Activation energy for pore opening in lipid membranes under an electric field.

The standard model of pore formation was introduced more than fifty years ago, and it has been since, despite some refinements, the cornerstone for interpreting experiments related to pores in membranes. A central prediction of the model concerning pore opening under an electric field is that the activation barrier for pore formation is lowered proportionally to the square of the electric potential. However, this has only been scarcely and inconclusively confronted to experiments. In this paper, we study the electropermeability of model lipid membranes composed of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) containing different fractions of POPC-OOH, the hydroperoxidized form of POPC, in the range 0 to 100 mol %. By measuring ion currents across a 50-µm-diameter black lipid membrane (BLM) with picoampere and millisecond resolution, we detect hydroperoxidation-induced changes to the intrinsic bilayer electropermeability and to the probability of opening angstrom-size or larger pores. Our results over the full range of lipid compositions show that the energy barrier to pore formation is lowered linearly by the absolute value of the electric field, in contradiction with the predictions of the standard model.

Resolving Isomeric Posttranslational Modifications Using a Biological Nanopore as a Sensor of Molecular Shape.

The chemical nature and precise position of posttranslational modifications (PTMs) in proteins or peptides are crucial for various severe diseases, such as cancer. State-of-the-art PTM diagnosis is based on elaborate and costly mass-spectrometry or immunoassay-based approaches, which are limited in selectivity and specificity. Here, we demonstrate the use of a protein nanopore to differentiate peptides─derived from human histone H4 protein─of identical mass according to the positions of acetylated and methylated lysine residues. Unlike sequencing by stepwise threading, our method detects PTMs and their positions by sensing the shape of a fully entrapped peptide, thus eliminating the need for controlled translocation. Molecular dynamics simulations show that the sensitivity to molecular shape derives from a highly nonuniform electric field along the pore. This molecular shape-sensing principle offers a path to versatile, label-free, and high-throughput characterizations of protein isoforms.

A chip-based array for high-resolution fluorescence characterization of free-standing horizontal lipid membranes under voltage clamp.

Optical techniques, such as fluorescence microscopy, are of great value in characterizing the structural dynamics of membranes and membrane proteins. A particular challenge is to combine high-resolution optical measurements with high-resolution voltage clamp electrical recordings providing direct information on e.g. single ion channel gating and/or membrane capacitance. Here, we report on a novel chip-based array device which facilitates optical access with water or oil-immersion objectives of high numerical aperture to horizontal free-standing lipid membranes while controlling membrane voltage and recording currents using individual micropatterned Ag/AgCl-electrodes. Wide-field and confocal imaging, as well as time-resolved single photon counting on free-standing membranes spanning sub-nanoliter cavities are demonstrated while electrical signals, including single channel activity, are simultaneously acquired. This optically addressable microelectrode cavity array will allow combined electrical-optical studies of membranes and membrane proteins to be performed as a routine experiment.

Pore-forming toxins as tools for polymer analytics: From sizing to sequencing.

We report here on the nanopore resistive pulse sensing (Np-RPS) method, involving pore-forming toxins as tools for polymer analytics at single molecule level. Np-RPS is an electrical method for the label-free detection of single molecules. A molecule interacting with the pore causes a change of the electrical resistance of the pore, called a resistive pulse, associated with a measurable transient current blockade. The features of the blockades, in particular their depth and duration, contain information on the molecular properties of the analyte. We first revisit the history of Np-RPS, then we discuss the effect of the configuration of the molecule/nanopore interaction on the molecular information that can be extracted from the signal, illustrated in two different regimes that either favor molecular sequencing or molecular sizing. Specifically, we focus on the sizing regime and on the use of two different pore-forming toxins, staphylococcal α-hemolysin (αHL) and aerolysin (AeL) nanopores, for the characterization of water-soluble polymers (poly-(ethylene glycol), (PEG)), homopeptides, and heteropeptides. We discuss how nanopore sizing of polymers could be envisioned as a new approach for peptide/protein sequencing.

Electrophysiology on Channel-Forming Proteins in Artificial Lipid Bilayers: Next-Generation Instrumentation for Multiple Recordings in Parallel.

Artificial lipid bilayers have been used for several decades to study channel-forming pores and ion channels in membranes. Until recently, the classical two-chamber setups have been primarily used for studying the biophysical properties of pore forming proteins. Within the last 10 years, instruments for automated lipid bilayer measurements have been developed and are now commercially available. This chapter focuses on protein purification and reconstitution of channel-forming proteins into lipid bilayers using a classic setup and on the commercially available systems, the Orbit mini and Orbit 16.

Retraction: Microelectrochemical cell arrays for whole-cell currents recording through ion channel proteins based on trans-electroporation approach.

Retraction of 'Microelectrochemical cell arrays for whole-cell currents recording through ion channel proteins based on trans-electroporation approach' by Tianyang Zheng et al., Analyst, 2020, 145, 197-205.

Electrical recognition of the twenty proteinogenic amino acids using an aerolysin nanopore.

Efforts to sequence single protein molecules in nanopores1-5 have been hampered by the lack of techniques with sufficient sensitivity to discern the subtle molecular differences among all twenty amino acids. Here we report ionic current detection of all twenty proteinogenic amino acids in an aerolysin nanopore with the help of a short polycationic carrier. Application of molecular dynamics simulations revealed that the aerolysin nanopore has a built-in single-molecule trap that fully confines a polycationic carrier-bound amino acid inside the sensing region of the aerolysin. This structural feature means that each amino acid spends sufficient time in the pore for sensitive measurement of the excluded volume of the amino acid. We show that distinct current blockades in wild-type aerolysin can be used to identify 13 of the 20 natural amino acids. Furthermore, we show that chemical modifications, instrumentation advances and nanopore engineering offer a route toward identification of the remaining seven amino acids. These findings may pave the way to nanopore protein sequencing.

Correction: Microelectrochemical cell arrays for whole-cell currents recording through ion channel proteins based on trans-electroporation approach.

Correction for 'Microelectrochemical cell arrays for whole-cell currents recording through ion channel proteins based on trans-electroporation approach' by Tianyang Zheng, et al., Analyst, 2020, DOI: 10.1039/c9an01737b.

Microelectrochemical cell arrays for whole-cell currents recording through ion channel proteins based on trans-electroporation approach.

High electrostability and long life-time of planar chip technology are crucial for electrophysiological measurements such as ionic current recording through ion channel proteins embedded in biological cell membrane. In this paper, we propose a novel planar microchip integrated with microelectrochemical cell array toward to a feasible solution for ion channel screening with high resolution and long life-time. In order to reduce the interference from the leakage currents, a synthetic lipid bilayer is applied to form a high sealing resistance. The whole-cell electrical access can be constructed via electroporating the lipid bilayer in close proximity to the cell membrane. Parameters of electroporation including amplitude and time scale are firstly optimized by using parallel electroporation to the lipid bilayers. In this approach, individual cells can be trapped to the target positions by applying dielectrophoresis (DEP) manipulation. Poly(ethylene glycol) (PEG) is employed with low concentration to facilitate the closed contact between the cells and the lipid bilayer to increase the efficiency of the whole-cell mode construction. Through this chip based method, stable current recordings through inward rectifier potassium (Kir) ion channels embedded in rat basophilic leukemia (RBL-1) cell membrane are achieved with high electrical sealing resistance (over 1 GΩ). In addition, without need for complex fluidic connections, this method allows for an easy operation and further miniaturization of the measuring system.

Activity of the Gramicidin A Ion Channel in a Lipid Membrane with Switchable Physical Properties.

Lipid bilayer membranes formed from the artificial 1,3-diamidophospholipid Pad-PC-Pad have the remarkable property that their hydrophobic thickness can be modified in situ: the particular arrangement of the fatty acid chains in Pad-PC-Pad allows them to fully interdigitate below 37 °C, substantially thinning the membrane with respect to the noninterdigitated state. Two stimuli, traversing the main phase transition temperature of the lipid or addition of cholesterol, have previously been shown to disable the interdigitated state. Both manipulations cause an increase in hydrophobic thickness of about 25 Å due to enhanced conformational entropy of the lipids. Here, we characterize the interdigitated state using electrophysiological recordings from free-standing lipid-membranes formed on micro structured electrode cavity arrays. Compared to standard membranes made from 1,2-diphytanoyl-sn-glycero-3-phosphocholin (DPhPC), pure Pad-PC-Pad membranes at room temperature had lowered electroporation threshold and higher capacitance. Ion channel formation by the peptide Gramicidin A was clearly facilitated in pure Pad-PC-Pad membranes at room temperature, with activity occurring at significantly lower peptide concentrations and channel dwell times increased by 2 orders of magnitude with respect to DPhPC-membranes. Both elevation of temperature beyond the phase transition and addition of cholesterol reduced channel dwell times, as expected if the reduced membrane thickness stabilized channel formation due to decreased hydrophobic mismatch.

Transgenic short-QT syndrome 1 rabbits mimic the human disease phenotype with QT/action potential duration shortening in the atria and ventricles and increased ventricular tachycardia/ventricular fibrillation inducibility.

AIMS: Short-QT syndrome 1 (SQT1) is an inherited channelopathy with accelerated repolarization due to gain-of-function in HERG/IKr. Patients develop atrial fibrillation, ventricular tachycardia (VT), and sudden cardiac death with pronounced inter-individual variability in phenotype. We generated and characterized transgenic SQT1 rabbits and investigated electrical remodelling. METHODS AND RESULTS: Transgenic rabbits were generated by oocyte-microinjection of ß-myosin-heavy-chain-promoter-KCNH2/HERG-N588K constructs. Short-QT syndrome 1 and wild type (WT) littermates were subjected to in vivo ECG, electrophysiological studies, magnetic resonance imaging, and ex vivo action potential (AP) measurements. Electrical remodelling was assessed using patch clamp, real-time PCR, and western blot. We generated three SQT1 founders. QT interval was shorter and QT/RR slope was shallower in SQT1 than in WT (QT, 147.8 ± 2 ms vs. 166.4 ± 3, P < 0.0001). Atrial and ventricular refractoriness and AP duration were shortened in SQT1 (vAPD90, 118.6 ± 5 ms vs. 154.4 ± 2, P < 0.0001). Ventricular tachycardia/fibrillation (VT/VF) inducibility was increased in SQT1. Systolic function was unaltered but diastolic relaxation was enhanced in SQT1. IKr-steady was increased with impaired inactivation in SQT1, while IKr-tail was reduced. Quinidine prolonged/normalized QT and action potential duration (APD) in SQT1 rabbits by reducing IKr. Diverse electrical remodelling was observed: in SQT1, IK1 was decreased-partially reversing the phenotype-while a small increase in IKs may partly contribute to an accentuation of the phenotype. CONCLUSION: Short-QT syndrome 1 rabbits mimic the human disease phenotype on all levels with shortened QT/APD and increased VT/VF-inducibility and show similar beneficial responses to quinidine, indicating their value for elucidation of arrhythmogenic mechanisms and identification of novel anti-arrhythmic strategies.

A Fully Integrated Current-Mode Continuous-Time Delta-Sigma Modulator for Biological Nanopore Read Out.

This paper presents a fully integrated low noise current-to-digital converter based on current-mode continuous-time delta-sigma modulators (CT DSMs). The circuit was realized in standard 0.35  µm complimentary metal-oxide-semiconductor process and achieves a noise floor as low as 200 fA rms within a bandwidth of 10 kHz. Thanks to the continuous-time architecture, the oversampling together with the implicit anti-aliasing behavior of the current-mode CT DSM prevents the back folding of any out-of-band signals and noises. Running at a sampling frequency of 1.6 MHz, the circuit achieves a measured worst case anti-aliasing filtering of -110 dB for an out-of-band signal at 1.59 MHz. This characteristic eliminates the need of an anti-aliasing filter, which reduces the complexity as well as the area and power consumption. Two feedback versions, a transistor based as well as a capacitor-based current division have been used to reduce the reference current noise. The transistor-based current division feedback modulator consumes a power of 6.73 mW and occupies an area of 0.63 mm 2, whereas the capacitor based consumes a power of 4.6 mW and occupies an area of 0.32 mm 2. Both of them operate at a supply voltage of ±1.5 V.

The Multifaceted Antibacterial Mechanisms of the Pioneering Peptide Antibiotics Tyrocidine and Gramicidin S.

Cyclic ß-sheet decapeptides from the tyrocidine group and the homologous gramicidin S were the first commercially used antibiotics, yet it remains unclear exactly how they kill bacteria. We investigated their mode of action using a bacterial cytological profiling approach. Tyrocidines form defined ion-conducting pores, induce lipid phase separation, and strongly reduce membrane fluidity, resulting in delocalization of a broad range of peripheral and integral membrane proteins. Interestingly, they also cause DNA damage and interfere with DNA-binding proteins. Despite sharing 50% sequence identity with tyrocidines, gramicidin S causes only mild lipid demixing with minor effects on membrane fluidity and permeability. Gramicidin S delocalizes peripheral membrane proteins involved in cell division and cell envelope synthesis but does not affect integral membrane proteins or DNA. Our results shed a new light on the multifaceted antibacterial mechanisms of these antibiotics and explain why resistance to them is virtually nonexistent.IMPORTANCE Cyclic ß-sheet decapeptides, such as tyrocidines and gramicidin S, were among the first antibiotics in clinical application. Although they have been used for such a long time, there is virtually no resistance to them, which has led to a renewed interest in this peptide class. Both tyrocidines and gramicidin S are thought to disrupt the bacterial membrane. However, this knowledge is mainly derived from in vitro studies, and there is surprisingly little knowledge about how these long-established antibiotics kill bacteria. Our results shed new light on the antibacterial mechanism of ß-sheet peptide antibiotics and explain why they are still so effective and why there is so little resistance to them.

Size-dependent interaction of a 3-arm star poly(ethylene glycol) with two biological nanopores.

We use two pore-forming proteins, alpha-hemolysin and aerolysin, to compare the polymer size-dependence of ionic current block by two types of ethyleneglycol polymers: 1) linear and 2) 3-arm star poly(ethylene glycol), both applied as a polydisperse mixture of average mass 1kDa under high salt conditions. The results demonstrate that monomer size sensitivity, as known for linear PEGs, is conserved for the star polymers with only subtle differences in the dependence of the residual conductance on monomer number. To explain this absence of a dominant effect of polymer architecture, we propose that PEG adsorbs to the inner pore wall in a collapsed, salted-out state, likely due to the effect of hydrophobic residues in the pore wall on the availability of water for hydration.

Translocation of Precision Polymers through Biological Nanopores.

Nanopore analysis, which is, currently, chiefly used for DNA sequencing, is also an appealing technique for characterizing abiotic polymers. As a first step toward this goal, nanopore detection of non-natural monodispersed poly(phosphodiester)s as candidate backbone structures is reported herein. Two model homopolymers containing phosphopropyl repeat units (i.e., 56 or 104 r.u.) and a short thymidine nucleotide sequence are analyzed in the present work. They are tested in two different biological nanopores, α-hemolysin from Staphylococcus aureus, and aerolysin from Aeromonas hydrophila. These recordings are performed in aqueous medium at different KCl concentrations and various driving voltages. The data show a complex interaction with evidence for voltage dependence and threading, and underline the influence of the molecular structure and orientation of the precision poly(phosphodiester)s on the observed residual current signal as well as on the translocation dynamics. In particular, they suggest a dominant entropic contribution due to the high flexibility of the phosphodiester homopolymer.

Interregional electro-mechanical heterogeneity in the rabbit myocardium.

BACKGROUND: Increased electrical heterogeneity has been causatively linked to arrhythmic disorders, yet the knowledge about physiological heterogeneity remains incomplete. This study investigates regional electro-mechanical heterogeneities in rabbits, one of the key animal models for arrhythmic disorders. METHODS AND FINDINGS: 7 wild-type rabbits were examined by phase-contrast magnetic resonance imaging in vivo to assess cardiac wall movement velocities. Using a novel data-processing algorithm regional contraction-like profiles were calculated. Contraction started earlier and was longer in left ventricular (LV) apex than base. Patch clamp recordings showed longer action potentials (AP) in LV apex compared to the base of LV, septum, and right ventricle. Western blots of cardiac ion channels and calcium handling proteins showed lower expression of Cav1.2, KvLQT1, Kv1.4, NCX and Phospholamban in LV apex vs. base. A single-cell in silico model integrating the quantitative regional differences in ion channels reproduced a longer contraction and longer AP in apex vs. base. CONCLUSIONS: Apico-basal electro-mechanical heterogeneity is physiologically present in the healthy rabbit heart. An apico-basal electro-mechanical gradient exists with longer APD and contraction duration in the apex and associated regionally heterogeneous expression of five key proteins. This pattern of apical mechanical dominance probably serves to increase pumping efficiency.

High Temperature Extends the Range of Size Discrimination of Nonionic Polymers by a Biological Nanopore.

We explore the effect of temperature on the interaction of polydisperse mixtures of nonionic poly(ethylene glycol) (PEG) polymers of different average molar masses with the biological nanopore α-hemolysin. In contrast with what has been previously observed with various nanopores and analytes, we find that, for PEGs larger than a threshold molar mass (2000 g/mol, PEG 2000), increasing temperature increases the duration of the PEG/nanopore interaction. In the case of PEG 3400 the duration increases by up to a factor of 100 when the temperature increases from 5 °C to 45 °C. Importantly, we find that increasing temperature extends the polymer size range of application of nanopore-based single-molecule mass spectrometry (Np-SMMS)-type size discrimination. Indeed, in the case of PEG 3400, discrimination of individual molecular species of different monomer number is impossible at room temperature but is achieved when the temperature is raised to 45 °C. We interpret our observations as the consequence of a decrease of PEG solubility and a collapse of PEG molecules with higher temperatures. In addition to expanding the range of application of Np-SMMS to larger nonionic polymers, our findings highlight the crucial role of the polymer solubility for the nanopore detection.

High-Resolution Size-Discrimination of Single Nonionic Synthetic Polymers with a Highly Charged Biological Nanopore.

Electrophysiological studies of the interaction of polymers with pores formed by bacterial toxins (1) provide a window on single molecule interaction with proteins in real time, (2) report on the behavior of macromolecules in confinement, and (3) enable label-free single molecule sensing. Using pores formed by the staphylococcal toxin α-hemolysin (aHL), a particularly pertinent observation was that, under high salt conditions (3-4 M KCl), the current through the pore is blocked for periods of hundreds of microseconds to milliseconds by poly(ethylene glycol) (PEG) oligomers (degree of polymerization approximately 10-60). Notably, this block showed monomeric sensitivity on the degree of polymerization of individual oligomers, allowing the construction of size or mass spectra from the residual current values. Here, we show that the current through the pore formed by aerolysin (AeL) from Aeromonas hydrophila is also blocked by PEG but with drastic differences in the voltage-dependence of the interaction. In contrast to aHL, AeL strongly binds PEG at high transmembrane voltages. This fact, which is likely related to AeL's highly charged pore wall, allows discrimination of polymer sizes with particularly high resolution. Multiple applications are now conceivable with this pore to screen various nonionic or charged polymers.

Antibiotic translocation through porins studied in planar lipid bilayers using parallel platforms.

In general, the method of choice to characterize the conductance properties of channel-forming bacterial porins is electrophysiology. Here, the classical method is to reconstitute single porins into planar lipid bilayers to derive functional information from the observed channel conductance. In addition to an estimated pore size, ion selectivity or transport properties in general are of importance. For the latter, measuring the ion current fluctuation can provide some information about the mode of transport of charged molecules penetrating the proteins. For instance, increasing the external voltage modifies the residence time in the channel: charged molecules with the ability to permeate through channels will travel faster whereas non-permeating molecules get pushed to the constriction zone with enhanced residence time. Here, we are interested in the ability of antibiotics to permeate channels and compare different techniques to reveal fast events.

Automated formation of lipid membrane microarrays for ionic single-molecule sensing with protein nanopores.

Efficient use of membrane protein nanopores in ionic single-molecule sensing requires technology for the reliable formation of suspended molecular membranes densely arrayed in formats that allow high-resolution electrical recording. Here, automated formation of bimolecular lipid layers is shown using a simple process where a poly(tetrafluoroethylene)-coated magnetic bar is remotely actuated to perform a turning motion, thereby spreading phospholipid in organic solvent on a nonpolar surface containing a <1 mm(2) 4 × 4 array of apertures with embedded microelectrodes (microelectrode cavity array). Parallel and high-resolution single-molecule detection by single nanopores is demonstrated on the resulting bilayer arrays, which are shown to form by a classical but very rapid self-assembly process. The technique provides a robust and scalable solution for the problem of reliable, automated formation of multiple independent lipid bilayers in a dense microarray format, while preserving the favorable electrical properties of the microelectrode cavity array.