Asymmetric Lipid Bilayers and Potassium Channels Embedded Therein in the Contact Bubble Bilayer.

Cell membranes are highly intricate systems comprising numerous lipid species and membrane proteins, where channel proteins, lipid molecules, and lipid bilayers, as continuous elastic fabric, collectively engage in multi-modal interplays. Owing to the complexity of the native cell membrane, studying the elementary processes of channel-membrane interactions necessitates a bottom-up approach starting from forming simplified synthetic membranes. This is the rationale for establishing an in vitro membrane reconstitution system consisting of a lipid bilayer with a defined lipid composition and a channel molecule. Recent technological advancements have facilitated the development of asymmetric membranes, and the contact bubble bilayer (CBB) method allows single-channel current recordings under arbitrary lipid compositions in asymmetric bilayers. Here, we present an experimental protocol for the formation of asymmetric membranes using the CBB method. The KcsA potassium channel is a prototypical model channel with huge structural and functional information and thus serves as a reporter of membrane actions on the embedded channels. We demonstrate specific interactions of anionic lipids in the inner leaflet. Considering that the local lipid composition varies steadily in cell membranes, we `present a novel lipid perfusion technique that allows rapidly changing the lipid composition while monitoring the single-channel behavior. Finally, we demonstrate a leaflet perfusion method for modifying the composition of individual leaflets. These techniques with custom synthetic membranes allow for variable experiments, providing crucial insights into channel-membrane interplay in cell membranes.

Probing membrane deformation energy by KcsA potassium channel gating under varied membrane thickness and tension.

This study investigated how membrane thickness and tension modify the gating of KcsA potassium channels when simultaneously varied. The KcsA channel undergoes global conformational changes upon gating: expansion of the cross-sectional area and longitudinal shortening upon opening. Thus, membranes impose differential effects on the open and closed conformations, such as hydrophobic mismatches. Here, the single-channel open probability was recorded in the contact bubble bilayer, by which variable thickness membranes under a defined tension were applied. A fully open channel in thin membranes turned to sporadic openings in thick membranes, where the channel responded moderately to tension increase. Quantitative gating analysis prompted the hypothesis that tension augmented the membrane deformation energy when hydrophobic mismatch was enhanced in thick membranes.

Different lateral packing stress in acyl chains alters KcsA orientation and structure in lipid membranes.

The molecular structures of the various intrinsic lipids in membranes regulate lipid-protein interactions. These different lipid structures with unique volumes produce different lipid molecular packing stresses/lateral stresses in lipid membranes. Most studies examining lipid packing effects have used phosphatidylcholine and phosphatidylethanolamine (PE), which are the main phospholipids of eukaryotic cell membranes. In contrast, Gram-negative or Gram-positive bacterial membranes are composed primarily of phosphatidylglycerol (PG) and PE, and the physical and thermodynamic properties of each acyl chain in PG at the molecular level remain unresolved. In this study, we used 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG, 16:0-18:1 PG) and 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (PAPG, 16:0-20:4 PG) to prepare lipid bilayers (liposome) with the rod-type fluorescence probe DPH. We measured the lipid packing conditions by determining the rotational freedom of DPH in POPG or PAPG bilayers. Furthermore, we investigated the effect of different monoacyl chains on a K+ channel (KcsA) structure when embedded in POPG or PAPG membranes. The results revealed that differences in the number of double bonds and carbon chain length in the monoacyl chain at sn-2 affected the physicochemical properties of the membrane and the structure and orientation of KcsA.

Cardiolipin binding enhances KcsA channel gating via both its specific and dianion-monoanion interchangeable sites.

KcsA is a potassium channel with a plethora of structural and functional information, but its activity in the KcsA-producing actinomycete membranes remains elusive. To determine lipid species involved in channel-modulation, a surface plasmon resonance (SPR)-based methodology, characterized by immobilization of membrane proteins under a membrane environment, was applied. Dianionic cardiolipin (CL) showed extremely higher affinity for KcsA than monoanionic lipids. The SPR experiments further demonstrated that CL bound not only to the N-terminal M0 helix, a lipid-sensor domain, but to the M0 helix-deleted mutant. In contrast, monoanionic lipids interacted primarily with the M0 helix. This indicates the presence of an alternative CL-binding site, plausibly in the transmembrane domain. Single-channel recordings demonstrated that CL enhanced channel opening in an M0-independent manner. Taken together, the action of monoanionic lipids is exclusively mediated by the M0 helix, while CL binds both the M0 helix and its specific site, further enhancing the channel activity.

Vectorial insertion of a ß-helical peptide into membrane: a theoretical study on polytheonamide B.

Spontaneous unidirectional, or vectorial, insertion of transmembrane peptides is a fundamental biophysical process for toxin and viral actions. Polytheonamide B (pTB) is a potent cytotoxic peptide with a ß6.3-helical structure. Previous experimental studies revealed that the pTB inserts into the membrane in a vectorial fashion and forms a channel with its single molecular length long enough to span the membrane. Also, molecular dynamics simulation studies demonstrated that the pTB is prefolded in aqueous solution. These are unique features of pTB because most of the peptide toxins form channels through oligomerization of transmembrane helices. Here, we performed all-atom molecular dynamics simulations to examine the dynamic mechanism of the vectorial insertion of pTB, providing underlying elementary processes of the membrane insertion of a prefolded single transmembrane peptide. We find that the insertion of pTB proceeds with only the local lateral compression of the membrane in three successive phases: "landing," "penetration," and "equilibration" phases. The free energy calculations using the replica-exchange umbrella sampling simulations present an energy cost of 4.3 kcal/mol at the membrane surface for the membrane insertion of pTB from bulk water. The trajectories of membrane insertion revealed that the insertion process can occur in two possible pathways, namely "trapped" and "untrapped" insertions; in some cases, pTB is trapped in the upper leaflet during the penetration phase. Our simulations demonstrated the importance of membrane anchoring by the hydrophobic N-terminal blocking group in the landing phase, leading to subsequent vectorial insertion.

Geometrical and electrophysiological data of the moving membrane method for the osmotic water permeability of a lipid bilayer.

Data of the osmotic water permeability of a lipid bilayer (diphytanoylphosphaticylcholin) in the presence of cholesterol (30 mole%) are shown under the simultaneous measurement of bilayer tension. Detailed methods and procedures for evaluating the water permeability using the moving membrane method (K. Yano, M. Iwamoto, T. Koshiji & S. Oiki: Visualizing the Osmotic Water Permeability of a Lipid Bilayer under Measured Bilayer Tension Using a Moving Membrane Method. Journal of Membrane Science, 627 (2021) 119231) are presented. The planar lipid bilayer is formed in a glass capillary, separating two aqueous compartments with different osmolarities, and osmotically-driven water flux is visualized as membrane movements along the capillary. The water permeability was evaluated under constant membrane area and tension after correcting for the unstirred layer effect. In these measurements, geometrical features, such as the edge of the planar lipid bilayer and the contact angle between bilayer and monolayer, were image-analyzed. The unstirred layer was evaluated electrophysiologically, in which gramicidin A channel was employed. In the presence of an osmotic gradient, the gramicidin channel generates the streaming potential, and the measured streaming potential data and the derived water-ion coupling ratio (water flux/ion flux) are shown. Detailed descriptions of the integrated method of the moving membrane allow researchers to reproduce the experiment and give opportunities to examine water permeability of various types of membranes, including those containing aquaporins. The present data of osmotic water permeability are compared with the previously published data, while they neglected the bilayer tension.

Hysteresis of a Tension-Sensitive K+ Channel Revealed by Time-Lapse Tension Measurements.

Various types of channels vary their function by membrane tension changes upon cellular activities, and lipid bilayer methods allow elucidation of direct interaction between channels and the lipid bilayer. However, the dynamic responsiveness of the channel to the membrane tension remains elusive. Here, we established a time-lapse tension measurement system. A bilayer is formed by docking two monolayer-lined water bubbles, and tension is evaluated via measuring intrabubble pressure as low as <100 Pa (Young-Laplace principle). The prototypical KcsA potassium channel is tension-sensitive, and single-channel current recordings showed that the activation gate exhibited distinct tension sensitivity upon stretching and relaxing. The mechanism underlying the hysteresis is discussed in the mode shift regime, in which the channel protein bears short "memory" in their conformational changes.

Physical and Chemical Interplay Between the Membrane and a Prototypical Potassium Channel Reconstituted on a Lipid Bilayer Platform.

Once membrane potential changes or ligand binding activates the ion channel, the activity of the channel is finely modulated by the fluctuating membrane environment, involving local lipid composition and membrane tension. In the age of post-structural biology, the factors in the membrane that affect the ion channel function and how they affect it are a central concern among ion channel researchers. This review presents our strategies for elucidating the molecular mechanism of membrane effects on ion channel activity. The membrane's diverse and intricate effects consist of chemical and physical processes. These elements can be quantified separately using lipid bilayer methods, in which a membrane is reconstructed only from the components of interest. In our advanced lipid bilayer platform (contact bubble bilayer, CBB), physical features of the membrane, such as tension, are freely controlled. We have elucidated how the specific lipid or membrane tension modulates the gating of a prototypical potassium channel, KcsA, embedded in the lipid bilayer. Our results reveal the molecular mechanism of the channel for sensing and responding to the membrane environment.

Conductance selectivity of Na+ across the K+ channel via Na+ trapped in a tortuous trajectory.

Ion selectivity of the potassium channel is crucial for regulating electrical activity in living cells; however, the mechanism underlying the potassium channel selectivity that favors large K+ over small Na+ remains unclear. Generally, Na+ is not completely excluded from permeation through potassium channels. Herein, the distinct nature of Na+ conduction through the prototypical KcsA potassium channel was examined. Single-channel current recordings revealed that, at a high Na+ concentration (200 mM), the channel was blocked by Na+, and this blocking was relieved at high membrane potentials, suggesting the passage of Na+ across the channel. At a 2,000 mM Na+ concentration, single-channel Na+ conductance was measured as one-eightieth of the K+ conductance, indicating that the selectivity filter allows substantial conduit of Na+ Molecular dynamics simulations revealed unprecedented atomic trajectories of Na+ permeation. In the selectivity filter having a series of carbonyl oxygen rings, a smaller Na+ was distributed off-center in eight carbonyl oxygen-coordinated sites as well as on-center in four carbonyl oxygen-coordinated sites. This amphipathic nature of Na+ coordination yielded a continuous but tortuous path along the filter. Trapping of Na+ in many deep free energy wells in the filter caused slow elution. Conversely, K+ is conducted via a straight path, and as the number of occupied K+ ions increased to three, the concerted conduction was accelerated dramatically, generating the conductance selectivity ratio of up to 80. The selectivity filter allows accommodation of different ion species, but the ion coordination and interactions between ions render contrast conduction rates, constituting the potassium channel conductance selectivity.

Queueing arrival and release mechanism for K+ permeation through a potassium channel.

The mechanism underlying ion permeation through potassium channels still remains controversial. K+ ions permeate across a narrow selectivity filter (SF) in a single file. Conventional scenarios assume that K+ ions are tightly bound in the SF, and, thus, they are displaced from their energy well by ion-ion repulsion with an incoming ion. This tight coupling between entering and exiting ions has been called the "knock-on" mechanism. However, this paradigm is contradicted by experimental data measuring the water-ion flux coupling ratio, demonstrating fewer ion occupancies. Here, the results of molecular dynamics simulations of permeation through the KcsA potassium channel revealed an alternative mechanism. In the aligned ions in the SF (an ion queue), the outermost K+ was readily and spontaneously released toward the extracellular space, and the affinity of the relevant ion was ~ 50 mM. Based on this low-affinity regime, a simple queueing mechanism described by loose coupling of entering and exiting ions is proposed.

In bulla functional channel expression systems that mimic bacterial synthetic membranes.

A functional characterization of channel proteins has been performed using planar lipid bilayers as the following procedure. For bacterial channels, such as the KcsA potassium channel, channel proteins were synthesized in Escherichia coli, followed by solubilization, purification, and incorporation into liposomes. Similarly, channel proteins were synthesized using an in vitro transcription/translation kit in the presence of liposomes. Then, these liposome-incorporated channels were served for electrophysiological recordings after liposome fusion into a preformed planar lipid bilayer. Here, we established a straightforward method for concurrent channel synthesis and functional measurement using a water-in-oil bubble bilayer system. Channel proteins were synthesized in vitro within a water-in-oil bubble, having a lipid bilayer at the contact with another bubble (in bulla synthesis). The channels were spontaneously incorporated into the lipid bilayer under application of the membrane potential, and we successfully detected nascent channel activities. This way our experiment has mimicked bacterial synthetic membrane in the presence of a resting membrane potential. Technical details for establishing the in bulla expression system are described.

Drop-in-well chamber for droplet interface bilayer with built-in electrodes.

Various methods have been developed for the formation of planar lipid bilayers, and recent techniques using water-in-oil droplets, such as droplet interface bilayer (DIB) and contact bubble bilayer (CBB) methods, allow the ready formation of bilayers with arbitrary lipid compositions. Here, we developed a simple and portable DIB system using drop-in-wells, shaping two merging wells for settling electrolyte droplets. An aliquot of the electrolyte solution (1µL) is dropped into an organic solvent, and the droplet sinks to the drop-in-well at the bottom, where two monolayer-lined droplets come in contact to form the bilayer. Pre-installed electrodes allow electrophysiological measurements. The detailed drop-in-well method is presented, and some variations of the method, such as the use of microelectrodes and a sheet with a small hole for low-noise recordings, are extended. Examples of single channel current recordings of the KcsA potassium channel are demonstrated.

A concise method for quantitative analysis of interactions between lipids and membrane proteins.

Although interactions between lipids and membrane proteins (MPs) have been considered crucially important for understanding the functions of lipids, lack of useful and convincing experimental methods has hampered the analysis of the interactions. Here, we developed a surface plasmon resonance (SPR)-based concise method for quantitative analysis of lipid-MP interactions, coating the sensor chip surface with self-assembled monolayer (SAM) with C6-chain. To develop this method, we used bacteriorhodopsin (bR) as an MP, and examined its interaction with various types of lipids. The merits of using C6-SAM-modified sensor chip are as follows: (1) alkyl-chains of SAM confer a better immobilization of MPs because of the efficient preconcentration due to hydrophobic contacts; (2) SAM provides immobilized MPs with a partial membranous environment, which is important for the stabilization of MPs; and (3) a thinner C6-SAM layer (1 nm) compared with MP size forces the MP to bulge outward from the SAM surface, allowing extraneously injected lipids to be accessible to the hydrophobic transmembrane regions. Actually, the amount of bR immobilized on C6-SAM is 10 times higher than that on a hydrophilic CM5 sensor chip, and AFM observations confirmed that bR molecules are exposed on the SAM surface. Of the lipids tested, S-TGA-1, a halobacterium-derived glycolipid, had the highest specificity to bR with a nanomolar dissociation constant. This is consistent with the reported co-crystal structure that indicates the formation of several intermolecular hydrogen bonds. Therefore, we not only reproduced the specific lipid-bR recognition, but also succeeded in its quantitative evaluation, demonstrating the validity and utility of this method.

Lipid Bilayer Experiments with Contact Bubble Bilayers for Patch-Clampers.

Lipid bilayers provide a unique experimental platform for functional studies of ion channels, allowing the examination of channel-membrane interactions under various membrane lipid compositions. Among them, the droplet interface bilayer has gained popularity; however, the large membrane size hinders the recording of low electrical background noise. We have established a contact bubble bilayer (CBB) method that combines the benefits of planar lipid bilayer and patch-clamp methods, such as the ability to vary the lipid composition and to manipulate the bilayer mechanics, respectively. Using the setup for conventional patch-clamp experiments, CBB-based experiments can be readily performed. In brief, an electrolyte solution in a glass pipette is blown into an organic solvent phase (hexadecane), and the pipette pressure is maintained to obtain a stable bubble size. The bubble is spontaneously lined with a lipid monolayer (pure lipids or mixed lipids), which is provided from liposomes in the bubbles. Next, the two monolayer-lined bubbles (~50 µm in diameter) at the tip of the glass pipettes are docked for bilayer formation. Introduction of channel-reconstituted liposomes into the bubble leads to the incorporation of channels in the bilayer, allowing for single-channel current recording with a signal-to-noise ratio comparable to that of patch-clamp recordings. CBBs with an asymmetric lipid composition are readily formed. The CBB is renewed repeatedly by blowing out the previous bubbles and forming new ones. Various chemical and physical perturbations (e.g., membrane perfusion and bilayer tension) can be imposed on the CBBs. Herein, we present the basic procedure for CBB formation.

Constitutive boost of a K+ channel via inherent bilayer tension and a unique tension-dependent modality.

Molecular mechanisms underlying channel-membrane interplay have been extensively studied. Cholesterol, as a major component of the cell membrane, participates either in specific binding to channels or via modification of membrane physical features. Here, we examined the action of various sterols (cholesterol, epicholesterol, etc.) on a prototypical potassium channel (KcsA). Single-channel current recordings of the KcsA channel were performed in a water-in-oil droplet bilayer (contact bubble bilayer) with a mixed phospholipid composition (azolectin). Upon membrane perfusion of sterols, the activated gate at acidic pH closed immediately, irrespective of the sterol species. During perfusion, we found that the contacting bubbles changed their shapes, indicating alterations in membrane physical features. Absolute bilayer tension was measured according to the principle of surface chemistry, and inherent bilayer tension was ∼5 mN/m. All tested sterols decreased the tension, and the nonspecific sterol action to the channel was likely mediated by the bilayer tension. Purely mechanical manipulation that reduced bilayer tension also closed the gate, whereas the resting channel at neutral pH never activated upon increased tension. Thus, rather than conventional stretch activation, the channel, once ready to activate by acidic pH, changes the open probability through the action of bilayer tension. This constitutes a channel regulating modality by two successive stimuli. In the contact bubble bilayer, inherent bilayer tension was high, and the channel remained boosted. In the cell membrane, resting tension is low, and it is anticipated that the ready-to-activate channel remains closed until bilayer tension reaches a few millinewton/meter during physiological and pathological cellular activities.

Thylakoid membranes contain a non-selective channel permeable to small organic molecules.

The thylakoid lumen is a membrane-enclosed aqueous compartment. Growing evidence indicates that the thylakoid lumen is not only a sink for protons and inorganic ions translocated during photosynthetic reactions but also a place for metabolic activities, e.g. proteolysis of photodamaged proteins, to sustain efficient photosynthesis. However, the mechanism whereby organic molecules move across the thylakoid membranes to sustain these lumenal activities is not well understood. In a recent study of Cyanophora paradoxa chloroplasts (muroplasts), we fortuitously detected a conspicuous diffusion channel activity in the thylakoid membranes. Here, using proteoliposomes reconstituted with the thylakoid membranes from muroplasts and from two other phylogenetically distinct organisms, cyanobacterium Synechocystis sp. PCC 6803 and spinach, we demonstrated the existence of nonselective channels large enough for enabling permeation of small organic compounds (e.g. carbohydrates and amino acids with Mr < 1500) in the thylakoid membranes. Moreover, we purified, identified, and characterized a muroplast channel named here CpTPOR. Osmotic swelling experiments revealed that CpTPOR forms a nonselective pore with an estimated radius of ∼1.3 nm. A lipid bilayer experiment showed variable-conductance channel activity with a typical single-channel conductance of 1.8 nS in 1 m KCl with infrequent closing transitions. The CpTPOR amino acid sequence was moderately similar to that of a voltage-dependent anion-selective channel of the mitochondrial outer membrane, although CpTPOR exhibited no obvious selectivity for anions and no voltage-dependent gating. We propose that transmembrane diffusion pathways are ubiquitous in the thylakoid membranes, presumably enabling rapid transfer of various metabolites between the lumen and stroma.

Lipid Bilayers Manipulated through Monolayer Technologies for Studies of Channel-Membrane Interplay.

Fluidity and mosaicity are two critical features of biomembranes, by which membrane proteins function through chemical and physical interactions within a bilayer. To understand this complex and dynamic system, artificial lipid bilayer membranes have served as unprecedented tools for experimental examination, in which some aspects of biomembrane features have been extracted, and to which various methodologies have been applied. Among the lipid bilayers involving liposomes, planar lipid bilayers and nanodiscs, recent developments of lipid bilayer methods and the results of our channel studies are reviewed herein. Principles and techniques of bilayer formation are summarized, which have been extended to the current techniques, where a bilayer is formed from lipid-coated water-in-oil droplets (water-in-oil bilayer). In our newly developed method, termed the contact bubble bilayer (CBB) method, a water bubble is blown from a pipette into a bulk oil phase, and monolayer-lined bubbles are docked to form a bilayer through manipulation by pipette. An asymmetric bilayer can be readily formed, and changes in composition in one leaflet were possible. Taking advantage of the topological configuration of the CBB, such that the membrane's hydrophobic interior is contiguous with the surrounding bulk organic phase, oil-dissolved substances such as cholesterol were delivered directly to the bilayer interior to perfuse around the membrane-embedded channels (membrane perfusion), and current recordings in the single-channel allowed detection of immediate changes in the channels' response to cholesterol. Chemical and mechanical manipulation in each monolayer (monolayer technology) allows the examination of dynamic channel-membrane interplay.

Concurrent In Vitro Synthesis and Functional Detection of Nascent Activity of the KcsA Channel under a Membrane Potential.

Processes involved in the functional formation of prokaryotic membrane proteins have remained elusive. Here, we developed a new in vitro membrane protein expression system to detect nascent activities of the KcsA potassium channel in lipid bilayers under an applied membrane potential. The channel was synthesized using a reconstituted Escherichia coli-based in vitro transcription/translation system (IVTT) in a water-in-oil droplet lined by a membrane. The synthesized channels spontaneously incorporated into the membrane even without the translocon machinery (unassisted pathway) and formed functional channels with the correct orientation. The single-channel current of the first appearing nascent channel was captured, followed by the subsequent appearance of multiple channels. Notably, the first appearance time shortened substantially as the membrane potential was hyperpolarized. Under a steadily applied membrane potential, this system serves as a production line of membrane proteins via the unassisted pathway, mimicking the bacterial synthetic membrane.

Structure and dynamics of solvent molecules inside the polytheonamide B channel in different environments: a molecular dynamics study.

The ß6.3-helical channel of the marine cytotoxic peptide, polytheonamide B (pTB), is examined in water, the POPC bilayer, and a 1 : 1 chloroform/methanol mixture using all-atom molecular dynamics simulations. The structures and fluctuations of the ß6.3-helix of pTB are investigated in the three environments. The average structure of pTB calculated in the mixed solvent is in good agreement with the NMR-resolved structure in the mixed solvent, indicating the validity of the parameters used for the non-standard groups in pTB. The configuration and dynamics of solvent molecules inside the pore are examined in detail. It is found that the motions of methanol molecules inside the pore are not correlated because of the absence of strong hydrogen bonds (HBs) between adjacent methanol molecules. On the other hand, the motions of water molecules inside the pore are highly correlated, both translationally and orientationally, due to the strong HBs between neighboring water molecules. It is suggested that the collective behavior of water molecules inside the pore in the membrane is crucial for the permeation of ions through the pTB channel.

Channel Formation and Membrane Deformation via Sterol-Aided Polymorphism of Amphidinol 3.

Amphidinol 3 (AM3) is an anti-fungal polyene extracted from a marine dinoflagellate. Here, we examined the ion channel activity and membrane-embedded structure of AM3 using a lipid bilayer method and atomic force microscopy (AFM). AM3 exhibited large-conductance (~1 nS) and non-selective single-channel activity only when sterols were present in the membrane leaflet of the AM3-added side. The variable conductance suggests the formation of a multimeric barrel-stave pore. At high AM3 concentrations, giant-conductance "jumbo" channels (~40 nS) emerged. AFM revealed a thicker raft-like membrane phase with the appearance of a wrinkled surface, in which phase pores (diameter: ~10 nm) were observed. The flip-flop of ergosterol occurred only after the appearance of the jumbo channel, indicating that the jumbo channel induced a continuity between the outer and inner leaflets of the membrane: a feature characteristic of toroidal-like pores. Thus, AM3 forms different types of sterol-aided polymorphic channels in a concentration dependent manner.