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At least 19 recordsLinked to original sources

PubMed1989

[Identification of human porins. II. Characterization and primary structure of a 31-lDa porin from human B lymphocytes (Porin 31HL)].

We characterize and describe for the first time the primary structure of a human porin with the molecular mass of 31 kDa derived from the plasmalemm of B-lymphocytes (Porin 31HL). Porin 31HL is shown to be a basic, channel forming membrane protein. The protein chain is composed of 282 amino acids with a relative molecular mass of 30641 Da without derivatisation. It is not a glycoprotein. The N-terminus is acetylated. Altogether the amino-acid sequence shows 56% hydrophilic or charged amino acids arranged in alternating regions of hydrophilic or hydrophobic character as it is typical for porins. In addition the 18 N-terminal amino acids of Porin 31HL can be arranged to an amphilic alpha-helix like in other porins. Porin 31HL shows approx. 29% or 24% identity to the primary structure of mitochondrial porins of Neurospora crassa and Saccharomyces cerevisiae. Partial data on mitochondrial porins from rat kidney and beef heart show sequence identity of about 90% to the human B cell porin elaborated here.

Amino Acid Sequence↗

PubMed1989

[Identification of human porins. I. Purification of a porin from human B-lymphocytes (Porin 31HL) and the topochemical proof of its expression on the plasmalemma of the progenitor cell].

We describe for the first time a porin (Porin 31HL) on the plasmalemm of an eukaryontic cell line, where porins have been found only on the outer mitochondrial membranes. The expression of the porin on the plasmalemm of transformed human B-lymphocytes is demonstrated by cytotoxicity- and indirect immunofluorescence techniques with living and fixed cells. The rabbit xenoantisera used were directed against purified Porin 31HL and free or acetylated synthetic peptides of its nineteen N-terminal amino acids. The three-step purification procedure for Porin 31HL started from a total membrane fraction of the B-cell line, followed by ion-exchange chromatography on CM- and DEAE-cellulose and a final gel filtration in SDS on Sephacryl S-300.

Amino Acids↗

PubMed1992

Studies on human porin. VII. The channel properties of the human B-lymphocyte membrane-derived "Porin 31HL" are similar to those of mitochondrial porins.

Porin 31HL was isolated and purified from total membrane preparations of a human B-lymphocyte cell line. The protein showed a single band of apparent molecular mass 31 kDa on SDS-PAGE. Reconstitution of the protein into artificial lipid bilayer membranes defines its function as a channel-forming protein. The distribution of single-channel conductances had two maxima of 2.4 and 4.3 nS in 1M KCl. The channel formed by Porin 31HL of human B-lymphocytes was found to be voltage-dependent and switched to ion-permeable substates at membrane voltage larger than 20mV. In the open state the pore exhibited the characteristics of a general diffusion pore because the mobility sequence of the ions inside the pore was similar to that in the bulk aqueous phase. The effective diameter was estimated to be about 1.7 nm. The properties of the low conductance state of the channel were studied in detail. In this state the pore favored the passage of cations, in contrast to the open state which favored anions slightly. Monoclonal antibodies against the N-terminal end of Porin 31HL blocked its reconstitution but had otherwise no influence on the channel properties. This result suggested that the amphipathic alpha-helical structure at the N-terminal end is probably not involved in channel gating. The channel-forming properties of Porin 31HL were compared to those of porins isolated from mitochondrial outer membranes and to those of the "maxi chloride channel" observed in the cytoplasmic membrane of several eukaryotic cells.

Animals↗

PubMed1994

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.

Amino Acid Sequence↗

PubMed1991

Studies on human porin. IV. The primary structures of "Porin 31HM" purified from human skeletal muscle membranes and of "Porin 31HL" derived from human B lymphocyte membranes are identical.

We report on the purification of "Porin 31HM" from the crude plasma membrane fraction of human skeletal muscle. Furthermore, all tryptic peptides of the molecule were purified and characterized by different methods. The alignment of the peptides with the complete primary structure of the human B lymphocyte plasma membrane-derived "Porin 31HL", published by us recently (Kayser, H. et al. (1989) this Journal 370, 1265-1278), proved both structures to be completely identical. Our data demonstrate that porin fractions from crude plasma membranes of different human cell types do not show any variation on the primary structure level.

Amino Acid Sequence↗

PubMed1996

Molecular analysis of the Rhodobacter capsulatus B10 porin (porCa) gene; purification and biochemical characterisation of the porin protein.

The pore-forming outer-membrane protein from Rhodobacter (R.) capsulatus (wild-type B10 strain) was isolated and purified under non-denaturing conditions. The monomer unit of the isolated porin has a molecular mass of about 28 kDa, as judged by SDS-PAGE, whereas the native protein migrates at 75 kDa. This suggests that the native porin from R. capsulatus B10 exists in a trimeric form. The N-terminal amino acid sequence was used to design an oligonucleotide which was utilised to screen a pBluescript library containing EcoRI fragments of R. capsulatus B10 DNA. A 5.3-kb DNA fragment, which included the entire structural porin gene (named porCa) and its flanking regions, was identified. A 945-bp open reading frame, coding for a mature protein of 295 amino acid residues (molecular mass 30,586 Da) plus a presequence of 20 amino acids, was found. The directly determined sequence of the amino-terminus and of four tryptic peptides of the purified porin matched perfectly with the deduced amino acid sequence. Northern blot analysis showed that the porin gene encodes an RNA transcript of 1050 nucleotides. In addition, there is no differential response in terms of either the size or abundance of the mRNA under different environmental conditions. Primer extension experiments confirmed a putative promoter upstream of the porin gene; and localised the RNA transcription start site 73 bp upstream of the ATG start codon, which is close to the putative promoter (-10/-35). As shown using a plasmid-borne porin-lacZ gene fusion, [in R. capsulatus] expression of the lacZ gene under the control of the porCa promoter is not regulated under the two different environmental conditions tested. The promoter of the porin gene was localised within 305 bp upstream of the ATG start codon. A model of the 3-D structure of porin B10 was deduced by comparative modelling with the R. capsulatus 37b4 and Rhodopseudomonas blastica porin crystallographic structures using the ProMod program on the Swiss-Model protein modelling e-mail Server. Analysis of the B10 sequence and comparison of a model of the B10 porin structure with the crystallographic structure of porin from the capsuleless strain 37B4 has revealed some important differences at the level of the protein surface, the pore and the putative ligand binding site.

Amino Acid Sequence↗

PubMed1996

Isolation of mitochondrial porin of the fly Protophormia: porin modification by the pesticide CGA 140'408 studied in lipid bilayer membranes.

Mitochondrial porin from the fly Protophormia was solubilized with detergent from whole mitochondria and purified by chromatography across a hydroxyapatite (HPT) column. The purified protein had an apparent molecular mass of about 30 kDa on SDS-PAGE. Partial sequencing of the protein confirmed that it is porin. When reconstituted in planar lipid bilayer membranes, porin formed ion-permeable channels with single-channel conductances of 2.4 and 4.5 nS in 1 M KCl. At low voltage, Protophormia porin displayed the properties of a general diffusion pore and had a small selectivity for anions over cations. At transmembrane potentials starting with about 20-30 mV, the channel switched in closed state, which is still ion-permeable. Our results suggest that Protophormia porin possesses functional properties similar to those of other mitochondrial porins. Porin was also isolated and purified from mitochondria, which were treated with the carbodiimide CGA 140'408 It represents the active derivative of diafenthiuron a new acaricide and insecticide. This carbodiimide labels both a F0-component of the inner membrane ATPase and outer membrane porin in a similar way as N,N'-dicyclohexylcarbodiimide (DCCD). Reconstitution experiments with the CGA 140'408-modified porin showed no significant effect of the modification on the single-channel conductance, suggesting that CGA 140'408 binds outside the channel. The voltage-dependence of the CGA 140'408-modified porin was changed with respect to the unmodified form. The closed configuration of the pesticide-modified channel was reached at smaller transmembrane potentials, suggesting a shift of the open to the closed state of Protophormia porin by pesticide binding. A possible contribution of this effect to the pesticide action is discussed.

Amino Acid Sequence↗

PubMed1994

Porins from plants. Molecular cloning and functional characterization of two new members of the porin family.

Porins are voltage-gated diffusion pores found in all eukaryotic kingdoms. Here we describe, for the first time, the identification and characterization of two cDNAs encoding porins from plants. Peptide sequences obtained from a 30-kDa protein of envelope membranes from pea root plastids allowed the isolation of two cDNA clones from pea and maize. On the protein level, both proteins are homologous by 58%. Sequence comparison against the Swiss-Prot sequence data base revealed a homology of about 25% to mitochondrial porins from fungi and human. Computer-aided predictions of the secondary structure of the plant porins revealed the presence of 16 antiparallel beta-strands that are also found in mitochondrial porins. Porins from non-green plastids and from the outer mitochondrial membrane were reconstituted into planar lipid bilayers. The proteins showed high pore-forming activities and similar single-channel conductances. In vitro translated porin was preferentially imported only into non-green plastids but not into chloroplasts. To our knowledge, this is the first example of selective import of a plastid protein into different types of plastids. This finding is in line with the observation that an immunoreactive 30-kDa band was only found in non-green plastids and mitochondria but not in chloroplasts. We conclude that mitochondria and non-green plastids possess homologous porin proteins, whereas chloroplasts are characterized by a different type of porin.

Amino Acid Sequence↗

PubMed1993

A mitochondrial porin cDNA predicts the existence of multiple human porins.

Pores formed in the outer membrane of mitochondria by mitochondrial porin make it permeable to water-soluble metabolites smaller than approximately 5 kDa. We have isolated a full-length cDNA encoding a human porin. This probe detects a single approximately 1.5-kilobase mRNA species on Northern blots, but multiple hybridizing bands on genomic Southern blots. The open reading frame predicts a 38.1-kDa protein with a pI of 6.59 that is homologous but not identical to a previously reported protein sequence of a 31-kDa porin isolated from human lymphocytes (porin 31HL). The most striking difference between the two porins is that the sequence predicted by the cDNA is longer than the 31HL porin by 27 amino acids at the amino terminus and 38 amino acids at the carboxyl terminus. The porin cDNA directs the in vitro translation of two protein species of approximately 32 and approximately 36 kDa, which appear to result from alternate usage of AUG initiation codons. The 32-kDa protein is the predominant species imported into both rat and yeast mitochondria in vitro. Taken together, these results suggest that multiple porin proteins can be expressed in humans. Additionally, a porin consensus protein sequence has been identified that is conserved in eukaryotic organisms as diverse as yeast and man.

Amino Acid Sequence↗

PubMed1991

Characterization of SH groups in porin of bovine heart mitochondria. Porin cysteines are localized in the channel walls.

Porin from bovine heart mitochondria contains probably two cysteines (Cys126 and Cys230 in human porin, Kayser, H., Kratzin, H. D., Thinnes, F. P., Götz, H., Schmidt, W. E., Eckart, K. & Hilschmann, N. (1989) Biol. Chem. Hoppe-Seyler 370, 1265-1278). Reduced and oxidized forms of these cysteines were investigated in purified protein and in intact mitochondria using the agents dithioerythritol, cuprous(II) phenantroline, diamide and performic acid. Furthermore, intact mitochondria were labelled with the sulfhydryl-alkylating agents N-[14C]ethylmaleimide, eosin-5-maleimide and N-(1-pyrenyl)-maleimide. Affinity chromatography of bovine heart porin was performed with cysteine-specific material. The results can be summarized as follows: (1) Porin has one reduced and two oxidized forms of apparent molecular masses between 30 and 35 kDa. The native form of porin is the reduced 33 kDa form. The oxidized forms only appear after denaturation with SDS. (2) The 35-kDa reduced and the 33.5-kDa oxidized forms of porin show the same pore-forming properties after reconstitution of the protein into lipid bilayer membranes. (3) Labelling of cysteines by eosin-5-maleimide and N-(1-pyrenyl)-maleimide suggested their location at a boundary between the water-phase and the lipid-phase. Incubation of intact mitochondria with N-ethylmaleimide prior to eosin-5-maleimide and N-(1-pyrenyl)maleimide treatment resulted in the inhibition of the fluorescent labelling. Among the cysteines present in the primary structure, Cys126 is the most sensitive to N-ethylmaleimide binding. (4) Bovine heart mitochondrial porin covalently bound to Affi-Gel 501 (with a 1.75 nm long spacer), but not to Thiopropyl-Sepharose 6B (with a 0.51 nm spacer). This suggests that at least one of the cysteines is localized between 0.51 nm and 1.75 nm deep in the protein micelle.

Animals↗

PubMed1993

Studies on human porin. IX. Immunolocalization of porin and CFTR channels in human surface respiratory epithelium.

The expression of the voltage-dependent anion channel (VDAC) "Porin 31 HL" and its cellular and subcellular immunocytochemical localization in the human respiratory epithelium were studied with monoclonal and polyclonal antibodies using immunofluorescence and immunogold labelling with light (LM) and transmission electron microscopy (TEM), respectively. Porin was identified in the apical domain of the ciliated cells and in the basal cells of the respiratory epithelium. Immunogold labelling was present in the apical plasma membrane and subapical vesicles of the ciliated cells. In pre-embedded freshly dissociated surface epithelial cells, porin could also be identified with TEM at the outer part of the plasma membrane of basal cells. By LM double immunolabelling, both porin and cystic fibrosis transmembrane conductance regulator (CFTR) were identified in the apical domain of ciliated cells but not in basal cells where CFTR was never identified. On Western blots of solubilized total membrane protein preparations from the same frozen surface epithelial respiratory cells, the antibodies recognized a group of 3 proteins of 31, 60 and 130-140 kDa with a strong reactivity for a 31 kDa protein, corresponding to the porin and a protein of 170 kDa which is consistent with mature CFTR. These results suggest that porin might be part of a multi-component chloride channel complex which could interact with CFTR.

Antibodies↗

PubMed1994

Refined structure of the porin from Rhodopseudomonas blastica. Comparison with the porin from Rhodobacter capsulatus.

The structure of the membrane channel porin from the phototrophic bacteria Rhodopseudomonas blastica has been refined at 1.96 A resolution yielding an R-factor of 17.6%. The final model consists of all 289 amino acid residues, 247 water molecules and three detergent molecules modelled as n-octyltetraoxyethylene. One of these detergent molecules binds together with its two symmetry-related molecules tightly in a pocket at the molecular 3-fold axis. This pocket may bind three alkyl chains of a lipopolysaccharide which in turn would stabilize the trimer and could possibly play a role in membrane insertion. The overall shape of this porin resembles OmpF of Escherichia coli more than the only known sequence-related porin from Rhodobacter capsulatus. The membrane contacting surface is similar in all structurally known porins; it shows exceptional frequencies of amino acid residues and side-chain rotamers. The 46-residue loop beta 5-beta 6 of the porin is shown to be tightly fastened to the beta-barrel, excluding an in vivo loop movement that closes the pore. The trimer interface region has the structure of a water-soluble protein with an extensive non-polar core and numerous hydrogen bonds at the surface. The loops at the external end of the barrel are long and rigid whereas those at the periplasmic barrel end are short and mobile. The crystal packing is discussed.

Amino Acid Sequence↗

PubMed2000

Studies on human porin XXI: gadolinium opens Up cell membrane standing porin channels making way for the osmolytes chloride or taurine-A putative approach to activate the alternate chloride channel in cystic fibrosis.

We recently proposed that cell-membrane-integrated vertebrate porin/voltage-dependent anion-selective channel (VDAC) forms part of the outwardly rectifying chloride channel (ORCC) complex that may be involved in volume regulation. The results we present here support this thesis. According to light scattering measurements micromolar concentrations of Gd(3+) induce cell swelling of human healthy and cystic fibrosis (CF) B-lymphocyte cell lines in isotonic Ringer solution. In high-potassium Ringer solution additional swelling is observed. Gd(3+) induces excessive cell swelling of cell lines in hypotonic Ringer solutions, containing 70 mM NaCl or 135 mM taurine, respectively. The gadolinium effect is lost when NaCl is replaced by Na-gluconate. Using video camera monitoring we show that HeLa cells also swell in micromolar concentrations of Gd(3+) in isotonic taurine Ringer solution. The dose-dependent effect of the agonist was always blocked by extracellular application of anti-human type-1 porin antibodies. Together with data on a decreasing effect of micromolar amounts of gadolinium on the voltage dependence of reconstituted human porin the results prove the involvement of porin channels in the swelling behavior in different cell lines. As a mechanism we propose that ionic gadolinium opens up plasmalemma-integrated porin channels, chloride or taurine then following their concentration gradients into the cells. Furthermore, our data argue for a single pathway for inorganic and organic osmolytes during regulatory volume decrease after cell swelling. There is indirect evidence that porin forms part of the cystic fibrosis relevant ORCC channel. Gadolinium thus may work to open the alternate chloride channel in CF.

Antibodies, Monoclonal↗

PubMed2001

Neisseria meningitidis lipopolysaccharide modulates the specific humoral immune response to neisserial porins but has no effect on porin-induced upregulation of costimulatory ligand B7-2.

The role of lipopolysaccharide (LPS) in the specific humoral response to meningococcal porins was investigated by measuring anti-PorA or -PorB antibody levels in mice immunized with wild-type meningococcal strain H44/76 or with its recently described LPS-negative mutant. Two murine strains were used for these immunizations: C3H/HeJ, which is LPS hyporesponsive, or C3H/HeOuJ, which is LPS responsive. A high level of anti-PorB immunoglobulin G (IgG) response was induced in both strains of mice immunized with either organism. The response induced by the wild-type strain was greater in C3H/HeOuJ mice than in C3H/HeJ mice, while the response induced by the LPS-negative mutant was similar in the two murine strains. Additionally, the anti-PorB response was similar in C3H/HeJ mice immunized with either bacterial strain. In general, the anti-PorA IgG response was lower than the anti-PorB response. These findings indicate that the presence of LPS is not essential for the induction of an antineisserial porin humoral response but can augment such a response. To determine whether LPS has any effect on the B-cell-stimulatory effect of neisserial porins (essential for the adjuvant activity of neisserial porins), B cells from both murine strains were incubated with outer membrane complexes (OMCs) prepared from strain H44/76 and its LPS-negative mutant. OMCs from either meningococcal strain were able to increase the surface expression of the costimulatory ligand B7-2 on B cells from either murine strain. Consistent with previously reported findings, LPS does not significantly affect the ability of neisserial porins to induce the costimulatory ligand B7-2.

Animals↗

PubMed1991

Nucleotide and derived amino acid sequences of the major porin of Comamonas acidovorans and comparison of porin primary structures.

The DNA sequence of the gene which codes for the major outer membrane porin (Omp32) of Comamonas acidovorans has been determined. The structural gene encodes a precursor consisting of 351 amino acid residues with a signal peptide of 19 amino acid residues. Comparisons with amino acid sequences of outer membrane proteins and porins from several other members of the class Proteobacteria and of the Chlamydia trachomatis porin and the Neurospora crassa mitochondrial porin revealed a motif of eight regions of local homology. The results of this analysis are discussed with regard to common structural features of porins.

Amino Acid Sequence↗

PubMed1992

Studies on human porin. VIII. Expression of "Porin 31HL" channels in the plasmalemma of the acute-lymphoblastic-leukemia cell line KM3 as revealed by light- and electron-microscopy.

First electron microscopy data on the expression of the VDAC "Porin 31HL" in the plasmalemma of a eucaryotic cell are presented. In a light and electron microscopic study we demonstrate the expression of the porin channel in the outer cell membrane of the pre-B lymphocyte type acute-lymphoblastic-leukemia cell line KM3. Monoclonal mouse anti-"Porin 31HL" antibodies were applied in indirect immunofluorescence or immunogold labelling experiments. The results confirm our early topological data on the expression of porin channels in the cytoplasmic membrane of different human cell types as revealed on the light microscopy level. Interestingly, only a pre-embedding immunoreaction approach was successful for gold particle labelling of the plasmalemma. This again is in agreement with our recent data on the accessibility of the acetylated N-terminal part of "Porin 31HL" molecules on the outer cell surface.

Antibodies, Monoclonal↗

PubMed2000

Characterization of the human porin isoform 1 (HVDAC1) gene by amplification on the whole human genome: A tool for porin deficiency analysis.

The deficiency of porin isoform 1 (HVDAC1) in human skeletal muscle has been associated with a pathological phenotype related to defects in the bioenergetic metabolism. In the best studied case, porin deficiency was not apparent in cultured fibroblasts: this observation raised the conclusion that no molecular defect was in the cDNA sequence coding for the protein. To get more insight in the pathogenetic mechanism that is involved in porin isoform 1 deficiency, we have determined the whole structure of the corresponding human gene. On the basis of the corresponding mouse gene structure and the human cDNA sequence, we designed long extension PCR amplifications using the whole genomic DNA as a template. Exonic/intronic regions were isolated and the exons and surrounding introns sequenced. The 5' and 3' extremities of the gene were determined by genome walking. The porin isoform 1 human gene is made up of 9 exons and spans about 33 kbp. A whole panel of PCR parameters was set and is now ready to be used for specific amplification upon patients' genomic DNA. The analysis of the putative promoter sequence was performed. It revealed the presence of a sterol Repressor element (SRE), an SRY, the testis-determining factor, and a nuclear respiratory factor 2 (NRF-2) binding site. These sites, according to results from literature, could be involved in the functional modulation of the gene expression.

3' Untranslated Regions↗

PubMed2000

Studies on human porin XXII: cell membrane integrated human porin channels are involved in regulatory volume decrease (RVD) of HeLa cells.

Cell volume regulation receives increasing attention not only as the basis of regulatory volume increase or regulatory volume decrease (RVD) of cells in surroundings of changing osmolarity, but also appears to be relevant in cell proliferation, differentiation, and apoptosis. A central event in RVD is the opening of a volume-sensitive chloride/anion channel(s), and blocking this pathway would abolish RVD. This is shown here with monoclonal mouse anti-human type-1 porin antibodies, proving that porin is involved in this process. HeLa cells preincubated with these antibodies dramatically increase their volume within about 1 min after a hypotonic stimulus by 70 mM NaCl Ringer solution, but do not move back toward their starting volume, thus indicating abolished RVD. Corresponding effects are induced by the established anion channel inhibitor DIDS. Video camera monitoring of cell size over time was used as a direct and noninvasive approach. We had already accumulated evidence that plasmalemma integrated eukaryotic porin channels form chloride/anion channels in this cell compartment and that they are involved in cell volume regulation. Finally, the present data again demonstrate the suitability of our anti-porin antibodies in physiological studies.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗