The cell wall of the pathogenic bacterium Rhodococcus equi contains two channel-forming proteins with different properties

We have identified in organic solvent extracts of whole cells of the gram-positive pathogen Rhodococcus equi two channel-forming proteins with different and complementary properties. The isolated proteins were able to increase the specific conductance of artificial lipid bilayer membranes made from phosphatidylcholine-phosphatidylserine mixtures by the formation of channels able to be permeated by ions. The channel-forming protein PorA(Req) (R. equi pore A) is characterized by the formation of cation-selective channels, which are voltage gated. PorA(Req) has a single-channel conductance of 4 nS in 1 M KCl and shows high permeability for positively charged solutes because of the presence of negative point charges. According to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein has an apparent molecular mass of about 67 kDa. The analysis (using the effect of negative charges on channel conductance) of the concentration dependence of the single-channel conductance suggested that the diameter of the cell wall channel is about 2.0 nm. The second channel (formed by PorB(Req) [R. equi pore B]) shows a preferred movement of anions through the channel and is not voltage gated. This channel shows a single-channel conductance of 300 pS in 1 M KCl and is characterized by the presence of positive point charges in or near the channel mouth. Based on SDS-PAGE, the apparent molecular mass of the channel-forming protein is about 11 kDa. Channel-forming properties of the investigated cell wall porins were compared with those of others isolated from mycolic acid-containing actinomycetes. We present here the first report of a fully characterized anion-selective cell wall channel from a member of the order Actinomycetales.     

The cell wall porin of the gram-positive bacterium Nocardia asteroides forms cation-selective channels that exhibit asymmetric voltage dependence

Detergent-solubilized cell wall extracts of the gram-positive, strictly aerobic bacterium Nocardia asteroides contain channel-forming activity as judged from reconstitution experiments using lipid bilayer membranes. The cell wall porin was identified as a protein with an apparent molecular mass of about 84 kDa based on SDS-PAGE. The porin was purified to homogeneity using preparative SDS-PAGE. The 84-kDa protein was no longer observed after heating in SDS buffer. The presumed dissociation products were not observed on SDS-polyacrylamide gels. The cell wall porin increased the specific conductance of artificial lipid bilayer membranes from phosphatidylcholine/phosphatidylserine mixtures by the formation of cation-selective channels, which had an average single-channel conductance of 3.0 nS in 1 M KCl. The single-channel conductance was only moderately dependent on the bulk aqueous KCl concentration, which indicated negative point charge effects on the channel properties. The analysis of the concentration dependence of the single-channel conductance using the effect of negative charges on channel conductance suggested that the diameter of the cell wall channel is about 1.4 nm. Asymmetric addition of the cell wall porin to lipid bilayer membranes resulted in an asymmetric voltage dependence. The cell wall channel switched into substates, when the cis side of the membrane, the side of the addition of the protein, had negative polarity. Positive potentials at the cis side had no influence on the conductance of the cell wall channel. Received: 23 September 1998 / Accepted: 9 December 1998     

The cell wall of Micromonospora purpurea contains a high conductance channel

In this communication it is demonstrated that the cell wall of the gram-positive bacterium Micromonospora purpurea contains a cell wall channel for the passage of hydrophilic solutes. The channel-forming protein was identified in sucrose step-density-gradient fractions of the cell envelope and in whole cell extracts using either organic solvent or detergent and the lipid bilayer technique. The fractions of the sucrose step-density centrifugation were assayed for NADH-oxidase activity and for the formation of ion-permeable channels in lipid bilayers. The highest NADH-oxidase activity and the highest channel-forming ability were found in different fractions. The cell wall fraction was identified by the presence of meso-diaminopimelic acid and contained an ion-permeable channel with the extremely high single-channel conductance of about 14 nS in 1 M KCl. The channel-forming unit was purified to homogeneity by FPLC on a HiTrap-Q column. It was identified as a heat- and SDS-resistant 200-kDa band on SDS-PAGE and formed the same general diffusion pores in lipid bilayer membranes as those formed by detergent extracts of the cell wall fraction of the sucrose step-density centrifugation. The channels were slightly selective for potassium ions over chloride, possibly caused by an excess of negative charges in or near the channel.     

Discovery of a novel channel-forming protein in the cell wall of the non-pathogenic Nocardia corynebacteroides

Detergent extracts of whole cells of the Gram-positive, non-pathogenic, strictly aerobic bacterium Nocardia corynebacteroides contain channel-forming activity. The protein responsible for channel formation was identified using lipid bilayer experiments. It was purified to homogeneity and had an apparent molecular mass of about 134 kDa on SDS-PAGE when it was solubilized at 40 degrees C. When the 134 kDa protein was heated to 100 degrees C for 10 min in sample buffer, it dissociated into subunits with a molecular mass of about 23 kDa and focused at pI of 4.5 during isoelectric focusing. The pure 134 kDa protein was able to increase the specific conductance of artificial lipid bilayer membranes from phosphatidylcholine-phosphatidylserine mixtures by the formation of ion-permeable channels. The channels had an average single-channel conductance of 5.5 nS in 1 M KCl and were found to be cation-selective. Asymmetric addition of the 134 kDa protein to lipid bilayer membranes resulted in an asymmetric voltage-dependence. The analysis of the single-channel conductance as a function of cation radii using the Renkin correction factor and the effect of negative charges on channel conductance suggested that the diameter of the cell wall porin is about 1.0 nm. The channel characteristics of the cell wall channel of N. corynebacteroides were compared with those of other members of the mycolata. They share common features because they are composed of small molecular mass subunits and form large and water-filled channels.     

The cell wall porin of Nocardia farcinica: biochemical identification of the channel-forming protein and biophysical characterization of the channel properties

A channel-forming protein was identified in cell wall extracts of the Gram-positive, strictly aerobic bacterium Nocardia farcinica . The cell wall porin was purified to homogeneity and had an apparent molecular mass of about 87 kDa on tricine-containing SDS–PAGE. When the 87 kDa protein was boiled for a longer time in sodium dodecylsulphate (SDS) it dissociated into two subunits with molecular masses of about 19 and 23 kDa. The 87 kDa form of the protein was able to increase the specific conductance of artificial lipid bilayer membranes from phosphatidylcholine (PC) phosphatidylserine (PS) mixtures by the formation of ion-permeable channels. The channels had on average a single-channel conductance of 3.0 nS in 1 M KCl, 10 mM Tris-HCl, pH 8, and were found to be cation selective. Asymmetric addition of the cell wall porin to lipid bilayer membranes resulted in an asymmetric voltage dependence. The single-channel conductance was only moderately dependent on the bulk aqueous KCl concentration, which indicated point charge effects on the channel properties. The analysis of the single-channel conductance data in different salt solutions using the Renkin correction factor, and the effect of negative charges on channel conductance suggested that the diameter of the cell wall porin is about 1.4–1.6 nm. Channel-forming properties of the cell wall porin of N. farcinica were compared with those of mycobacteria and corynebacteria. The cell wall porins of these members of the order Actinomycetales share common features because they form large and water-filled channels that contain negative point charges.     

Biochemical identification and biophysical characterization of a channel-forming protein from Rhodococcus erythropolis

Organic solvent extracts of whole cells of the gram-positive bacterium Rhodococcus erythropolis contain a channel-forming protein. It was identified by lipid bilayer experiments and purified to homogeneity by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The pure protein had a rather low molecular mass of about 8.4 kDa, as judged by SDS-PAGE. SDS-resistant oligomers with a molecular mass of 67 kDa were also observed, suggesting that the channel is formed by a protein oligomer. The monomer was subjected to partial protein sequencing, and 45 amino acids were resolved. According to the partial sequence, the sequence has no significant homology to known protein sequences. To check whether the channel was indeed localized in the cell wall, the cell wall fraction was separated from the cytoplasmic membrane by sucrose step gradient centrifugation. The highest channel-forming activity was found in the cell wall fraction. The purified protein formed large ion-permeable channels in lipid bilayer membranes with a single-channel conductance of 6.0 nS in 1 M KCl. Zero-current membrane potential measurements with different salts suggested that the channel of R. erythropolis was highly cation selective because of negative charges localized at the channel mouth. The correction of single-channel conductance data for negatively charged point charges and the Renkin correction factor suggested that the diameter of the cell wall channel is about 2.0 nm. The channel-forming properties of the cell wall channel of R. erythropolis were compared with those of other members of the mycolata. These channels have common features because they form large, water-filled channels that contain net point charges.     

haemolysin of Escherichia coli: Comparison of pore-forming properties between chromosome and plasmid-encoded haemolysins

Abstract Lipid bilayer experiments were performed with chromosome-encoded haemolysin of Escherichia coli . The addition of the toxin to the aqueous phase bathing lipid bilayer membranes of asolectin resulted in the formation of transient ion-permeable channels with two states at small transmembrane voltages. One is prestate (single-channel conductance 40 pS in 0.15 M KCl) of the open state, which had a single-channel conductance of 420 pS in 0.15 M KCl and a mean lifetime of 30 s. Membranes formed of pure lipids were rather inactive targets for this haemolysin. Experiments with different salts suggested that the haemolysin channel was highly cation-selective at neutral pH. The mobility sequence of the cations in the channel was similar if not identical to their mobility sequence in the aqueous phase. The single-channel data were consistent with a wide, water-filled channel with an estimated minimal diameter of about 1 nm. The pore-forming properties of chromosome-encoded haemolysin were compared with those of plasmid-encoded haemolysin. Both toxins share common features, oligomerize probably to form pores in lipid bilayer membranes. Both types of haemolysin channels have similar properties but different lifetimes.     

Corynebacterium diphtheriae: identification and characterization of a channel-forming protein in the cell wall

The cell wall fraction of the gram-positive, nontoxic Corynebacterium diphtheriae strain C8_r(−) Tox⁻ (= ATCC 11913) contained a channel-forming protein, as judged from reconstitution experiments with artificial lipid bilayer experiments. The channel-forming protein was present in detergent-treated cell walls and in extracts of whole cells obtained using organic solvents. The protein had an apparent molecular mass of about 66 kDa as determined on Tricine-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and consisted of subunits having a molecular mass of about 5 kDa. Single-channel experiments with the purified protein suggested that the protein formed channels with a single-channel conductance of 2.25 nS in 1 M KCl. Further single-channel analysis suggested that the cell wall channel is wide and water filled because it has only slight selectivity for cations over anions and its conductance followed the mobility sequence of cations and anions in the aqueous phase. Antibodies raised against PorA, the subunit of the cell wall channel of Corynebacterium glutamicum, detected both monomers and oligomers of the isolated protein, suggesting that there are highly conserved epitopes in the cell wall channels of C. diphtheriae and PorA. Localization of the protein on the cell surface was confirmed by an enzyme-linked immunosorbent assay. The prospective homology of PorA with the cell wall channel of C. diphtheriae was used to identify the cell wall channel gene, cdporA, in the known genome of C. diphtheriae. The gene and its flanking regions were cloned and sequenced. CdporA is a protein that is 43 amino acids long and does not have a leader sequence. cdporA was expressed in a C. glutamicum strain that lacked the major outer membrane channels PorA and PorH. Organic solvent extracts of the transformed cells formed in lipid bilayer membranes the same channels as the purified CdporA protein of C. diphtheriae formed, suggesting that the expressed protein is able to complement the PorA and PorH deficiency of the C. glutamicum strain. The study is the first report of a cell wall channel in a pathogenic Corynebacterium strain.     

Study of the Properties of a Channel-forming Protein of the Cell Wall of the Gram-positive Bacterium Mycobacterium phlei

The gram-positive bacterium Mycobacterium phlei was treated with detergents. Reconstitution experiments using lipid bilayers suggested that the detergent extracts contain a channel forming protein. The protein was purified to homogeneity by preparative SDS-PAGE and identified as a protein with an apparent molecular mass of about 135 kDa. The channel-forming unit dissociated into subunits with a molecular mass of about 22 kDa when it was boiled in 80% dimethylsulfoxid (DMSO). The channel has on average a single channel conductance of 4.5 nS in 1 m KCl and is highly voltage-dependent in an asymmetric fashion when the protein is added to only one side of the membrane. Zero-current membrane potential measurements with different salts implied that the channel is highly cation-selective because of negative point charges in or near the channel mouth. Analysis of the single-channel conductance as a function of the hydrated cation radii using the Renkin correction factor and the effect of the negative point charges on the single-channel conductance suggest that the diameter of the cell wall channel is about 1.8 to 2.0 nm. The channel properties were compared with those of other members of the mycolata and suggest that these channels share common features. Southern blots demonstrated that the chromosome of M. phlei and other mycolata tested contain homologous sequences to mspA (gene of the cell wall porin of Mycobacterium smegmatis). Received: 22 December 2000/Revised: 10 April 2001     

The N-terminal domain of OmpATb is required for membrane translocation and pore-forming activity in mycobacteria

OmpATb is the prototype of a new family of porins in Mycobacterium tuberculosis and Mycobacterium bovis BCG. Although the pore-forming activity of this protein has been clearly established by using recombinant protein produced in Escherichia coli, characterization of the native porin has been hampered by the scarce amount of protein present in the M. tuberculosis detergent extracts. To this aim, we have developed a protocol to overproduce and obtain high yields of OmpATb in both Mycobacterium smegmatis and M. bovis BCG. The protein could be extracted and purified from the cell wall fraction and subsequently used for analysis of the pore-forming activity in multichannel and single-channel conductance experiments. Our results indicate that OmpATb produced in mycobacteria presents an average conductance value of 1,600+/-100 pS, slightly higher than that of OmpATb produced in E. coli, suggesting the occurrence of OmpATb in a highly ordered organization within the mycobacterial cell wall. In contrast to OmpATb, a truncated form lacking the first 72 amino acids (OmpATb73-326) was essentially found in the cytosol and was not active in planar lipid bilayers. This suggested that the N-terminal domain of OmpATb could participate in targeting of OmpATb to the cell wall. This was further confirmed by analyzing M. smegmatis clones expressing a chimeric protein consisting of a fusion between the N-terminal domain of OmpATb and the E. coli PhoA reporter. The present study shows for the first time that the N terminus of OmpATb is required for targeting the porin to the cell wall and also appears to be essential for its pore-forming activity.     

Identification of a cell wall channel of Streptomyces griseus: the channel contains a binding site for streptomycin

Cells of the Gram-positive actinomycete Streptomyces griseus were disrupted and the cell envelope was subjected to sucrose step-gradient centrifugation. The different fractions were analysed for NADH-oxidase activity and the formation of ion-permeable channels in lipid bilayers. Highest channel-forming activity and highest NADH-oxidase activity were found in different fractions. The cell wall fraction contained an ion-permeable channel with a single-channel conductance of 850 pS in 1 M KCl. The channel-forming protein, with an apparent molecular mass of 28 kDa, was purified to homogeneity using fast protein liquid chromatography after the extraction of whole cells with detergent. Single-channel experiments suggest that the cell wall channel is wide and water-filled. Titration experiments with streptomycin produced by S. griseus suggested that the cell wall channel binds this antibiotic with a half saturation constant of about 6 mM in 1 M KCl. The binding of streptomycin was found to be ionic strength dependent and the half saturation constant decreased to 60 microM at 0.1 M KCl. The results indicate that the 28 kDa protein represents the hydrophilic pathway through the cell wall of the Gram-positive bacterium S. griseus.     

Corynebacterium jeikeium jk0268 Constitutes for the 40 Amino Acid Long PorACj,Which Forms a Homooligomeric and Anion-Selective Cell Wall Channel

Corynebacterium jeikeium, a resident of human skin, is often associated with multidrug resistant nosocomial infections in immunodepressed patients. C. jeikeium K411 belongs to mycolic acid-containing actinomycetes, the mycolata and contains a channel-forming protein as judged from reconstitution experiments with artificial lipid bilayer experiments. The channel-forming protein was present in detergent treated cell walls and in extracts of whole cells using organic solvents. A gene coding for a 40 amino acid long polypeptide possibly responsible for the pore-forming activity was identified in the known genome of C. jeikeium by its similar chromosomal localization to known porH and porA genes of other Corynebacterium strains. The gene jk0268 was expressed in a porin deficient Corynebacterium glutamicum strain. For purification temporarily histidine-tailed or with a GST-tag at the N-terminus, the homogeneous protein caused channel-forming activity with an average conductance of 1.25 nS in 1M KCl identical to the channels formed by the detergent extracts. Zero-current membrane potential measurements of the voltage dependent channel implied selectivity for anions. This preference is according to single-channel analysis caused by some excess of cationic charges located in the channel lumen formed by oligomeric alpha-helical wheels. The channel has a suggested diameter of 1.4 nm as judged from the permeability of different sized hydrated anions using the Renkin correction factor. Surprisingly, the genome of C. jeikeium contained only one gene coding for a cell wall channel of the PorA/PorH type found in other Corynebacterium species. The possible evolutionary relationship between the heterooligomeric channels formed by certain Corynebacterium strains and the homooligomeric pore of C. jeikeium 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.     

MspA provides the main hydrophilic pathway through the cell wall of Mycobacterium smegmatis

MspA is an extremely stable, oligomeric porin from Mycobacterium smegmatis that forms water-filled channels in vitro. Immunogold electron microscopy and an enzyme-linked immunosorbent assay demonstrated that MspA is localized in the cell wall. An mspA deletion mutant did not synthesize detectable amounts of mspA mRNA, as revealed by amplification using mspA-specific primers and reverse-transcribed RNA. Detergent extracts of the DeltamspA mutant exhibited a significantly lower porin activity in lipid bilayer experiments and contained about fourfold less porin than extracts of wild-type M. smegmatis. The chromosome of M. smegmatis encodes three proteins very similar to MspA. Sequence analysis of the purified porin revealed that mspB or mspC or both genes are expressed in the DeltamspA mutant. The properties of this porin, such as single channel conductance, extreme stability against denaturation, molecular mass and composition of 20 kDa subunits, are identical to those of MspA. Deletion of mspA reduced the cell wall permeability towards cephaloridine and glucose nine- and fourfold respectively. These results show that MspA is the main general diffusion pathway for hydrophilic molecules in M. smegmatis and was only partially replaced by fewer porins in the cell wall of the DeltamspA mutant [corrected] This is the first experimental evidence that porins are the major determinants of the exceptionally low permeability of mycobacteria to hydrophilic molecules.     

The role of specific surface loop regions in determining the function of the imipenem-specific pore protein OprD of Pseudomonas aeruginosa.

Pseudomonas aeruginosa OprD is a specific porin which facilitates the uptake of basic amino acids and imipenem across the outer membrane. In this study, we examined the effects of deletions in six of the proposed eight surface loops of OprD on the in vivo and in vitro functions of this protein. Native OprD formed very small channels in planar lipid bilayers, with an average single-channel conductance in 1.0 M KCl of 20 pS. When large numbers of OprD channels were incorporated into lipid bilayer membranes, addition of increasing concentrations of imipenem to the bathing solutions resulted in a progressive blocking of the membrane conductance of KCl, indicating the presence of a specific binding site(s) for imipenem in the OprD channel. From these experiments, the concentration of imipenem value of resulting in 50% inhibition of the initial conductance was calculated as approximately 0.6 microM. In contrast, no decrease in channel conductance was observed for the OprDdeltaL2 channel upon addition of up to 2.4 microM imipenem, confirming that external loop 2 was involved in imipenem binding. Deletion of four to eight amino acids from loops 1 and 6 had no effect on antibiotic susceptibility, whereas deletion of eight amino acids from loops 5, 7, and 8 resulted in supersusceptibility to beta-lactams, quinolones, chloramphenicol, and tetracycline. Planar lipid bilayer analysis indicated that the OprDdeltaL5 channel had a 33-fold increase in single-channel conductance in 1 M KCl but had retained its imipenem binding site. The disposition of these loop regions in the interior of the OprD channel is discussed.     

Purification, functional characterization, and cDNA sequencing of mitochondrial porin from Dictyostelium discoideum.

Porin of Dictyostelium discoideum was extracted from mitochondria with Genapol X-80 and was purified by hydroxyapatite and CM-cellulose chromatography. The purified protein displayed a single band of 30 kDa in SDS-polyacrylamide gel electrophoresis. The formation of channels in artificial lipid bilayer membranes defined its function as a channel-forming component. Its average single-channel conductance was 3.9 nanosiemens in 1 M KCl, which suggested that the effective diameter of the channel is approximately 1.7 nm at small transmembrane potentials. The channel displayed a characteristic voltage dependence for potentials higher than 20 mV. It switched to substates of smaller conductance and a selectivity different to that of the open state. The closed state was stabilized at low ionic strength. The cDNA sequence of mitochondrial porin from D. discoideum was determined. It showed little sequence similarities to other known mitochondrial porins. The functional similarity, however, was striking. Localization of the porin in the mitochondrial outer membrane was confirmed by immunogold labeling of cryosections of fixed cells.     

Interaction of Clostridium perfringens iota-toxin with lipid bilayer membranes. Demonstration of channel formation by the activated binding component Ib and channel block by the enzyme component Ia.

The interaction between model lipid membranes and the binding component (Ib) of the ADP-ribosylating iota-toxin of Clostridium perfringens was studied in detail. Ib had to be activated by trypsin to result in channel formation in artificial lipid bilayers. The channels formed readily by Ib had a small single-channel conductance of about 85 picosiemens in 1 m KCl. Channel function was blocked in single-channel and multichannel experiments by the enzymatic component Ia in a pH-dependent manner. The strong Ia-mediated channel block of Ib occurred only when the pH was at least lowered to pH 5.6. The single-channel conductance showed a linear dependence on the bulk aqueous KCl concentration, which indicated that the channel properties were more general than specific. Zero current membrane potential measurements suggested the Ib channel has an approximately 6-fold higher permeability for potassium ions than for chloride. The selectivity ratio changed for salts composed of cations and anions of different mobility in the aqueous phase, again suggesting that Ib formed a water-filled general diffusion pore. Asymmetric addition of activated Ib to lipid bilayer membranes resulted in an asymmetric voltage dependence, indicating its full orientation within the membrane. Titration experiments with chloroquine and different tetraalkylammonium ions suggested that the Ib channel was blocked by these compounds but had only a weak affinity to them. In vivo measurements using Vero cells demonstrate that chloroquine and related molecules also did not efficiently block intoxication of the cells by iota-toxin. The possible role of Ib in the translocation of iota-toxin across the target cell membrane is discussed.     

pH-dependent pore-forming activity of OmpATb from Mycobacterium tuberculosis and characterization of the channel by peptidic dissection

Mycobacteria are characterized by an unusual cell wall that controls nutrient and small hydrophilic compound permeability. Porin-like proteins are necessary to ensure the transport of molecules into the cell. Here, we investigated the pore-forming properties of OmpATb, a porin from Mycobacterium tuberculosis, in lipid bilayers. Multi-channel experiments showed an asymmetric behaviour with channel closures at negative critical voltages (Vc) and a strong decrease in Vc at acidic pH. Single-channel experiments gave conductance values of about 850 +/- 80 pS in 1 M KCl and displayed a weak cationic selectivity in 4-8 pH range. The production and characterization of a series of truncated OmpATb proteins, showed that the central domain (OmpATb73-220) was sufficient to induce the ion channel properties of the native protein in lipid bilayers, i.e. asymmetric insertion, pH-dependent voltage closure, cationic selectivity and similar conductance values in 1 M KCl. Western blot analysis suggests that the presence of OmpATb is only restricted to certain pathogenic species. Therefore, the propensity of channels of native OmpATb to close at low pH may represent an intrinsic property allowing pathogenic mycobacteria to adapt and survive to mildly acidic conditions, such as those encountered within the macrophage phagosome.     

Ion channels in human endothelial cells.

Ion channels were studied in human endothelial cells from umbilical cord by the patch clamp technique in the cell attached mode. Four different types of ion channels were recorded: i) potassium channel current that rectifies at positive potentials in symmetrical potassium solutions (inward rectifier); ii) low-conductance non-selective cation channel with a permeability ratio K:Na:Ca = 1:0.9:0.2; iii) high-conductance cation-selective channel that is about 100 times more permeable for calcium than for sodium or potassium; iv) high-conductance potassium channel with a permeability ratio K:Na = 1:0.05. The extrapolated reversal potential of the inwardly rectifying current was near to the potassium equilibrium potential. The slope conductance decreased from 27 pS in isotonic KCl solution to 7 pS with 5.4 mmol/l KCl and 140 mmol/l NaCl in the pipette but 140 mmol/l KCl in the bath. The low-conductance non-selective cation channel showed a single-channel conductance of 26 pS with 140 mmol/l Na outside, 28 pS with 140 mmol/l K outside, and rectified in inward direction in the presence of Ca (60 mmol/l Ca, 70 mmol/l Na, 2.7 mmol/l K in the pipette) at negative potentials. The current could be observed with either chloride or aspartate as anion. The high-conductance non-selective channel did not discriminate between Na and K. The single-channel conductance was about 50 pS. The extrapolated reversal potential was more positive than +40 mV (140 K or 140 Na with 5 Ca outside). Both the 26 and 50 pS channel showed a run-down, and they rapidly disappeared in excised patches. The high-conductance potassium channel with a single-channel conductance of 170 pS was observed only rarely. It reversed near the expected potassium equilibrium potential. The 26 pS channel could be stimulated with histamine and thrombin from outside in the cell-attached mode. Both the 26 pS as well as the 50 pS channel can mediate calcium flux into the endothelial cell.     

Porins are required for uptake of phosphates by Mycobacterium smegmatis

Phosphorus is an essential nutrient, but how phosphates cross the mycobacterial cell wall is unknown. Phosphatase activity in whole cells of Mycobacterium smegmatis was significantly lower than that in lysed cells, indicating that access to the substrate was restricted. The loss of the outer membrane (OM) porin MspA also reduced the phosphatase activity in whole cells compared to that in lysed cells. A similar result was obtained for M. smegmatis that overexpressed endogenous alkaline phosphatase, indicating that PhoA is not a surface protein, contrary to a previous report. The uptake of phosphate by a mutant lacking the porins MspA and MspC was twofold lower than that by wild-type M. smegmatis. Strikingly, the loss of these porins resulted in a severe growth defect of M. smegmatis on low-phosphate plates. We concluded that the OM of M. smegmatis represents a permeability barrier for phosphates and that Msp porins are the only OM channels for the diffusion of phosphate in M. smegmatis. However, phosphate diffusion through Msp pores is rather inefficient as shown by the 10-fold lower permeability of M. smegmatis for phosphate compared to that for glucose. This is likely due to the negative charges in the constriction zone of Msp porins. The phosphatase activity in whole cells of Mycobacterium bovis BCG was significantly less than that in lysed cells, indicating a similar uptake pathway for phosphates in slow-growing mycobacteria. However, porins that could mediate the diffusion of phosphates across the OM of M. bovis BCG and Mycobacterium tuberculosis are unknown.