Polarity-dependent voltage-gated porin channels from Escherichia coli in lipid bilayer membranes.

A porin preparation from Escherichia coli 0111:B4 consisting of Omp F and Omp C (with Omp F in excess) was purified by salt extraction procedures and investigated in bilayer lipid membranes formed according to the Montal-Mueller technique. The porin preparation was added to the KCl electrolyte compartment of the Montal-Mueller cell which was connected to the voltage source. As the porin incorporated into the membrane, asymmetric, voltage-gated ion channels were formed. Transmembrane voltages greater than +50 mV (measured with respect to the side of porin addition) caused channel closing, while negative voltages, on the other hand, had no effect on channel behaviour but did increase the rate of porin incorporation at higher voltages. With porin added to both compartments voltage gating no longer occurred. Single-channel conductances corresponded to effective pore diameters of 1.5 nm for opening events and 1.18 nm for channel closing events. The number of charges involved in gating was approximately 2.     

Molecular basis of voltage gating of OmpF porin.

OmpF and PhoE from Escherichia coli and related homologous proteins from other Gram-negative bacteria allow the passive transport of small polar molecules across the bacterial outer membrane. In vitro, they exhibit voltage gating depending on the experimental conditions. We review current hypotheses on the underlying molecular mechanism of voltage gating of OmpF porin and show how computer simulations can be used to examine each of the proposed mechanisms.     

Charged Residues in Surface-located Loops Influence Voltage Gating of Porin from Haemophilus influenzae Type b

Porin of Haemophilus influenzae type b (341 amino acids; M _r 37782) determines the permeability of the outer membrane to low molecular mass compounds. Purified Hib porin was subjected to chemical modification of lysine residues by succinic anhydride. Electrospray ionization mass spectrometry identified up to 12 modifications per porin molecule. Tryptic digestion of modified Hib porin followed by reverse phase chromatography and matrix assisted laser desorption ionization time-of-flight mass spectrometry mapped the succinylation sites. Most modified lysines are positioned in surface-located loops, numbers 1 and 4 to 7. Succinylated porin was reconstituted into planar lipid bilayers, and biophysical properties were analyzed and compared to Hib porin: there was an increased average single channel conductance compared to Hib porin (1.24+/−0.41 vs. 0.85+/−0.40 nanosiemens). The voltage-gating activity of succinylated porin differed considerably from that of Hib porin. The threshold voltage for gating was decreased from 75 to 40 mV. At 80 mV, steady-state conductance for succinylated porin was 50–55% of the instantaneous conductance. Hib porin at 80 mV showed a decrease to 89–91% of the instantaneous current levels. We propose that surface-located lysine residues are determinants of voltage gating for porin of Haemophilus influenzae type b. Received: 11 August 2000/Revised: 8 September 2000     

Targeting of the pro-apoptotic VDAC-like porin (PorB) of Neisseria gonorrhoeae to mitochondria of infected cells

Infection of cell cultures with Neisseria gonorrhoeae results in apoptosis that is mediated by the PorB porin. During the infection process porin translocates from the outer bacterial membrane into host cell membranes where its channel activity is regulated by nucleotide binding and voltage-dependent gating, features that are shared by the mitochondrial voltage-dependent anion channel (VDAC). Here we show that porin is selectively and efficiently transported to mitochondria of infected cells. Prevention of porin translocation also blocked the induction of apoptosis. Mitochondria of cells treated with porin both in vitro and in vivo were depleted of cytochrome c and underwent permeability transition. Overexpression of Bcl-2 blocked porin-induced apoptosis. The release of cytochrome c occurred independently of active caspases but was completely prevented by Bcl-2. Our data suggest that the Neisseria porin can, like its eukaryotic homologue, function at the mitochondrial checkpoint to mediate apoptosis.     

Understanding Voltage Gating of Providencia stuartii Porins at Atomic Level

Bacterial porins are water-filled β-barrel channels that allow translocation of solutes across the outer membrane. They feature a constriction zone, contributed by the plunging of extracellular loop 3 (L3) into the channel lumen. Porins are generally in the open state, but undergo gating in response to external voltages. To date the underlying mechanism is unclear. Here we report results from molecular dynamics simulations on the two porins of Providenica stuartii, Omp-Pst1 and Omp-Pst2, which display distinct voltage sensitivities. Voltage gating was observed in Omp-Pst2, where the binding of cations in-between L3 and the barrel wall results in exposing a conserved aromatic residue in the channel lumen, thereby halting ion permeation. Comparison of Omp-Pst1 and Omp-Pst2 structures and trajectories suggests that their sensitivity to voltage is encoded in the hydrogen-bonding network anchoring L3 onto the barrel wall, as we observed that it is the strength of this network that governs the probability of cations binding behind L3. That Omp-Pst2 gating is observed only when ions flow against the electrostatic potential gradient of the channel furthermore suggests a possible role for this porin in the regulation of charge distribution across the outer membrane and bacterial homeostasis.     

Altered channel properties of porins from Haemophilus influenzae: isolates from cystic fibrosis patients

Changes in amino-acid sequence of the unique pore-forming protein of H. influenzae (OmpP2; porin) have been associated with increased antimicrobial resistance in H. influenzae strains isolated from cystic fibrosis patients. From patients who were subjected to long-term antimicrobial therapy, H. influenzae strains 67d and 69a (patient 27) and strains 77a and 77f (patient 30) were isolated. Strains 67d and 77a were previously shown to have elevated values for minimal inhibitory concentrations of antibiotics compared to strains 69a and 77f. Porins were extracted from all four H. influenzae strains by detergent treatment and purified to homogeneity by ion exchange chromatography. By reconstitution of the clinical Hi porins into planar lipid bilayers, single-channel conductance, ionic selectivity, and voltage-gating characteristics were assessed. Porins 77a and 77f displayed similar single-channel conductance and ionic selectivity. Current-voltage relationships were determined for the different porins: porin 77f displayed substantial voltage gating at both positive and negative polarity; porin 77a gated at negative polarity only. Porins 67d and 69a showed substantial differences in their pore-forming properties: the single-channel conductance of porin 69a was significantly increased (1.05 nS) relative to porin 67d (0.73 nS). Porin 67d was twice as permeable to cations as porin 69a, and at both positive and negative polarities the extent of voltage gating was greater for porin 67d relative to porin 69a. Expression of the porins in an isogenic, porin-deleted H. influenzae background allowed for assessment of the contribution of each porin to the minimum inhibitory concentrations of various antimicrobial compounds. Porin 67d was found to have a decreased susceptibility to the antimicrobials novobiocin and streptomycin. This decreased susceptibility of porin 67d to novobiocin and streptomycin correlates with its decrease in single-channel conductance.     

The gate of mitochondrial porin channel is controlled by a number of negative and positive charges

Negatively charged carboxyl groups of mitochondrial porin have been converted into positively charged ones by means of reaction with water-soluble carbodiimide in the presence of ethylenediamine. Properties of channels formed in a planar lipid bilayer by native and modified porins are compared. Amidation has only little influence on the porin channel-forming activity as well as on the open-state conductance of the channel. However, the modification results in a significant enhancement of the voltage dependence of the channel gating and in an increase of the anionic selectivity. It is suggested that the voltage sensor of the porin channel gate is composed of a number of negative (greater than 14) and positive (greater than 22) charges.     

Mutagenesis identifies amino acid residues in extracellular loops and within the barrel lumen that determine voltage gating of porin from Haemophilus influenzae type b.

Porin (341 amino acids; M(r) 37 782) of Haemophilus influenzae type b mediates exchange of solutes between the external environment and the periplasm of this Gram-negative bacterium. Positively charged residues in the extracellular loops have been shown to be involved in the voltage gating of this protein. To further elucidate our observations on the functional properties of this channel, we mutated seven lysines (Lys(48), Lys(161), Lys(165), Lys(170), Lys(248), Lys(250), and Lys(253)) to glutamic acid. The selected residues were previously shown to be accessible to chemical modification, and they map to three locations: loop 4 and loop 6, and within the barrel lumen. The seven mutant proteins were purified, and each was reconstituted into planar lipid bilayers to characterize its channel forming properties. The single substitution mutant porins displayed increased single channel conductances in 1 M KCl ranging between 134 and 178% of the single channel conductance for wild-type Hib porin. Six of the seven mutant porins also displayed altered current-voltage relationships when compared to wild-type Hib porin. Whereas Lys(170)Glu had activity similar to wild-type Hib porin, Lys(48)Glu, Lys(248)Glu, and Lys(253)Glu showed substantial voltage gating at both positive and negative polarities. Lys(161)Glu and Lys(250)Glu gated only at negative potentials, and Lys(165)Glu gated only at positive potentials. Rather than ascribing one specific loop in gating, our analyses of these mutant Hib porins suggest that voltage gating can be attributed to contributions from loops 4 and 6 and a residue within the barrel lumen.     

Electromagnetic field (EMF) effects on channel activity of nanopore OmpF protein

In this study, the effects of nonionizing electromagnetic fields (EMF; 925 MHz) on the OmpF porin channel have been characterized at the single-channel level. Channel activity was recorded in real time by the voltage clamp method. Our results showed an increase in the frequency of channel gating and voltage sensitivity. The effects of EMF lasted for several milliseconds after the field source was terminated. However, the conductance levels of channels did not change significantly. Thermal effects of EMF on single-channel properties are a possible cause, based on theoretical evaluation of results that were comparable to those seen in conventional experiments at different temperatures. We conclude that EMF affects both the dynamics and conformation of the channel, either directly by affecting critical amino acid side-chain arrangement, or indirectly, via the electrolyte or the lipid membrane.     

Properties of the large ion-permeable pores formed from protein F of Pseudomonas aeruginosa in lipid bilayer membranes

The incorporation of porin protein F from the outer membrane of Pseudomonas aeruginosa into artificial lipid bilayers results in an increase of the membrane conductance by many orders of magnitude. The membrane conductance is caused by the formation of large ion-permeable channels with a single-channel conductance in the order of 5 nS for 1 M alkali chlorides. The conductance has an ohmic current vs. voltage relationship. Further information on the structure of the pore formed by protein F was obtained by determining the single-channel conductance for various species differing in charge and size, and from zero-current potential measurements. The channel was found to be permeable for large organic ions (Tris+, N(C2H5)4+, Hepes-) and a channel diameter of 2.2 nm could be estimated from the conductance data (pore length of 7.5 nm). At neutral pH the pore is about two times more permeable for cations than for anions, possibly caused by negative charges in the pore. The consistent observation of large water filled pores formed by porin protein F in model membrane systems is discussed in the light of the known low permeability of the Ps. aeruginosa outer membrane towards antibiotics. It is suggested that this results from a relatively low proportion of open functional porin protein F pores in vivo.     

Positive residues involved in the voltage-gating of the mitochondrial porin-channel are localized in the external moiety of the pore.

The role of positive charges located on the hydrophilic surface of the mitochondrial outer membrane channel was investigated by studying the interaction between LDAO-solubilized porin and a cation-exchanger column. The binding of porin to the column material was inhibited when the elution buffer had a pH of 9 or when 2 mM dextran sulfate was added to the buffer at neutral pH. Interestingly, the addition of a synthetic copolymer of methacrylate, maleate and styrene known as a potent modulator of the voltage-dependence, did not influence the interaction between column material and porin. Incubation of porin with fluorescein isothiocyanate (FITC) resulted in the isolation of a porin fraction in which on average two lysines located on the surface of the pore-forming complex per 35 kDa polypeptide were modified. The voltage-dependence of the fluorescein isothiocyanate modified porin was strongly decreased as compared with the unmodified porin. The experiments presented here give the first biochemical evidence that positively charged lysine residues located on the surface of the channel-forming complex are responsible for the gating of the mitochondrial porin-channel.     

Asymmetric electrostatic effects on the gating of rat brain sodium channels in planar lipid membranes.

The effects of ionic strength (10-1,000 mM) on the gating of batrachotoxin-activated rat brain sodium channels were studied in neutral and in negatively charged lipid bilayers. In neutral bilayers, increasing the ionic strength of the extracellular solution, shifted the voltage dependence of the open probability (gating curve) of the sodium channel to more positive membrane potentials. On the other hand, increasing the intracellular ionic strength shifted the gating curve to more negative membrane potentials. Ionic strength shifted the voltage dependence of both opening and closing rate constants of the channel in analogous ways to its effects on gating curves. The voltage sensitivities of the rate constants were not affected by ionic strength. The effects of ionic strength on the gating of sodium channels reconstituted in negatively charged bilayers were qualitatively the same as in neutral bilayers. However, important quantitative differences were noticed: in low ionic strength conditions (10-150 mM), the presence of negative charges on the membrane surface induced an extra voltage shift on the gating curve of sodium channels in relation to neutral bilayers. It is concluded that: (a) asymmetric negative surface charge densities in the extracellular (1e-/533A2) and intracellular (1e-/1,231A2) sides of the sodium channel could explain the voltage shifts caused by ionic strength on the gating curve of the channel in neutral bilayers. These surface charges create negative electric fields in both the extracellular and intracellular sides of the channel. Said electric fields interfere with gating charge movements that occur during the opening and closing of sodium channels; (b) the voltage shifts caused by ionic strength on the gating curve of sodium channels can be accounted by voltage shifts in both the opening and closing rate constants; (c) net negative surface charges on the channel's molecule do not affect the intrinsic gating properties of sodium channels but are essential in determining the relative position of the channel's gating curve; (d) provided the ionic strength is below 150 mM, the gating machinery of the sodium channel molecule is able to sense the electric field created by surface changes on the lipid membrane. I propose that during the opening and closing of sodium channels, the gating charges involved in this process are asymmetrically displaced in relation to the plane of the bilayer. Simple electrostatic calculations suggest that gating charge movements are influenced by membrane electrostatic potentials at distances of 48 and 28 A away from the plane of the membrane in the extracellular sides of the channel, respectively.     

The voltage-dependent activity of Escherichia coli porins in different planar bilayer reconstitutions

Because of conflicting results from differing techniques, the degree of voltage sensitivity of Escherichia coli porins in planar bilayers is still a matter of debate. In order to provide the first comparative study, OmpF porin was purified in three ways; firstly as native outer membrane vesicles, secondly as salt-extracted porin trimers in sodium dodecyl sulphate and thirdly as solubilised trimers extracted with octyl-polyoxyethylene (Octyl-POE). These methods represent the major approaches to porin isolation and purification. All three were reconstituted into Schindler-type bilayers. Detergent-solubilised OmpF was also reconstituted into Montal-Mueller- and Mueller-Rudin-type bilayers. In all cases voltage-dependent closing of OmpF was observed. Octyl-POE-extracted PhoE porin was similarly investigated in all three types of planar bilayer. Two membrane-formation techniques appeared genuinely to alter the voltage sensitivity of the porins they contained. Firstly, porins in membranes formed by the Montal-Mueller technique sometimes showed an increase in voltage sensitivity during the first 30 min after bilayer formation. Secondly, membranes formed by the Mueller-Rudin technique on thick polyethylene septa showed both poor solvent drainage and a significantly reduced porin voltage sensitivity.     

Tracking S4 movement by gating pore currents in the bacterial sodium channel NaChBac

Voltage-gated sodium channels mediate the initiation and propagation of action potentials in excitable cells. Transmembrane segment S4 of voltage-gated sodium channels resides in a gating pore where it senses the membrane potential and controls channel gating. Substitution of individual S4 arginine gating charges (R1–R3) with smaller amino acids allows ionic currents to flow through the mutant gating pore, and these gating pore currents are pathogenic in some skeletal muscle periodic paralysis syndromes. The voltage dependence of gating pore currents provides information about the transmembrane position of the gating charges as S4 moves in response to membrane potential. Here we studied gating pore current in mutants of the homotetrameric bacterial sodium channel NaChBac in which individual arginine gating charges were replaced by cysteine. Gating pore current was observed for each mutant channel, but with different voltage-dependent properties. Mutating the first (R1C) or second (R2C) arginine to cysteine resulted in gating pore current at hyperpolarized membrane potentials, where the channels are in resting states, but not at depolarized potentials, where the channels are activated. Conversely, the R3C gating pore is closed at hyperpolarized membrane potentials and opens with channel activation. Negative conditioning pulses revealed time-dependent deactivation of the R3C gating pore at the most hyperpolarized potentials. Our results show sequential voltage dependence of activation of gating pore current from R1 to R3 and support stepwise outward movement of the substituted cysteines through the narrow portion of the gating pore that is sealed by the arginine side chains in the wild-type channel. This pattern of voltage dependence of gating pore current is consistent with a sliding movement of the S4 helix through the gating pore. Through comparison with high-resolution models of the voltage sensor of bacterial sodium channels, these results shed light on the structural basis for pathogenic gating pore currents in periodic paralysis syndromes.     

Characterization of different reactive lysines in bovine heart mitochondrial porin

Incubation of mitochondrial outer membrane porin with citraconic anhydride prior to treatment with fluorescein isothiocyanate (FITC) resulted in the labeling of a set of lysines located at a boundary between the water phase and lipid phase. The elution pattern of porin from the cation exchanger has been considered as indicative for the location of lysines. Electrical measurements after reconstitution of the modified protein in lipid bilayer membranes revealed that certain specific lysine residues are more susceptible to alterations. The innermost positive residues were only slightly influenced, while the outermost lysines exhibited a substantial change in channel properties. These results suggest the presence of critical charged residues in mitochondrial outer membrane porin that may be responsible for both the channel's selectivity and its voltage dependence.     

Subconductance states in OmpF gating

Discrepancies were noted in the published conductance of the Escherichia coli porin OmpF. Results from various papers are hard to compare because of the use of different channel preparations, salt types and concentrations, and electrophysiological techniques (black lipid membrane (BLM) vs. patch clamp). To reconcile these data, we present a side-by-side comparison of OmpF activity studied with the two techniques on the same preparation of pure protein, and in the same low salt concentrations (150 mM KCl). The novel aspect of OmpF porin behavior revealed by this comparison is the ubiquitous existence of states of smaller conductance than the monomeric conductance (subconductance states), regardless of the techniques or experimental conditions used, and the drastic enhancement of subconductance gating by polyamines. Transitions to subconductance states have received little attention in previous publications, in particular when BLM electrophysiology was used. Monomeric closures are rare in recordings at clamped potentials, at least at voltages lower than ∼100-120 mV. Most closing activity is in the form of subconductance gating, which becomes more dominant in the presence of spermine, with a more frequent and prolonged occupation of these substates. A discussion of the molecular basis for this hallmark behavior of porin is presented.     

Deletion variants of Neurospora mitochondrial porin: electrophysiological and spectroscopic analysis

Mitochondrial porins are predicted to traverse the outer membrane as a series of beta-strands, but the precise structure of the resulting beta-barrel has remained elusive. Toward determining the positions of the membrane-spanning segments, a series of small deletions was introduced into several of the predicted beta-strands of the Neurospora crassa porin. Overall, three classes of porin variants were identified: i), those producing large, stable pores, indicating deletions likely outside of beta-strands; ii), those with minimal pore-forming ability, indicating disruptions in key beta-strands or beta-turns; and iii), those that formed small unstable pores with a variety of gating and ion-selectivity properties. The latter class presumably results from a subset of proteins that adopt an alternative barrel structure upon the loss of stabilizing residues. Some variants were not sufficiently stable in detergent for structural analysis; circular dichroism spectropolarimetry of those that were did not reveal significant differences in the overall structural composition among the detergent-solubilized porin variants and the wild-type protein. Several of the variants displayed altered tryptophan fluorescence profiles, indicative of differing microenvironments surrounding these residues. Based on these results, modifications to the existing models for porin structure are proposed.     

A tetrameric porin limits the cell wall permeability of Mycobacterium smegmatis

Mycobacteria protect themselves with an outer lipid bilayer, which is the thickest biological membrane hitherto known and has an exceptionally low permeability rendering mycobacteria intrinsically resistant against many antibiotics. Pore proteins mediate the diffusion of hydrophilic nutrients across this membrane. Electron microscopy revealed that the outer membrane of Mycobacterium smegmatis contained about 1000 protein pores per microm(2), which are about 50-fold fewer pores per microm(2) than in Gram-negative bacteria. The projection structure of the major porin MspA of M. smegmatis was determined at 17 A resolution. MspA forms a cone-like tetrameric complex of 10 nm in length with a single central pore. Thus, MspA is drastically different from the trimeric porins of Gram-negative bacteria and represents a new class of channel proteins. The formation of MspA micelles indicated that the ends of MspA have different hydrophobicities. Oriented insertion of MspA into membranes was demonstrated in lipid bilayer experiments, which revealed a strongly asymmetrical voltage gating of MspA channels at -30 mV. The length of MspA is sufficient to span the outer membrane and contributes in combination with the tapering end of the pore and the low number of pores to the low permeability of the cell wall of M. smegmatis for hydrophilic compounds.     

Properties of the large ion-permeable pores formed from protein F of Pseudomonas aeruginosa in lipid bilayer membranes

The incorporation of porin protein F from the outer membrane of Pseudomonas aeruginosa into artificial lipid bilayers results in an increase of the membrane conductance by many orders of magnitude. The membrane conductance is caused by the formation of large ion-permeable channels with a single-channel conductance in the order of 5 nS for 1 M alkali chlorides. The conductance has an ohmic current vs. voltage relationship. Further information on the structure of the pore formed by protein F was obtained by determining the single-channel conductance for various species differing in charge and size, and from zero-current potential measurements. The channel was found to be permeable for large organic ions (Tris⁺, N(C₂H₅)₄⁺, Hepes^?) and a channel diameter of 2.2 nm could be estimated from the conductance data (pore length of 7.5 nm). At neutral pH the pore is about two times more permeable for cations than for anions, possibly caused by negative charges in the pore. The consistent observation of large water filled pores formed by porin protein F in model membrane systems is discussed in the light of the known low permeability of the Ps. aeruginosa outer membrane towards antibiotics. It is suggested that this results from a relatively low proportion of open functional porin protein F pores in vivo.