Single Pore Conductance Research Articles

Real-time imaging of individual electropores proves their longevity in cells

With over 50 years of electroporation research, the nature of cell membrane permeabilization remains elusive. The lifetime of electropores in molecular models is limited to nano- or microseconds, whereas the permeabilization of electroporated cells can last minutes. This study aimed at resolving a longstanding debate on whether the prolonged permeabilization is due to the formation of long-lived pores in cells. We developed a method for dynamic monitoring and conductance measurements of individual electropores. This was accomplished by time-lapse total internal reflection fluorescence (TIRF) imaging in HEK cells loaded with CAL-520 dye and placed on an indium tin oxide (ITO) surface. Applying a 1-ms, 0 to −400 mV pulse between the patch pipette and ITO evoked focal Ca2+ transients that identified individual electropores. Some transients disappeared in milliseconds but others persisted for over a minute. Persistent transients (“Ca2+ plumes”) faded over time to a stable or a randomly fluctuating level that could include periods of full quiescence. Single pore conductance, measured by 0 to −50 mV, 50 ms steps at 30 and 60 s after the electroporation, ranged from 80 to 200 pS. These experiments proved electropore longevity in cells, in stark contrast to molecular simulations and many findings in lipid bilayers.

R-Type Fonticins Produced by Pragia fontium Form Large Pores with High Conductance.

Fonticins are phage tail-like bacteriocins produced by the Gram-negative bacterium Pragia fontium from the family Budviciaceae. This bacterium produces contractile-type particles that adsorb on the surface of sensitive bacteria and penetrate the cell wall, probably during contraction, in a way similar to the type VI secretion system. We characterized the pore-forming activity of fonticins using both living cells and in vitro model membranes. Using a potassium leakage assay, we show that fonticins are able to permeabilize sensitive cells. On black lipid membranes, single-pore conductance is about 0.78 nS in 1 M NaCl and appears to be linearly dependent on the increasing molar strength of NaCl solution, which is a property of considerably large pores. In agreement with these findings, fonticins are not ion selective for Na+, K+, and Cl-. Polyethylene glycol 3350 (PEG 3350) molecules of about 3.5 nm in diameter can enter the fonticin pore lumen, whereas the larger molecules cannot pass the pore. The size of fonticin pores was confirmed by transmission electron microscopy. The terminal membrane-piercing complex of the fonticin tube probably creates a selective barrier restricting passage of macromolecules. IMPORTANCE Phage tail-like bacteriocins are now the subject of research as potent antibacterial agents due to their narrow host specificity and single-hit mode of action. In this work, we focused on the structure and mode of action of fonticins. According to some theories, related particles were initially adapted for passage of double-stranded DNA (dsDNA) molecules, but fonticins changed their function during the evolution; they are able to form large pores through the bacterial envelope of Gram-negative bacteria. As various pore-forming proteins are extensively used for nanopore sequencing and stochastic sensing, we decided to investigate the pore-forming properties of fonticin protein complexes on artificial lipid membranes. Our research revealed remarkable structural properties of these particles that may have a potential application as a nanodevice.

Colicin U from Shigella boydii Forms Voltage-Dependent Pores.

Colicin U is a protein produced by the bacterium Shigella boydii (serovars 1 and 8). It exerts antibacterial activity against strains of the enterobacterial genera Shigella and Escherichia Here, we report that colicin U forms voltage-dependent pores in planar lipid membranes; its single-pore conductance was found to be about 22 pS in 1 M KCl at pH 6 under 80 mV in asolectin bilayers. In agreement with the high degree of homology between their C-terminal domains, colicin U shares some pore characteristics with the related colicins A and B. Colicin U pores are strongly pH dependent, and as we deduced from the activity of colicin U in planar membranes at different protein concentrations, they have a monomeric pore structure. However, in contrast to related colicins, we observed a very low cationic selectivity of colicin U pores (1.5/1 of K+/Cl- at pH 6) along with their atypical voltage gating. Finally, using nonelectrolytes, we determined the inner diameter of the pores to be in the range of 0.7 to 1 nm, which is similar to colicin Ia, but with a considerably different inner profile.IMPORTANCE Currently, a dramatic increase in antibiotic resistance is driving researchers to find new antimicrobial agents. The large group of toxins called bacteriocins appears to be very promising from this point of view, especially because their narrow killing spectrum allows specific targeting against selected bacterial strains. Colicins are a subgroup of bacteriocins that act on Gram-negative bacteria. To date, some colicins are commercially used for the treatment of animals (1) and tested as a component of engineered species-specific antimicrobial peptides, which are studied for the potential treatment of humans (2). Here, we present a thorough single-molecule study of colicin U which leads to a better understanding of its mode of action. It extends the range of characterized colicins available for possible future medical applications.

Dilation of Fusion Pores by Synaptotagmin-1 C2AB Domains

Synaptotagmin-1 is the calcium sensor for rapid, synchronous neurotransmitter release, but how calcium binding leads to fusion is not understood. We tested the effects of Synaptotagmin's cytoplasmic C2 domains (C2AB) on fusion pore opening and dilation using a recently established single-pore conductance assay which probes fusion between nanodiscs reconstituted with neuronal v-SNAREs and engineered cells that express the complementary t-SNAREs in a flipped configuration on the cell surface. Fusion results in a nm-sized fusion pore that can be detected in cell-attached, voltage-clamped recordings. Compared to fusion induced by SNAREs alone, in the presence of C2AB the fusion rate, the mean single-pore conductance, and the pore lifetime increased, but only in the presence of calcium and PI(4,5)P2. The assay recapitulated effects of previously characterized mutations on Synaptotagmin. In particular, abolishing binding of Synaptotagmin to the SNARE complex or calcium via mutagenesis eliminated Synaptotagmin'ss effects on pore dilation. Binding of calcium to Synaptotagmin does not alter its interactions with the SNARE complex, suggesting rapid calcium-dependent Synaptotagmin-bilayer interactions may be key to triggering fusion. We probed interactions between single C2AB domains and lipid bilayers using high-resolution optical tweezers. It was recently shown that in the presence of calcium, Synaptotagmin-1 C2AB domains bound and unbound the membrane rapidly, releasing ∼8-13 kT per C2AB molecule, depending on the lipid composition. Here we show that disrupting binding of C2AB to PI(4,5)P2 through mutagenesis reduced the binding energy, while disrupting binding to calcium abolished C2AB-membrane interactions. Together, our results indicate that Synaptotagmin-1's effects on fusion pore opening and dilation require it to interact both with membranes and SNAREs. Additionally they suggest the role of Synaptotagmin-1 is not confined to triggering fusion pore opening; it also plays a post-fusion role in pore dilation in the absence of additional factors.

Macroscopic and microscopic electrochemical investigation of Clostridium botulinum C2IIa embedded in supported lipid membranes

A hybrid membrane system based on 1-octadecanethiol (ODT) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) has been investigated in respect to the incorporation of the transport component C2IIa of Clostridium botulinum C2 toxin into such bilayers. Such model systems are particularly interesting surrogates for studying fundamental transport phenomena across cell membranes. Here, we present structural and electrochemical studies at a model ODT-POPC hybrid membrane system established at a solid gold support substrate before and after the incorporation of pores. Pore incorporation was investigated via macroscopic electrochemical characterization using cyclic voltammetry (CV), and microscopic studies using atomic force microscopy (AFM) and AFM-scanning electrochemical microscopy (SECM) recording current-distance curves at the supported lipid membranes (SLMs) before and after the incorporation of pores. Recently developed AFM-SECM probes with integrated conical platinum composite electrodes were used for these spatially resolved studies. Finally, control experiments blocking the pores were performed using chloroquine (CQ), which is a known inhibitor for C2IIa proteins. The number of C2IIa pores incorporated in an ODT-POPC hybrid membrane was determined as 6.6*104 pores/mm2 based on the single pore conductance model. Blocking with chloroquine has significantly reduced the number of active pores to 2.6*104 pores/mm2. To the best of our knowledge, the associated visualization via AFM was accomplished for the first time.

Insertion of an Anionic Analogue of the Antimicrobial Peptide PGLa in Lipid Architectures Including S-Layer Supported Lipid Bilayers

The membrane – peptide insertion behavior of an artificial antimicrobial peptide analogue in liposomes, planar free-standing bilayer and planar lipid membranes supported by a crystalline bacterial surface layer, termed S-layer, was investigated. The template for this peptide was peptidyl-glycine-leucine-carboxyamide (PGLa) where all lysine residues were replaced by glutamic acid resulting in a negatively charged analogue termed PGLa(-). Zeta potential measurements and calcein release experiments on liposomes revealed that the insertion of PGLa(-) can be compared to that of native antimicrobial peptides. Patch clamp recordings on free-standing lipid membranes provided evidence of pore formation at a lipid to peptide ratio (L/P) of 1600 with a single pore conductance of 25 pS. However, also a lower conductance at a high L/P (3200) was observed which might be explained by membrane disordering effects caused by PGLa(-) interaction. In line with other studies on the action of membrane active peptides, the rupture of the lipid membrane was strongly influenced by the peptide concentration. S-layer supported lipid membranes were utilized to perform combined surface-sensitive (quartz crystal microbalance with dissipation measurements) and electrical (impedance spectroscopy) measurements. These data evidenced not only the attachment and/or insertion of PGLa(-) in the supported lipid membrane but also indicated toroidal pore formation in a concentration dependent fashion. Hence, S-layer supported lipid membranes constitute a promising platform for studying the interaction and insertion of antimicrobial peptides. Keywords: Antimicrobial peptide, electrochemical quartz crystal microbalance with dissipation monitoring, model lipid membranes, nanobiotechnology, patch-clamp, peptidyl-glycine-leucine-carboxyamide, S-layer supported bilayer.

Single-Channel Electrophysiology Reveals a Distinct and Uniform Pore Complex Formed by α-Synuclein Oligomers in Lipid Membranes

Synucleinopathies such as Parkinson's disease, multiple system atrophy and dementia with Lewy bodies are characterized by deposition of aggregated α-synuclein. Recent findings indicate that pathological oligomers rather than fibrillar aggregates may represent the main toxic protein species. It has been shown that α-synuclein oligomers can increase the conductance of lipid bilayers and, in cell-culture, lead to calcium dyshomeostasis and cell death. In this study, employing a setup for single-channel electrophysiology, we found that addition of iron-induced α-synuclein oligomers resulted in quantized and stepwise increases in bilayer conductance indicating insertion of distinct transmembrane pores. These pores switched between open and closed states depending on clamped voltage revealing a single-pore conductance comparable to that of bacterial porins. Pore conductance was dependent on transmembrane potential and the available cation. The pores stably inserted into the bilayer and could not be removed by buffer exchange. Pore formation could be inhibited by co-incubation with the aggregation inhibitor baicalein. Our findings indicate that iron-induced α-synuclein oligomers can form a uniform and distinct pore species with characteristic electrophysiological properties. Pore formation could be a critical event in the pathogenesis of synucleinopathies and provide a novel structural target for disease-modifying therapy.

Pore formation by the Bordetella adenylate cyclase toxin in lipid bilayer membranes: Role of voltage and pH

The bifunctional adenylate cyclase toxin (ACT or CyaA) of Bordetella pertussis invades target cells via transport through the cytoplasmic membrane. The membrane potential represents thereby an important factor for the uptake in vivo. Previous studies demonstrated that adenylate cyclase (AC) delivery into cells requires a negative membrane potential inside the cells. The results of lipid bilayer experiments with ACT presented here indicated that two different types of pore-like structures are formed by ACT dependent on the orientation of the electrical potential across the membranes. Pore formation at a positive potential at the cis side of the membranes, the side of the addition of the toxin, was fast and its conductance had a defined size, whereas at negative potential the pores were not defined, had a reduced pore-forming activity and a very short lifetime. Fluctuations inserted at positive potentials showed asymmetric current–voltage relationships for positive and negative voltages. Positive potentials at the cis side resulted in an increasing current, whereas at negative potentials the current decreased or remained at a constant level. Calcium ions enhanced the voltage dependence of the ACT pores when they were added to the cis side. The single-pore conductance was strongly affected by the variation of the pH value and increased in 1M KCl with increasing pH from about 4 pS at pH 5 to about 60 pS at pH 9. The ion selectivity remained unaffected by pH. Experiments with ACT mutants revealed, that the adenylate cyclase (AC) and repeat (RT) domains were not involved in voltage and pH sensing.

Segments Crucial for Membrane Translocation and Pore-forming Activity of Bordetella Adenylate Cyclase Toxin

Bordetella adenylate cyclase toxin-hemolysin (CyaA, AC-Hly, or ACT) permeabilizes cell membranes by forming small cation-selective (hemolytic) pores and subverts cellular signaling by delivering into host cells an adenylate cyclase (AC) enzyme that converts ATP to cAMP. Both AC delivery and pore formation were previously shown to involve a predicted amphipathic alpha-helix(502-522) containing a pair of negatively charged Glu(509) and Glu(516) residues. Another predicted transmembrane alpha-helix(565-591) comprises a Glu(570) and Glu(581) pair. We examined the roles of these glutamates in the activity of CyaA. Substitutions of Glu(516) increased specific hemolytic activity of CyaA by two different molecular mechanisms. Replacement of Glu(516) by positively charged lysine residue (E516K) increased the propensity of CyaA to form pores, whereas proline (E516P) or glutamine (E516Q) substitutions extended the lifetime of open single pore units. All three substitutions also caused a drop of pore selectivity for cations. Substitutions of Glu(570) and Glu(581) by helix-breaking proline or positively charged lysine residue reduced (E570K, E581P) or ablated (E570P, E581K) AC membrane translocation. Moreover, E570P, E570K, and E581P substitutions down-modulated also the specific hemolytic activity of CyaA. In contrast, the E581K substitution enhanced the hemolytic activity of CyaA 4 times, increasing both the frequency of formation and lifetime of toxin pores. Negative charge at position 570, but not at position 581, was found to be essential for cation selectivity of the pore, suggesting a role of Glu(570) in ion filtering inside or close to pore mouth. The pairs of glutamate residues in the predicted transmembrane segments of CyaA thus appear to play a key functional role in membrane translocation and pore-forming activities of CyaA.

Pore formation by tetanus toxin, its chain and fragments in neuronal membranes and evaluation of the underlying motifs in the structure of the toxin molecule.

The pore-forming activity of tetanus toxin, its chains and fragments was studied on membrane patches from spinal cord neurons of fetal mice using the outside-out patch-clamp configuration. 1. The dichain tetanus toxin forms pores at pH 5, but not at pH 7.4. The elementary pore conductance is 38.4 +/- 1.1 pS and nonselective for small cations. The open probability of the pores is voltage-dependent and increases with membrane depolarisation. The pores activate at +80 mV with a time constant of about 20 ms and deactivate at -80 mV with two time constants of about 2 ms and 10 ms. Besides the elementary pore conductance, larger pore conductances which are multiples of the elementary conductance were observed. With increasing conductances, their frequency of occurrence decreases exponentially. 2. The light chain of tetanus toxin alone does not form pores in neuronal membranes at pH 5 or at pH 7.4. 3. The heavy chain of tetanus toxin forms pores at pH 5 as well as at pH 7.4. The single pore conductance increases from 35.0 +/- 1.2 pS at pH 5 to 43.2 +/- 1.8 pS at pH 7.4. The pores allow mono- and divalent cations and chloride ions to pass. Only at pH 5 do they have a voltage dependence with time constants identical to those obtained with tetanus toxin. 4. Secondary structure predictions show a high density of presumably helically organized elements in fragment beta 2 (45 kDa) of the heavy chain between residues 700-850.(ABSTRACT TRUNCATED AT 250 WORDS)

The 35 kDa DCCD-binding protein from pig heart mitochondria is the mitochondrial porin

The protein which can be labelled by low concentrations of dicyclohexylcarbodiimide in the M r region of 30000–35000 has been purified from pig heart mitochondria with a high yield and as a single band of apparent M r 35000 in dodecyl sulphate-containing gels. The protein is not identical with the phosphate carrier as suggested before, since the two proteins behave differently during isolation. Incorporation of the isolated 35 kDa dicyclohexylcarbodiimide-binding protein into lipid bilayer membranes causes an increase of the membrane conductance in definite steps, due to the formation of pores. The specific pore-forming activity increases during the purification procedure. The single pore conductance is about 4.0 nS, suggesting a diameter of 1.7 nm of the open pore. The pore conductance is dependent on the voltage across the membrane. Anion permeability of the pore is higher than cation permeability. These properties are similar to those described for isolated mitochondrial and bacterial porins. It is concluded that the 35 kDa dicyclohexylcarbodiimide-binding protein from pig heart mitochondria is identical with porin from outer mitochondrial membrane.

Incorporation of a Voltage Sensitive Pore from Guinea Pig Heart Mitochondria into Black Lipid Membranes and Characterization of Electrical Properties

A pore from guinea pig heart mitochondria has been incorporated into BLM's (Black lipid membranes) in a highly oriented manner and its electrical properties studied. The pore shows multistate behaviour, the distribution of the pore between different conducting states being very sensitive to voltage. This has been proven by computation of single-pore experiments. Highest single pore conductance was 4.5 nSi in 1 M KCl, independent of voltage and with no detectable preference for cations or anions. The pore from guinea pig heart mitochondria reacts more sensitively to voltage than pores of mitochondria from other tissues so far incorporated into BLM's.

Purification and characterisation of a pore protein of the outer mitochondrial membrane from Neurospora crassa.

The major protein of the outer mitochondrial membrane of Neurospora was purified. On dodecylsulfate-containing gels it displayed a single band with an apparent molecular weight of 31 000. Reconstitution experiments with artificial lipid bilayers showed that this protein forms pores. Pore conductance was dependent on the voltage across the membrane. The protein inserted into the membrane in an oriented fashion, the membrane current being dependent on the sign of the voltage. Single pore conductance was 5nS, suggesting a diameter of 2 nm of the open pore. This mitochondrial protein shows a number of similarities to the outer membrane porins of gram-negative bacteria.

Determination of single-pore conductance from noise analysis. Influence of distribution in pore-amplitudes

For pores which can adopt only the open and closed states, the influence of the amplitude distribution of the single-pore conductance (γ, open state) on the covariance function is derived. It is shown that reliable mean values of γ ( E(γ)) can be derived from noise analysis only if the variance in the amplitude distribution (σ γ 2) is known. In the past, σ γ 2 was set always to zero, leading to an overestimation of E(γ). In the case of gramicidin-doped lipid bilayer membranes, this overestimation amounts to as much as 15% of the true value of E(γ).

Conductance noise of monazomycin-doped bilayer membranes

The conductance noise of the monazomycin pore has been studied by autocorrelation analysis in multi-pore systems. The autocorrelation function could be described by a superposition of two single exponential functions of different time- and voltage-dependence. The slow voltage-dependent correlation time in the range of seconds is assigned to the formation of nonconducting pore precursors. The fast voltage-independent correlation time in the msec range is related to fluctuations in the number of open pores whereby each pore adopts only two conducting states (open and closed). The corresponding correlation amplitude depends on monazomycin concentration and could be related to the single pore conductance. With increasing voltage, a slight increase of the single pore conductance was obtained which is explained on the basis of an electrostatic barrier within the pore. The pore was found to be virtually unselective for different alkali ions (Li, K, Cs).

Ionic selectivity of pores formed by the matrix protein (porin) of Escherichia coli

Incorporation of the matrix protein (porin) from the outer membrane of Escherichia coli into black lipid films results in the formation of ion-permeable pores with a single-pore conductance of the order of 2 nS (in 1 M KCl). Information on the structure of this pore has been obtained by determining the selectivity for various species differing in charge and size. From the permeability of the pore for large organic ions (Tris+, glucosamine+, Hepes-) a minimum pore diameter of 0.8 nm is estimated. At neutral pH the pore is two to four times more permeable for alkali ions than for chloride. On the basis of the observed pH dependence of permeability, this cationic selectivity is explained by the assumption that the pore contains fixed negative charges.

Analysis of the multi-pore system of alamethicin in a lipid membrane

The properties of the alamethicin pore in multi-pore systems have been studied by autocorrelation analysis and by power spectral density. In agreement with the relaxation studies of part I, we found two processes of different time behavior and voltage dependency. The slow (sec) and fast (msec) correlation times could be related to fluctuations in the number of pores and to transitions between different levels of conductance within a pore, respectively. The interpretation of the correlation amplitudes in terms of a simplified three-state model used for the calculation of the corresponding unit step conductances confirms the assignment. The correlation amplitude of the slow process could be related to the mean single-pore conductance. The mean conductance difference of neighboring conducting pore states could be estimated using the product of the correlation amplitudes of the slow and fast process. In the range of weakly voltage-dependent conductance a strongly conductance-dependent increase of the correlation times as well as of the correlation amplitudes was observed. A conductance dependent on the size of the preaggregate from which the pore is formed was proposed for an interpretation of these findings. The mean single-pore conductance and the mean conductance difference of neighboring conducting pore states vary approximately linearly (on the log/log scale) on ion concentration in the range 0.1 to 1m KCl. The salt concentration dependence of the macroscopic conductance was found to increase with decreasing temperature. An influence of the ionic strength on both correlation times could be observed.