog oocytes are often used to study this basic cell function. Here we document the localisation of endogenous porin voltage-dependent anion-selective channels in Xenopus laevis oocyte plasma membranes. In confocal laser microscopy images a disjunctive pattern of fluorescing spots appear about 10 microm apart. Labelling was prevented by preabsorption of the antibodies with synthetic peptides comprising the epitope of the antigen. Immuno-gold marking of oocyte surfaces followed by silver enhancement of the gold particles lead to a plasma membrane labelling corresponding to that obtained by the confocal laser approach. The data suggests the presence of voltage-dependent, anion-selective channels in oocyte plasma membranes. This data should be borne in mind when frog oocytes are used to study the characteristics of endogenous or heterologously expressed ion channels or regulatory proteins.

LENTIL LECTIN ENRICHED MICROSOMES FROM THE PLASMA MEMBRANE OF THE HUMAN B-LYMPHOCYTE CELL LINE H2LCL CARRY A HEAVY LOAD OF TYPE-1 PORIN

Using an established biochemical approach, five subcellular fractions of human B lymphocytes were prepared by differential centrifugation. Crude membranes were passed over a lentil lectin column to enrich carbohydrate-coated cell surface microsomes. The lectin-bound fraction contained a high amount of plasma membrane-derived microsomes as indicated by cell surface markers. All subcellular fractions in Western blots proved to contain distinct but variable amounts of porin. There was a strong increase in porin content from crude membranes to plasma membrane-derived vesicles. The porin content of this fraction appeared to be higher than that of mitochondria. In the final step the plasma membrane-derived microsome fraction proved to be devoid of contamination by outer mitochondrial membranes, as revealed by antibodies against the established markers MAO B and Tom20 applied in Western blots. These data prove the extramitochondrial expression of human type-1 porin/ type-1 VDAC.

Endosomes: another extra-mitochondrial location of type-1 porin/voltage-dependent anion-selective channels (VDAC)

Abstract Endocytotic vesicles (EV) isolated from rat renal cortex were subjected to SDS-polyacrylamide gel electrophoresis and Western blotting. A monoclonal antibody against human type-1 porin (31 kDa) detected a strong band of 31 kDa. The same antibody has been used as the primary antibody in indirect immunocytochemistry. Light microscopy of cryostat sections of rat renal cortex showed a heavy staining of EV underneath the brush-border membrane. Electron microscopy was performed by ”preembedding immunogold staining” of rat renal cortex, the sections of which showed an extensive labelling of EV with gold particles. These results demonstrate that the expression of type-1 porin is not restricted to outer mitochondrial membranes. The biological function of endosomal type-1 porin has as yet to be ascertained.

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

Abstract 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.