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.

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.

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.

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.

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.

pH-dependent promotion of phospholipid flip-flop by the KcsA potassium channel.

KcsA is a pH-dependent potassium channel that is activated at acidic pH. The channel undergoes global conformational changes upon activation. We hypothesized that the open-close conformational changes of the transmem brane region could promote the flip-flop of phosphoiipids. Based on this hypothesis, we measured the flip-flop ofNBD-labeled phospholipids in KcsA-incorporated proteoliposomes. Both flip and flop rates of ~NBD-PC were significantly enhanced in the presence of KcsA and were several times higher at pH 4.0 than at pH 7.4, suggesting that KcsA promotes the phospholipid flip in a conformation-dependent manner. Phospholipids were nonselectively flipped with respect to the glycerophospholipid structure. In the active state of KcsA channel,tetrabutylammonium locks the channel in the open conformation at acidic pH; however, it did not alter the fliprate of CGNBD-PC. Thus, the open-close transition of the transmembrane region did not affect the flip-flop of phospholipids. In addition, the KcsA mutant that lacked anN-terminal amphipathic helix (MO-helix) was found to show reduced ability to fl ip C6NBD-phospholipids at acidic pH. The closed conformation is stabilized in the absence of MO-heli x, and thus the attenuated flip could be explained by the reduced prevalence of the open conformation.These results suggest that the open conformation of KcsA can disturb the bilayer integrity and facilitate the flip-flop of phospholipids.

Digitalized K(+) Occupancy in the Nanocavity Holds and Releases Queues of K(+) in a Channel.

The mechanisms of ion permeation through potassium channels have been extensively examined. Molecular dynamics (MD) simulations have demonstrated that rapidly permeating ions collide near the selectivity filter (SF) ("knock-on" mechanism), but this oversimplified mechanism is insufficient to account for the experimentally observed single-channel current amplitudes. Here, we analyzed the MD-simulated ion trajectories through a Kv1.2 potassium channel using an event-oriented analysis method, and surprisingly, we found that the nanocavity (NC) governs ion permeation in a digital fashion. The NC has a maximal diameter of 10 Å and stands between the intracellular bulk solution and the SF, which holds only up to one K(+) during permeation. Accordingly, the K(+) concentration in the intracellular solution is translated as a digitalized zero or one K(+) in the NC. When the ion number in the NC is zero, the multiple ions in the SF are mostly immobilized. By contrast, when the number of ions in the NC is one, the structured water in the NC mediates the ion-occupied status to the queueing ions in the SF, and the ions then initiate a collective outward motion. Accordingly, the one ion in the NC serves as a catalytic intermediate for permeation, which quantitatively accounts for the experimentally obtained conductance-concentration relationships. We conclude that the ion movements are coherent across the entire pore.

Rectified Proton Grotthuss Conduction Across a Long Water-Wire in the Test Nanotube of the Polytheonamide B Channel.

A hydrogen-bonded water-chain in a nanotube is highly proton conductive, and examining the proton flux under electric fields is crucial to understanding the one-dimensional Grotthuss conduction. Here, we exploited a nanotube-forming natural product, the peptide polytheonamide B (pTB), to examine proton conduction mechanisms at a single-molecule level. The pTB nanotube has a length of ∼40 Å that spans the membrane and a uniform inner diameter of 4 Å that holds a single-file water-chain. Single-channel proton currents were measured using planar lipid bilayers in various proton concentrations and membrane potentials (±400 mV). We found, surprisingly, that the current-voltage curves were asymmetric with symmetric proton concentrations in both solutions across the membrane (rectification). The proton flux from the C-terminal to the N-terminal end was 1.6 times higher than that from the opposite. At lower proton concentrations, the degree of rectification was attenuated, but with the addition of a pH-buffer (dichloroacetate) that supplies protons near the entrance, the rectification emerged. These results indicate that the permeation processes inside the pore generate the rectification, which is masked at low concentrations by the diffusion-limited access of protons to the pore entrance. The permeation processes were characterized by a discrete-state Markov model, in which hops of a proton followed by water-chain turnovers were implemented. The optimized model revealed that the water-chain turnover exhibited unusual voltage dependence, and the distinct voltage-dependencies of the forward and backward transition rates yielded the rectification. The pTB nanotube serves as a rectified proton conductor, and the design principles can be exploited for proton-conducting materials.

Amphipathic antenna of an inward rectifier K+ channel responds to changes in the inner membrane leaflet.

Membrane lipids modulate the function of membrane proteins. In the case of ion channels, they bias the gating equilibrium, although the underlying mechanism has remained elusive. Here we demonstrate that the N-terminal segment (M0) of the KcsA potassium channel mediates the effect of changes in the lipid milieu on channel gating. The M0 segment is a membrane-anchored amphipathic helix, bearing positively charged residues. In asymmetric membranes, the M0 helix senses the presence of negatively charged phospholipids on the inner leaflet. Upon gating, the M0 helix revolves around the axis of the helix on the membrane surface, inducing the positively charged residues to interact with the negative head groups of the lipids so as to stabilize the open conformation (i.e., the "roll-and-stabilize model"). The M0 helix is thus a charge-sensitive "antenna," capturing temporary changes in lipid composition in the fluidic membrane. This unique type of sensory device may be shared by various types of membrane proteins.

Channel function reconstitution and re-animation: a single-channel strategy in the postcrystal age.

The most essential properties of ion channels for their physiologically relevant functions are ion-selective permeation and gating. Among the channel species, the potassium channel is primordial and the most ubiquitous in the biological world, and knowledge of this channel underlies the understanding of features of other ion channels. The strategy applied to studying channels changed dramatically after the crystal structure of the potassium channel was resolved. Given the abundant structural information available, we exploited the bacterial KcsA potassium channel as a simple model channel. In the postcrystal age, there are two effective frameworks with which to decipher the functional codes present in the channel structure, namely reconstitution and re-animation. Complex channel proteins are decomposed into essential functional components, and well-examined parts are rebuilt for integrating channel function in the membrane (reconstitution). Permeation and gating are dynamic operations, and one imagines the active channel by breathing life into the 'frozen' crystal (re-animation). Capturing the motion of channels at the single-molecule level is necessary to characterize the behaviour of functioning channels. Advanced techniques, including diffracted X-ray tracking, lipid bilayer methods and high-speed atomic force microscopy, have been used. Here, I present dynamic pictures of the KcsA potassium channel from the submolecular conformational changes to the supramolecular collective behaviour of channels in the membrane. These results form an integrated picture of the active channel and offer insights into the processes underlying the physiological function of the channel in the cell membrane.

Surface-enhanced IR absorption spectroscopy of the KcsA potassium channel upon application of an electric field.

Surface-enhanced IR absorption spectroscopy (SEIRAS) is a powerful tool for studying the structure of molecules adsorbed on an electrode surface (ATR-SEIRA). Coupled with an electrochemical system, structural changes induced by changes in the electric field can be detected. All the membrane proteins are subjected to the effect of membrane electric field, but conformational changes at different membrane potentials and their functional relevance have not been studied extensively except for channel proteins. In this contribution, background information of potential-dependent functional and structural changes of a prototypical channel, the KcsA channel, is summarized, and SEIRAS applied to the KcsA channel under the application of the potential is shown. The potassium channels allow K(+) to permeate selectively through the structural part called the selectivity filter, in which dehydrated K(+) ions interact with backbone carbonyls. In the absence of K(+), the selectivity filter undergoes conformational changes to the non-conductive collapsed conformation. To apply the electric field, the KcsA channels were fixed on the gold surface in either upside or reverse orientation. The SEIRA spectrum in K(+) or Na(+) solution revealed both backbone structural changes and local changes in the OCO-carboxylate groups. Upon application of the negative electric field, the spectrum of OCO was enhanced only in the K(+) solution. These results indicate that the negative electric field accumulates local K(+) concentration, which turned the collapsed filter to the conductive conformation. ATR-SEIRA serves as an unprecedented experimental system for examining membrane proteins under an electric field.

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.

Functional equilibrium of the KcsA structure revealed by NMR.

KcsA is a tetrameric K(+) channel that is activated by acidic pH. Under open conditions of the helix bundle crossing, the selectivity filter undergoes an equilibrium between permeable and impermeable conformations. Here we report that the population of the permeable conformation (p(perm)) positively correlates with the tetrameric stability and that the population in reconstituted high density lipoprotein, where KcsA is surrounded by the lipid bilayer, is lower than that in detergent micelles, indicating that dynamic properties of KcsA are different in these two media. Perturbation of the membrane environment by the addition of 1-3% 2,2,2-trifluoroethanol increases p(perm) and the open probability, revealed by NMR and single-channel recording analyses. These results demonstrate that KcsA inactivation is determined not only by the protein itself but also by the surrounding membrane environments.

The synergic modeling for the binding of fluoroquinolone antibiotics to the hERG potassium channel.

The fluoroquinolone antibiotic binding site in the hERG potassium channel was examined for the residues involved and their position in the tetrameric channel. The blocking effect of the two fluoroquinolones levofloxacin and sparfloxacin to tandem dimers of the hERG mutants were evaluated electrophysiologically. The results indicated that two Tyr652s in the neighboring subunits and one or two Phe656s in the diagonal subunits contributed to the blockade in the case of both compounds, and Ser624 was also involved. The docking studies suggested that the protonated carboxyl group in the compounds strongly interacts with Phe656 as a π acceptor.

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.

Counting ion and water molecules in a streaming file through the open-filter structure of the K channel.

The mechanisms underlying the selective permeation of ions through channel molecules are a fundamental issue related to understanding how neurons exert their functions. The "knock-on" mechanism, in which multiple ions in the selectivity filter are hit by an incoming ion, is one of the leading concepts. This mechanism has been supported by crystallographic studies that demonstrated ion distribution in the structure of the Streptomyces lividans (KcsA) potassium channel. These still pictures under equilibrium conditions, however, do not provide a snapshot of the actual, ongoing permeation processes. To understand the dynamics of permeation, we determined the ratio of the ion and water flow [the water-ion coupling ratio (CR(w-i))] through the KcsA channel by measuring the streaming potential (V(stream)) electrophysiologically. The V(stream) value was converted to the CR(w-i) value, which reveals how individual ion and water molecules are queued in the narrow and short filter during permeation. At high K(+) concentrations, the CR(w-i) value was 1.0, indicating that turnover between the alternating ion and water arrays occurs in a single-file manner. At low K(+), the CR(w-i) value was increased to a point over 2.2, suggesting that the filter contained mostly one ion at a time. These average behaviors of permeation were kinetically analyzed for a more detailed understanding of the permeation process. Here, we envisioned the permeation as queues of ion and water molecules and sequential transitions between different patterns of arrays. Under physiological conditions, we predicted that the knock-on mechanism may not be predominant.

Oriented reconstitution of a membrane protein in a giant unilamellar vesicle: experimental verification with the potassium channel KcsA.

We report a method for the successful reconstitution of the KcsA potassium channel with either an outside-out or inside-out orientation in giant unilamellar vesicles, using the droplet-transfer technique. The procedure is rather simple. First, we prepared water-in-oil droplets lined with a lipid monolayer. When solubilized KcsA was encapsulated in the droplet, it accumulated at monolayers of phosphatidylglycerol (PG) and phosphoethanolamine (PE) but not at a monolayer of phosphatidylcholine (PC). The droplet was then transferred through an oil/water interface having a preformed monolayer. The interface monolayer covered the droplet so as to generate a bilayer vesicle. By creating chemically different lipid monolayers at the droplet and oil/water interface, we obtained vesicles with asymmetric lipid compositions in the outer and inner leaflets. KcsA was spontaneously inserted into vesicles from the inside or outside, and this was accelerated in vesicles that contained PE or PG. Integrated insertion into the vesicle membrane and the KcsA orientation were examined by functional assay, exploiting the pH sensitivity of the opening of the KcsA when the pH-sensitive cytoplasmic domain (CPD) faces toward acidic media. KcsA loaded from the inside of the PG-containing vesicles becomes permeable only when the intravesicular pH is acidic, and the KcsA loaded from the outside becomes permeable when the extravesicular pH is acidic. Therefore, the internal or external insertion of KcsA leads to an outside-out or inside-out configuration so as to retain its hydrophilic CPD in the added aqueous side. The CPD-truncated KcsA exhibited a random orientation, supporting the idea that the CPD determines the orientation. Further application of the droplet-transfer method is promising for the reconstitution of other types of membrane proteins with a desired orientation into cell-sized vesicles with a targeted lipid composition of the outer and inner leaflets.