Angela Schmid

MECHANISM OF SUGAR-TRANSPORT THROUGH THE SUGAR-SPECIFIC LAMB CHANNEL OF ESCHERICHIA-COLI OUTER-MEMBRANE

SummaryLipid bilayer experiments were performed with the sugar-specific LamB (maltoporin) channel ofEscherichia coli outer membrane. Single-channel analysis of the conductance steps caused by LamB showed that there was a linear relationship between the salt concentration in the aqueous phase and the channel conductance, indicating only small or no binding between the ions and the channel interior. The total or the partial blockage of the ion movement through the LamB channel was not dependent on the ion concentration in the aqueous phase. Both results allowed the investigation of the sugar binding in more detail, and the stability constants of the binding of a large variety of sugars to the binding site inside the channel were calculated from titration experiments of the membrane conductance with the sugars. The channel was highly cation selective, both in the presence and absence of sugars, which may be explained by the existence of carbonyl groups inside the channel. These carbonyl groups may also be involved in the sugar binding via hydrogen bonds. The kinetics of the sugar transport through the LamB channel were estimated relative to maltose by assuming a simple one-site, two-barrier model from the relative rates of permeation taken from M. Luckey and H. Nikaido (Proc. Natl. Acad. Sci. USA77:165–171 (1980a)) and the stability constants for the sugar binding given in this study.

Modulation of Neisseria Porin (PorB) by Cytosolic ATP/GTP of Target Cells: Parallels between Pathogen Accommodation and Mitochondrial Endosymbiosis

PorB of the pathogenic Neisseria species belongs to the large family of pore-forming proteins (porins) produced by gram-negative bacteria. PorB is exceptional in that it is capable of translocating vectorially into membranes of infected target cells and functions in the infection process. Here we report on an unexpected similarity between Neisserial PorB and mitochondrial porins. Both porin classes interact with purine nucleoside triphosphates, which down-regulate pore size and cause a shift in voltage dependence and ion selectivity. Patch-clamp analyses indicate that PorB channel activity is tightly regulated in intact epithelial cells. In light of recent findings on the pivotal role of PorB in virulence and the prevention of phagosome lysosome fusion, these data provide important mechanistic clues on the intracellular pathogen accommodation reminiscent of mitochondrial endosymbiosis.

Mutations affecting pore formation by haemolysin from Escherichia coli

SummaryBy introduction of site-specific deletions, three regions in HlyA were identified, which appear to be involved in pore formation by Escherichia coli haemolysin. Deletion of amino acids 9–37 at the N-terminus led to a haemolysin which had an almost threefold higher specific activity than wild-type and formed pores in an artificial asolectin lipid bilayer with a much longer lifetime than those produced by wild-type haemolysin. The three hydrophobic regions (DI–DIII) located between amino acids 238–410 contributed to pore formation to different extents. Deletion of DI led to a mutant haemolysin which was only slightly active on erythrocyte membranes and increased conductivity of asolectin bilayers without forming defined pores. Deletions in the two other hydrophobic regions (DII and DIII) completely abolished the pore-forming activity of the mutant haemolysin. The only polar amino acid in DI, Asp, was shown to be essential for pore formation. Removal of this residue led to a haemolysin with a considerably reduced capacity to form pores, while replacement of Asp by Glu or Asn had little effect on pore formation. A deletion mutant which retained all three hydrophobic domains but had lost amino acids 498–830 was entirely inactive in pore formation, whereas a shorter deletion from amino acids 670–830 led to a mutant haemolysin which formed abnormal minipores. The conductivity of these pores was drastically reduced compared to pores introduced into an asolectin bilayer by wild-type haemolysin. Based on these data and structural predictions, a model for the pore-forming structure of E. coli haemolysin is proposed.

Molecular basis of porin selectivity: membrane experiments with OmpC-PhoE and OmpF-PhoE hybrid proteins of Escherichia coli K-12.

Lipid bilayer experiments were performed with one OmpF-PhoE and several OmpC-PhoE hybrid porins of Escherichia coli K-12. All hybrid pores had approximately the same pore-forming activity, which indicated that the structure of the pores remained essentially unchanged by the genetic manipulation. This result was supported by single-channel experiments because all pores had similar single-channel conductances in potassium chloride. Measurements with other salts indicated a drastic change in the ionic selectivity when the fusion site in the ompC-phoE hybrid genes passed along the sequence of the porins from the N-terminal to the C-terminal end. Selectivity measurements using zero-current membrane potentials showed that the selectivity suddenly changed from anion to cation selectivity when a relatively short portion from the N-terminal end of PhoE was replaced by the corresponding part of OmpC. The replacement of increasing portions led to an increase in the cation selectivity until that of OmpC was reached. The change in the anion to cation selectivity is correlated with exchange of lysine-18 and serine-28 by aspartic acids. The anion selectivity of the phosphate starvation-inducible PhoE porin is closely related to the presence of several lysines spread along the primary sequence of the polypeptide chain.

Pores from mitochondrial outer membranes of yeast and a porin-deficient yeast mutant: A comparison

Reconstitution experiments were performed on lipid bilayer membranes in the presence of purified mitochondrial porin from yeast and of detergent-solubilized mitochondrial outer membranes of a porin-free yeast mutant. The addition of the porin resulted in a strong increase of the membrane conductance, which was caused by the formation of ion-permeable channels in the membranes. Yeast porin has a single-channel conductance of 4.2 nS in 1 M KCl. In the open state it behaves as a general diffusion pore with an effective diameter of 1.7 nm and possesses properties similar to other mitochondrial porins. Surprisingly, the membrane conductance also increased in the presence of detergent extracts of the mitochondrial outer membrane of the mutant. Single-channel recordings of lipid bilayer membranes in the presence of small concentration of the mutant membranes suggested that this membrane also contained a pore. The reconstituted pores had a single-channel conductance of 2.0 nS in 1 M KCl and the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. This means that the pores present in the mitochondrial outer membranes of the yeast mutant have a much smaller effective diameter than “normal” mitochondrial porins. Zero-current membrane potential measurements suggested that the second mitochondrial porin is slightly cation-selective, while yeast porin is slightly anion-selective in the open state but highly cation-selective in the closed state. The possible role of these pores in the metabolism of mitochondria is discussed.

Identification of two general diffusion channels in the outer membrane of pea mitochondria

Reconstitution experiments were performed on lipid bilayer membranes in the presence of detergent solubilized mitochondrial membranes of pea seedlings (Pisum sativum). The addition of the detergent-solubilized material to the membranes resulted in a strong increase of the membrane conductance. To identify the proteins responsible for membrane activity the detergent extracts were applied to a hydroxyapatite (HTP) column and the fractions were tested for channel formation. The eluate of the column contained a protein which migrated as a single band with an apparent molecular mass of 30 kDa on SDS-PAGE. This channel was identified as the porin of pea mitochondria since it formed voltage-dependent channels with single-channel conductances of 1.5 and 3.7 nS in 1 M KCl and an estimated effective diameter of about 1.7 nm. Further elution of the column with KCl containing solutions yielded fractions which resulted in the formation of transient channels in lipid bilayer membranes. These channels had a single-channel conductance of 2.2 nS in 1 M KCl and had also the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. Zero-current membrane potential measurements suggested that pea porin was anion-selective in the open state. The selectivity of the second channel was investigated by the measurement of the reversal potential. It was also slightly anion-selective. Its possible role in the metabolism of mitochondria is discussed.

Single-channel analysis of the conductance fluctuations induced in lipid bilayer membranes by complement proteins C5b-9

SummarySingle-channel analysis of electrical fluctuations induced in planar bilayer membranes by the purified human complement proteins C5b6, C7, C8, and C9 have been analyzed. Reconstitution experiments with lipid bilayer membranes showed that the C5b-9 proteins formed pores only if all proteins were present at one side of the membrane. The complement pores had an average single-channel conductance of 3.1 nS at 0.15m KCl. The histogram of the complement pores suggested a substantial variation of the size of the single channel. The linear relationship between single-channel conductance at fixed ionic strength and the aqueous mobility of the ions in the bulk aqueous phase indicated that the ions move inside the complement pore in a manner similar to the way they move in the aqueous phase. The minimum diameter of the pores as judged from the conductance data is approximately 3 nm. The complement channels showed no apparent voltage control or regulation up to transmembrane potentials of 100 mV. At neutral pH the pore is three to four times more permeable for alkali ions than for chloride, which may be explained by the existence of fixed negatively charged groups in or near the pore. The significance of these observations to current molecular models of the membrane lesion formed by these cytolytic serum proteins is considered.

Haemolysin‐derived synthetic peptides with pore‐forming and haemolytic activity

Escherichia coli haemolysin (Hlya) is a pore‐forming protein which belongs to the family of ‘Repeat‐toxins’ (RTX)(Lo et al., 1989; Lally et al., 1989; Kraig et al., (1990). A model for the pore‐forming structure of HlyA has been proposed (Ludwig et al., 1991) which consists of eight transmembrane segments all present in this hydrophobic region of HlyA. We report here that two synthetic peptides of 10 and 8 amino acids in length (Pep1 and Pep2, respectively), which are derived from transmembrane segment V, are able to form pores in an artificial lipid bilayer. In addition, Pep1 exhibits strong haemolytic activity when tested on human red blood cells (HRBCs). The haemolytic activity of Pep1 and of E. coli haemolysin is completely inhibited by antibodies raised against Pep1.

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.

Characterization of Channel-Forming Activity in Muscle Biopsy from a Porin-Deficient Human Patient

A bioptic specimen from the muscles of a patient suffering from severe myopathy was inspected for the presence of human porin 31HL. Western blotting suggested that the specimen was free of the most abundant eukaryotic porin 31HL (HVDAC1). The specimen was treated with detergent and the soluble protein fraction was passed through a dry hydroxyapatite column. The passthrough of this column was inspected for channel formation in artificial lipid-bilayer membranes. The channel observed under these conditions had a single-channel conductance of about 2.5 nS in 1 M KCl, was cation selective, and was found to be virtually voltage independent. Experiments with a control specimen from a healthy human being, without any indication for muscle myopathy, revealed the presence of the voltage-dependent porin 31HL in the sample. It is discussed whether the patients bioptic specimen contained another human porin, which has not been studied to date in its natural environment.