A Lipid Bilayer Formed on a Hydrogel Bead for Single Ion Channel Recordings

micromachines

Article A Lipid Bilayer Formed on a Hydrogel Bead for Single Ion Channel Recordings

Minako Hirano 1, Daiki Yamamoto 2, Mami Asakura 2, Tohru Hayakawa 2 , Shintaro Mise 3, Akinobu Matsumoto 3 and Toru Ide 2,*

1 Bio Photonics Laboratory, The Graduate School for the Creation of New Photonics Industries, Shizuoka 431-1202, Japan; [email protected] 2 Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, Okayama 700-8530, Japan; [email protected] (D.Y.); [email protected] (M.A.); [email protected] (T.H.) 3 Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan; [email protected] (S.M.); [email protected] (A.M.) * Correspondence: [email protected]; Tel.: +81-86-251-8203

Received: 13 November 2020; Accepted: 29 November 2020; Published: 1 December 2020

Abstract: Ion channel proteins play important roles in various cell functions, making them attractive drug targets. Artificial lipid bilayer recording is a technique used to measure the ion transport activities of channel proteins with high sensitivity and accuracy. However, the measurement efficiency is low. In order to improve the efficiency, we developed a method that allows us to form bilayers on a hydrogel bead and record channel currents promptly. We tested our system by measuring the activities of various types of channels, including gramicidin, alamethicin, α-hemolysin, a voltage-dependent anion channel 1 (VDAC1), a voltage- and calcium-activated large conductance potassium channel (BK channel), and a potassium channel from Streptomyces lividans (KcsA channel). We confirmed the ability for enhanced measurement efficiency and measurement system miniaturizion.

Keywords: ion channel; lipid bilayer; single-channel recording

1. Introduction Ion channel proteins are membrane proteins that regulate various biological functions by controlling ion flow across cell membranes in response to stimuli [1,2]. Ion channel proteins play important roles in a wide variety of cell functions, making them important drug targets [3,4]. For example, in nerve cells, the influx of Na+ ions thorough voltage-gated Na+ channels produces a nerve signal [1,2], while voltage-gated calcium channels regulate gene expressions [5]. At the same time, the dysfunctional activity of ion channels can cause serious and even fatal diseases; the activity of the P2X purinoceptor 4 (P2X4) channel is related to neuropathic pain [6,7], malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) channel causes cystic fibrosis [8,9], and disorder of the human ether-a-go-go related gene (HERG) channel leads to serious arrhythmia [10]. The artificial lipid bilayer recording technique measures the ion transport activities of channel proteins along with other ion transporting proteins and peptides [11]. This technique allows researchers to determine the biophysical and pharmacological properties of the channel with high sensitivity and accuracy. However, the measurement efficiency of the technique is low. In fact, more than several minutes and sometimes tens of minutes are required to measure ion currents due to the time-consuming process of making artificial lipid bilayer membranes and the low incorporation rate of channels into the membranes [11,12].

Micromachines 2020, 11, 1070; doi:10.3390/mi11121070 www.mdpi.com/journal/micromachines Micromachines 2020, 11, 1070 2 of 15

The physical processes that occur during lipid bilayer formation have been extensively studied and are described in standard textbooks [11]. Principally, the lipid bilayer membrane forms when organic solvent and inverted lipid micelles are removed between the two lipid monolayers. Bilayers cannot be made from a lipid solution without contacting the two monolayers. In the most frequently used bilayer forming technique, i.e., the painting technique, a lipid solution is painted over a small hole in a thin plastic sheet under the aqueous solution and the organic solvent is spontaneously removed between the two lipid monolayers. The removal takes several tens of minutes at most. Membrane proteins, including channel proteins, are generally not inserted into bilayers directly from the aqueous solution, with certain exceptions such as voltage-dependent anion channels (VDACs). The most efficient method used to incorporate channel proteins into bilayers is vesicle fusion. However, because vesicles stochastically fuse with the bilayer, the process cannot be controlled, and thus sometimes takes a long time. In order to improve the measurement efficiency, several approaches have been proposed. For example, bilayer membranes can be promptly made in a lipid solution at the droplet–droplet interface [13–15], droplet–aqueous solution interface [16], droplet–agarose gel interface [14], or agarose gel–agarose gel interface [17]. In addition, in order to enhance the incorporation rate of the channel proteins into artificial membranes, we developed a technique whereby the incorporation happens simultaneously with the formation of the membrane on a supporting substrate [18,19]. We reported a method that measures the ion current of potassium channels from Streptomyces lividans (KcsA) or from Methanobacterium thermoautotrophicum (MthK) channels by binding them to cobalt affinity gel beads with a histidine tag and then forming a lipid bilayer membrane with the channels on the bead by pushing the lipid layer onto the bead [18]. In another study, we used a gold electrode covered with polyethylene glycol (PEG), on which the ion channels were immobilized [19]. By contacting the lipid monolayer at the surface of the probe with another lipid monolayer formed at the interface between the lipid solution and aqueous recording solution, a lipid bilayer membrane was formed at the same time as the channels were incorporated into the membrane. Since lipid bilayer membranes containing ion channels were formed in a single step, channel currents could be measured with high efficiency [19–21]. In general, we have developed techniques for channel current recordings using various types of substrate-supported bilayers to improve the measurement efficiency of conventional artificial bilayer methods. Here, we report further enhanced efficiency by simplifying the bilayer formation technique and shortening the required time for the measurement preparation. This component technology could potentially lead to the miniaturization of a measurement apparatus.

2. Materials and Methods

2.1. Materials Asolectin, alamethicin, and gramicidin were purchased from Sigma (St. Louis, MO, USA), α-hemolysin was from Abcam plc (Cambridge, UK), Co2+ aﬃnity gel beads (TALON Metal Aﬃnity Resins) from Clontech (Mountain View, CA, USA), and Sepharose 4B beads were from Cytiva Japan (Tokyo, Japan). All other chemicals were commercial products of analytical grade.

2.2. Constructs and Mutants The cDNA of mouse voltage-dependent anion channel 1 (VDAC1) (ENSMUST00000102758.7) was subcloned into a multi-cloning site (NdeI-XhoI) of the pET21b vector. The potassium channels from Streptomyces lividans (KcsA channel) gene cloned into pQE-30 vectors, including an N-terminal hexahistidine tag, was a gift from Dr. Kubo of the National Institute of Advanced Industrial Science and Technology. We used a KcsA mutant (E71A) made by using the QuickChangeTM site-directed mutagenesis kit (Stratagene), because the E71A mutation is known to inhibit inactivation. Micromachines 2020, 11, 1070 3 of 15

2.3. Protein Expression and Purification of Voltage-Dependent Anion Channel 1 (VDAC1) The pET21b plasmids containing the VDAC1 sequence were transformed into Escherichia coli BL21(DE3) and then overexpressed in the presence of 1 mM isopropyl-d-thiogalactopyranoside (IPTG) for 4 h. The BL21(DE3) pellets were incubated in BugBuster Protein Extraction Reagent (Merck, Kenilworth, NJ, USA) containing protease inhibitors (cOmplete™ Protease Inhibitor Cocktail EDTA-free, Roche, Basel, Switzerland) and benzonase (Merck) for 5 min at room temperature, and then incubated in BugBuster Protein Extraction Reagent containing 250 µg/mL lysozyme (Merck) for 5 min at room temperature. After they were centrifuged at 16,000 g for 20 min, the precipitates were × sonicated in binding buffer (6 M GHCl, 300 mM NaCl, 10 mM imidazole, 50 mM Tris-HCl (pH 8.0)) and then centrifuged at 16,000 g for 10 min. The supernatant was mixed with nickel resin (Thermo × Fisher, Waltham, MA, USA) and rotated at 4 ◦C for 60 min. Nonspecific bound proteins were removed with 5-fold volume of binding buffer, 5-fold volume of wash buffer (6 M GHCl, 300 mM NaCl, 20 mM imidazole, 50 mM Tris-HCl (pH 8.0)), and 5-fold volume of refolding buffer (100 mM NaCl, 0.4% n-dodecyl-N,N-Dimethylamine-N-oxide (LDAO, Affymetrix), 50 mM Tris-HCl (pH 8.0)). Then, VDAC1 was eluted with elution buffer (100 mM NaCl, 0.4% LDAO, 500 mM imidazole, 50 mM Tris-HCl (pH 8.0)) and dialyzed with a dialysis membrane (Slide-A-Lyzer MINI Dialysis Device, 10K MVCO, Thermo Fisher). VDAC1 protein was further purified by cation exchange using ÄKTA explorer 10S (GE Healthcare, Chicago, IL, USA) and RESOURCE™ S column (GE Healthcare).

2.4. Protein Expression and Purification of KcsA The pQE-30 plasmids containing the KcsA sequence were transformed into Escherichia coli XL1-Blue and overexpressed by the addition of IPTG to a final concentration of 0.5 mM for 2 h. The expressed channels were extracted from membrane fractions by 10 mM n-dodecyl-d-maltoside (DM, Dojin, Kumamoto, Japan). Co2+ affinity gel beads (TALON Metal Affinity Resins, Clontech) equilibrated with normal buffer (5 mM DM, 20 mM Tris-HCl (pH7.6), 100 mM KCl) were added to the extracted channel protein solution and incubated for 30 min at 4 ◦C in order to bind histidine tags to the gel beads. Nonspecific bound proteins were removed with washing buffer (normal buffer containing 20 mM imidazol).

2.5. Isolation of Plasma Membrane Vesicles from Myometrium Myometrium plasma membrane vesicles containing BK channels (a voltage- and calcium-activated large conductance potassium channel) were prepared according to the method of Toro et al. [22]. Membrane vesicles were isolated from pig uteri. Connective tissue and endometrium were removed from the uteri. The tissue was homogenized in sucrose solution (300 mM sucrose and 20 mM Tris-HEPES, pH 7.2) in the presence of protease inhibitors. The homogenate was centrifuged for 30 min at 700 g. The supernatant was centrifuged for 40 min at 14,000 g and then for 20 min at 9500 g. × × × The pellet was resuspended in solution (600 mM KC1 and 5 mM Na-PIPES, pH 6.8) with a Teﬂon pestle homogenizer. Samples were incubated on ice for 1 h and centrifuged for 1 h at 42,000 g. The pellets × were resuspended in 400 mM KCl, 10% sucrose (w/w), and 5 mM Na-PIPES, pH 6.8. The microsomes were placed on top of a discontinuous sucrose gradient (w/w): 20:25:30:35:40%. The gradient was centrifuged at 67,500 g in a swinging rotor for 18 h. Membranes obtained from the 20:25% sucrose × interface were used in this study.

2.6. Formation of Bilayers on a Gel Bead and Current Recordings 1 A hydrogel bead was immobilized to the aperture at the glass pipette tip by suction. A Co2+ aﬃnity gel bead, on which KcsA proteins were immobilized, was used in the case of KcsA channel recordings and a Sepharose 4B bead was used for other types of channels. A glass capillary (GC150T-10, Harvard Apparatus, Holliston, MA, USA) was pulled to make a pipette with a very ﬁne tip (<1 µm) using a pipette puller (P-97, Sutter Instrument, Novato, CA, USA), then the tip was cut to make a Micromachines 2020, 11, x 4 of 15

AnMicromachines aqueous2020 recording, 11, 1070 solution was poured into the bath chamber and lipid solution (30 mg/ml4 of 15 asolectin in n-decane or hexadecane) was layered over the recording solution layer. Bilayers were made by moving the bead fixed at the pipette tip through this lipid solution layer into the recording µ solutionlarge aperture (Figure (> 101C).m). The The bead tip waswas fabricatedmoved downwa using ard microforge until gently (MF-900, contacting Narishige, the lipid/aqueous Tokyo, Japan) 2+ solutionin order interface to smooth by the using surface. a manipulator. Figure1A Contact shows abetween Co a ffithenity bead gel and bead the fixed interface at the could tip of be a easily glass pipetteseen with by suction.a microscope. The glass The pipette depth wasat which set with the abead pipette was holder dipped and into a bead the wasaueous fixed solution at the tip was of usuallythe pipette 10–100 by suctioningμm. However, it through it was not a tube necessary from the to holderbe precise, with such a syringe. that using In Figure a coarse1A, manipulator the bead is µ µ toapproximately move the bead 80 wasm in sufficient. diameter A and lipid the bilayer pipette membrane aperture is was 30 spontaneouslym in diameter. formed Prior to immediately the electrical afterrecording, the contact. the beads were incubated in order to be equilibrated with the recording solution for 30 min.

Figure 1. Bilayer formation on a hydrogel bead. (A) A micrographmicrograph ofof aa CoCo22++ affinityaffinity gel bead fixed fixed at the pipette tip byby suction.suction. (B) The experimentalexperimental apparatus. The The pipette solution and the recording solution were connected with an Ag–AgCl wire to the the patch patch clamp clamp amplifier amplifier and and virtual virtual ground ground level, level, respectively. ( (CC)) The The bilayer bilayer formation formation process. process. A A gel gel bead bead fixed fixed at at the the tip tip of of a glass pipette was plunged into a lipid solution layer and inserted in intoto an aqueous recording solutionsolution through the lipid solution layer. The bilayer is spontaneously formed at the gel–water interface.interface.

OneFigure of1 Btwo shows channel a schematic forming drawing peptides of the(gramici experimentaldin and apparatusalamethicin), (see the also hemolytic Figures A1 protein and A2 α).- Ahemolysin, cut glass or tube detergent-solubilized (internal diameter, VDAC1 6 mm) gluedwas added to a slide to the glass recording was used solution as the at bath appropriate chamber. Anconcentrations aqueous recording before the solution bilayer wasformation. poured These into themolecules bath chamber were spontaneously and lipid solution incorporated (30 mg /intomL theasolectin bilayer in formedn-decane on orthe hexadecane) Sepharose 4B was bead layered to make over ionic the recording channels. solutionKcsA channel layer. proteins Bilayers were spontaneouslymade by moving incorporated the bead fixed into at the the bilayers pipette tipby throughforming thisbilayers lipid with solution a Co layer2+ affinity intothe gelrecording bead, on whichsolution the (Figure channel1C). proteins The bead were was immobilized moved downward through until a histidine gently contacting tag. BK channels the lipid were/aqueous incorporated solution intointerface the bilayers by using by a vesicle manipulator. fusion. Prior Contact to the between bilayer the formation, bead and a theSepharose interface 4B could immobilized be easily at seen the withglass apipette microscope. tip was The dipped depth into at whicha membrane the bead vesicle was dippedsuspension into that the aueouscontained solution BK channels. was usually The 10–100aqueousµ m.recording However, solution it was was not held necessary at virtual to beground precise, level such so that that the using voltage a coarse at the manipulator solution level to inmove the thepipette bead connected was sufficient. to a patch A lipid clamp bilayer amplifie membraner (CEZ-2400, was spontaneously Nihonkohden, formed Tokyo, immediately Japan) by after an Ag–AgClthe contact. electrode defined the membrane potential. Unless otherwise noted, the signal was filtered at 1 kHz,One sampled of two channel at 10 kHz, forming digitized, peptides then stored (gramicidin on a PC. and alamethicin), the hemolytic protein α-hemolysin, or detergent-solubilized VDAC1 was added to the recording solution at appropriate 2.7.concentrations Formation of before Bilayers the on bilayer a Gel Bead formation. and Current These Recordings molecules 2 were spontaneously incorporated into the bilayerAs shown formed in Figure on the 2, Sepharosea Sepharose 4B 4B bead bead to was make fixed ionic by channels.suction at KcsAthe tip channel of a glass proteins pipette were in a 2+ recordingspontaneously solution. incorporated Then, the into chamber the bilayers was by filled forming with bilayers a lipid with solution a Co (30affi mg/mLnity gel bead,asolectin on which in n- decane).the channel The proteins aqueous were recording immobilized solution through was extr a histidineuded out tag. from BK a channels polyethylene were tube incorporated (0.1–0.5 mm into innerthe bilayers diameter) by vesicle placedfusion. near the Prior bead to by the manually bilayer formation,exerting positive a Sepharose pressure 4B immobilizedwith a syringe, at such the glass that pipetteit contacted tip was the dipped bead in into the a lipid membrane solution. vesicle The suspensioncontact was that observed contained with BK a channels.microscope. The A aqueous bilayer membranerecording solution was promptly was held formed at virtual at the ground water–gel level inte sorface. that the The voltage solution at thein the solution tube was level held in the at pipette connected to a patch clamp amplifier (CEZ-2400, Nihonkohden, Tokyo, Japan) by an Ag–AgCl 4 Micromachines 2020, 11, 1070 5 of 15 electrode defined the membrane potential. Unless otherwise noted, the signal was filtered at 1 kHz, sampled at 10 kHz, digitized, then stored on a PC.

2.7. Formation of Bilayers on a Gel Bead and Current Recordings 2 As shown in Figure2, a Sepharose 4B bead was fixed by suction at the tip of a glass pipette in a recording solution. Then, the chamber was filled with a lipid solution (30 mg/mL asolectin in n-decane). The aqueous recording solution was extruded out from a polyethylene tube (0.1–0.5 mm inner diameter) placed near the bead by manually exerting positive pressure with a syringe, such that itMicromachines contacted 2020 the, bead11, x in the lipid solution. The contact was observed with a microscope. A bilayer5 of 15 membrane was promptly formed at the water–gel interface. The solution in the tube was held at virtual groundvirtual ground level so level that theso that voltage the voltage at the solution at the solu leveltion in level the glass in the pipette glass pipette connected connected to a patch to a clamp patch amplifierclamp amplifier by an Ag–AgCl by an Ag–AgCl electrode electrode defined defined the membrane the membrane potential potential (see also (see Figure also A3Figure). A3).

FigureFigure 2.2. A bilayer was formed at the water–gelwater–gel interface by pushing the recording solutionsolution outout fromfrom aa tube.tube.

3.3. Results and Discussion

3.1.3.1. Formation of Bilayers on a Gel Bead TheThe bilayerbilayer formationformation technique technique developed developed in in this this study study allowed allowed us us to maketo make artificial artificial bilayers bilayers for channelfor channel recordings recordings much much more quicklymore quickly than with than conventional with conventional techniques, techniques, significantly significantly increasing theincreasing measurement the measurement efficiency. Figure efficiency.1C shows Figure the formation1C shows processthe formation for the artificialprocess bilayersfor the onartificial a gel bead.bilayers As on described a gel inbead. the MaterialsAs described and Methodsin the Materials section, bilayers and Methods were spontaneously section, bilayers formed were on aspontaneously gel bead by inserting formed on the a bead gel bead into by an aqueousinserting solutionthe bead frominto an the aqueous lipid solution solution layer. from Figure the lipid1A showssolution a micrographlayer. Figure of 1A a hydrogel shows a beadmicrograph fixed at of the a tiphydrogel of a glass bead pipette fixed by at suction.the tip of In a orderglass topipette prevent by thesuction. lipid In solution order to from prevent flowing the lipid inside solution the pipette from throughflowing inside a gap betweenthe pipette the through bead and a gap the between pipette surface,the bead the and tip the of thepipette pipette surface, was melted the tip so of as th toe bepipette smoothed was bymelted a microforge so as to apparatus be smoothed for patch by a clampmicroforge pipettes. apparatus Even slight for patch roughness clamp on pipettes. the pipette Even surface slight complicated roughness the on current the pipette measurement, surface 2+ particularlycomplicated when the current using ameasurement, Co affinity gelparticularly bead, which when is a using cross-linked a Co2+ affinity agarose gel bead. bead, which is a cross-linkedWe measured agarose gramicidin bead. channel currents to confirm if the membrane formed at the gel–water interfaceWe wasmeasured a lipid gramicidin bilayer. In thechannel bilayer currents formation to co process,nfirm if two the lipid membrane monolayers, formed one at at the the gel–water gel–lipid interfaceinterface andwas the a lipid other bilayer. at the lipid–water In the bilayer interface, format wereion process, folded togethertwo lipid to monolayers, make a bilayer. one This at the means gel– organiclipid interface solvent and and the lipid other reversed at the lipid–water micelles should interf beace, removed were folded between together the two to make lipid monolayersa bilayer. This to makemeans the organic bilayer. solvent We easily and observed lipid reversed contact betweenmicelles theshould bead be and removed the lipid–water between interface the two using lipid a microscope.monolayers However,to make the we bilayer. could notWe confirm easily obse if arved bilayer contact was formed, between even the ifbead the beadand the was lipid–water in contact withinterface theinterface. using a microscope. Therefore, we However, measured we the could gramicidin not confirm current, if a because bilayer gramicidinwas formed, must even make if the a tandembead was dimer in contact in a membrane with the in interface. order to showTherefor currente, we activity, measured which the can gramicidin be measured current, only when because the membranegramicidin becomesmust make thin a enoughtandem todimer form in a bilayera membrane [23]. in order to show current activity, which can be measured only when the membrane becomes thin enough to form a bilayer [23]. Figure 3 shows repeated bilayer formations, which were formed by plunging the gel bead into a lipid solution. The current was recorded by moving the gel bead up and down near the lipid–water interface. As shown in the figure, the bead was moved using a piezo-micromanipulator with an amplitude of 10 μm at approximately 1 Hz. The aqueous recording solution contained 250 mM KCl, 5 mM Tris-HCl pH 7.4, and 100 nM gramicidin. As the bead moved downward, the current did not increase until just after the bead reached the interface between the lipid and aqueous solutions, which marked the formation of the bilayer membrane and incorporation of gramicidin. On the other hand, when the bead was moved upward, the current decreased back to zero, indicating that the membrane became thick as lipids and organic solvent flowed into the space between the two lipid monolayers. We incorporated gramicidin into the bilayers by adding it to the recording solution.

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Figure3 shows repeated bilayer formations, which were formed by plunging the gel bead into a lipid solution. The current was recorded by moving the gel bead up and down near the lipid–water interface. As shown in the ﬁgure, the bead was moved using a piezo-micromanipulator with an amplitude of 10 µm at approximately 1 Hz. The aqueous recording solution contained 250 mM KCl, 5 mM Tris-HCl pH 7.4, and 100 nM gramicidin. As the bead moved downward, the current did not increase until just after the bead reached the interface between the lipid and aqueous solutions, which marked the formation of the bilayer membrane and incorporation of gramicidin. On the other hand, when the bead was moved upward, the current decreased back to zero, indicating that the membrane became thick as lipids and organic solvent ﬂowed into the space between the two lipid monolayers. We incorporated gramicidin into the bilayers by adding it to the recording solution. Micromachines 2020, 11, x 6 of 15

Figure 3. Repeated formation of bilayers on a gel bead. The bead was moved up and down near the water–lipid interface in the presence of gramicidin in the recording solution. The height of the bead was changed by 10 µμm at approximately 11 Hz.Hz.

Figure3 3 demonstratesdemonstrates thatthat wewe werewere ableable to quicklyquickly repeat current measurements using the bead. This ability can increase the throughput of pharmacological measurements. As As shown shown in in the the figure, figure, a bilayer was formed almost instantly after bringing bringing the the bead bead into into contact contact with with the the aqueous aqueous solution. solution. The greatestgreatest advantageadvantage of of this this method method is thatis that we werewe were able toable form to artificialform artificial bilayers bilayers much moremuch rapidly more rapidlythan with than conventional with conventional methods. methods. In conventional In conventional artificial artificial bilayer methods,bilayer methods, it takes severalit takes minutes several minutesor longer or to longer form bilayers to form [24 bilayers]. Furthermore, [24]. Furtherm in conventionalore, in conventional artificial bilayer artificial methods, bilayer extreme methods, care extrememust be takencare must not tobe rupture taken not the to bilayer rupture when the the bilayer recording when solutions the recording are exchanged. solutions are Perfusion exchanged. often Perfusionresults in aoften ruptured results bilayer in a membrane, ruptured thusbilayer requiring membrane, the measurement thus requiring to be the recommenced. measurement With to thebe recommenced.present technique, With however, the present ionic currenttechnique, measurement however, ionic can be current restarted measurement using the same can bead be restarted within a usingfew seconds, the same saving bead muchwithin time a few and seconds, labor. saving much time and labor.

3.2. Spontaneous Spontaneous Incorporation Incorporation of Channels into Bilayers and Current Recordings The bilayerbilayer formationformation and and insertion insertion processes processes for for the the channel-forming channel-forming proteins proteins and peptidesand peptides were werealways always reproducible. reproducible. As shown As shown in Figure in Figure3, the 3, bilayers the bilayers could could be repeatedly be repeatedly formed formed any any number number of oftimes, times, while while the the channel-forming channel-forming peptides peptides and and proteins proteins were were invariably invariably inserted inserted into theinto bilayers the bilayers from fromthe aqueous the aqueous solution. solution.

3.2.1. Gramicidin Figure 4A is a typical current trace of a gramicidin channel. The membrane voltage was held at 50 mV, and the aqueous solution contained 150 mM NaCl, 100 nM gramicidin, and 10 mM MOPS- Tris, pH 7.4. Each step in the current recording corresponds to the unitary current of the gramicidin channel. The single-channel conductance was determined as 10 ± 0.1 pS (n = 5), which agreed well with the value obtained by conventional methods [25].

3.2.1. Gramicidin Figure4A is a typical current trace of a gramicidin channel. The membrane voltage was held at 50 mV, and the aqueous solution contained 150 mM NaCl, 100 nM gramicidin, and 10 mM MOPS-Tris, pH 7.4. Each step in the current recording corresponds to the unitary current of the gramicidin channel. The single-channel conductance was determined as 10 0.1 pS (n = 5), which agreed well with the ± value obtained by conventional methods [25].

3.2.2. Alamethicin Figure4B represents a current recording for alamethicin channels. Alamethicin is a channel-forming peptide antibiotic produced by fungi. It is known to form voltage-dependent ion channels by aggregating four to six molecules in bilayers. The current trace in the ﬁgure was taken with a solution containing 1 KCl and 500 nM alamethicin. The single-channel conductance calculated from the I–V relationship was 1.1 0.11 nS (n = 5). The current ﬂuctuation showed voltage dependency, as reported ± in previous papers [26]. The open probability of the channel changed from 0.4 at +100 mV to <0.01 at 100 mV. The properties of the single-channel conductance and voltage dependency observed in this − study agreed with conventional experiments, validating our novel technique.

3.2.3. α-Hemolysin Figure4C shows the α-hemolysin channel current recorded with our method. α-Hemolysin is a hemolytic protein secreted by Staphylococcus aureus, which is known to form a heptamer in bilayers, forming a large ion channel pore [27,28]. The trace in Figure4C was recorded with a solution containing 100 mM KCl and 10 mM Hepes-Tris, pH 7.3, in the presence of 1 µM α-hemolysin. From the single-channel I–V relationship, the single-channel conductance was calculated to be 245 16 pS (n = 5), ± which was identical to the values obtained by conventional bilayer methods [29], again validating our method. Our technique could potentially be used to measure other types of channel-forming proteins, such as γ-hemolysin and Cry toxins.

3.2.4. Voltage-Dependent Anion Channel 1 (VDAC1) Figure4D shows a current tracing of mouse VDAC 1 taken with 1 M KCl and 10 mM MOPS-Tris, pH 7.4, and 1 µg/mL VDAC1. VDAC1 solubilized with detergent is known to be directly incorporated into artificial bilayers from an aqueous solution [30]. From the slope of the I–V relationship of single-channel currents, the single-channel conductance was calculated to be 1.4 nS (n = 5), and the channel showed voltage dependent gating as reported previously [30]. The above confirmed that our method can be used to measure the currents of several types of channels. Importantly, the channel current properties observed were in good agreement with the results obtained from other techniques, such as the conventional planar bilayer technique and patch clamp analysis. Thus, our method can be used to study a wide range of channels. Moreover, the channel current measurements were relatively easy, as we only needed to form bilayers with recording solutions containing the channel peptides or proteins. All of the channels shown in Figure4, except for VDAC1, were channel-forming peptides or pore-forming proteins, which spontaneously incorporate into bilayer membranes from an aqueous solution to make ion channel pores. Detergent-solubilized VDAC1 proteins can be easily reconstituted into artificial bilayers from aqueous solution, but most other biological channel proteins cannot be reconstituted in this way. Micromachines 2020, 11, 1070 8 of 15

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FigureFigure 4. 4.Channel Channel current recordings recordings via via the the spontaneou spontaneouss incorporation incorporation of peptides of peptides and proteins. and proteins. (A) (AThe) The gramicidin gramicidin channel channel current current was was recorded recorded with with a recording a recording solution solution containing containing 150 mM 150 NaCl, mM 100 NaCl, 100nM nM gramicidin, gramicidin, and and 10 mM 10 mMMOPS-Tris, MOPS-Tris, pH 7.4. pH The 7.4. trace The was trace low-pass-filtered was low-pass-ﬁltered at 0.5 kHz. at (B 0.5) The kHz. (B)alamethicin The alamethicin channel channel current was current recorded was recordedat 100 mV atwith 100 a mVsolution with containing a solution 1 M containing KCl and 500 1 M nM KCl andalamethicin. 500 nM alamethicin. (C) The α-hemolysin (C) The αcurrent-hemolysin was recorded current wasat 60 recordedmV with at100 60 mM mV KCl with and 100 1 μ mMM α KCl- andhemolysin, 1 µM α-hemolysin, 10 mM Hepes-Tris, 10 mM pH Hepes-Tris, 7.3. (D) A pH channel 7.3. (Dcurrent) A channel recording current of mouse recording VDAC1. of mouseThe recording solution contained 1 M KCl and 1 μg/ml VDAC1, 10 mM MOPS-Tris, pH 7.4. The recording VDAC1. The recording solution contained 1 M KCl and 1 µg/mL VDAC1, 10 mM MOPS-Tris, pH 7.4. was taken at 30 mV. The recording was taken at 30 mV.

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3.3.3.3. Incorporation Incorporation of of Channel Channel Proteins Proteins Immobilized Immobilized onon aa GelGel BeadBead Next, we immobilized physiological channel proteins (which cannot be directly inserted into Next, we immobilized physiological channel proteins (which cannot be directly inserted into bilayers) from an aqueous solution on the surface of a hydrogel bead and reconstituted them into bilayers from an aqueous solution) on the surface of a hydrogel bead and reconstituted them into bilayers to measure their currents. Figure 5 shows the results for a single KcsA channel. KcsA is a bilayers to measure their currents. Figure5 shows the results for a single KcsA channel. KcsA is a potassium-selective channel from the soil bacteria Streptomyces lividans. It is one of the best- potassium-selective channel from the soil bacteria Streptomyces lividans. It is one of the best-characterized characterized channel proteins because its structure is very simple. As described in the Materials and channel proteins because its structure is very simple. As described in the Materials and Methods Methods section, purified KcsA channels were solubilized with detergent and immobilized on a gel section,bead via puriﬁed histidine KcsA tags. channels The bead were was solubilized moved to with contact detergent the aqueous and immobilized solution through on a gel the bead lipid via histidinesolution tags. layer, The and bead immobilized was moved KcsA to contact channels the were aqueous reconstituted solution through immediately the lipid after solution the bilayer layer, andformation. immobilized KcsA channels were reconstituted immediately after the bilayer formation.

FigureFigure 5. Recording5. Recording of a singleof a single KcsA channelKcsA channel immobilized immobilized on a hydrogel on a bead.hydrogel (A) Direct bead. reconstitution (A) Direct ofreconstitution the KcsA channel of the into KcsA a bilayer. channel Theinto KcsAa bilayer. channel The proteinKcsA channel was solubilized protein was with solubilized detergent with and immobilizeddetergent and on a immobilized gel bead via aon histidine a gel bead tag. via The a channelhistidine was tag. incorporated The channel intowas the incorporated bilayer immediately into the afterbilayer the bilayer immediately formation. after (theB) Abilayer typical formation. current time(B) A course typical of current the KcsA time channel course immobilizedof the KcsA channel on a gel bead.immobilized (C) The single-channel on a gel bead. current (C) The amplitude single-channel was plotted current against amp thelitude membrane was plotted voltage. against From the the slope of this line, the single-channel conductance was determined to be 200 pS. 9 Micromachines 2020, 11, 1070 10 of 15

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Figuremembrane5A illustrates voltage. theFrom incorporation the slope of this of line, the th KcsAe single-channel channel conductanc immobilizede was on determined a gel bead to be in a bilayer. Figure5B200 shows pS. the current fluctuation of the KcsA channel recorded in a solution containing 200 mM KCl and 20 mM succinate-KOH, pH 4.0. The membrane potential was held at 50 mV. Figure5C shows the I–V relationFigure 5A of theillustrates unitary the current. incorporation From of this the relationship, KcsA channel the immobilized single-channel on a gel conductance bead in a was determinedbilayer. toFigure be 200 5B shows pS, which the current agreed fluctuation well with of values the KcsA obtained channel using recorded the in conventional a solution containing planar bilayer 200 mM KCl and 20 mM succinate-KOH, pH 4.0. The membrane potential was held at 50 mV. Figure method [31–33]. 5C shows the I–V relation of the unitary current. From this relationship, the single-channel As we previously reported, some types of physiological channel proteins that have been conductance was determined to be 200 pS, which agreed well with values obtained using the solubilizedconventional with detergentplanar bilayer and method immobilized [31–33]. on a gel [18] or solid [19] substrate have been successfully reconstitutedAs we directly previously into reported, artificial bilayerssome types for of single-channel physiological channel current proteins measurements. that have In been previous studies,solubilized we incorporated with detergent channel and proteinsimmobilized into on bilayers a gel without[18] or solid any additional[19] substrate processes, have been such as vesiclesuccessfully fusion. Here, reconstituted we further directly improved into artificial this method bilayers by for simplifying single-channel the current measurement measurements. to enhance In the measurementprevious studies, efficiency. we incorporated Of note, one channel limitation proteins was into the bilayers manual without movement any additional of the bead. processes, Automating this stepsuch isas expected vesicle fusion. to further Here, we improve further the improved efficiency. this method Furthermore, by simplifying our system the measurement anchored the to KcsA enhance the measurement efficiency. Of note, one limitation was the manual movement of the bead. channel proteins to the gel at a protein terminal; however, the N- and C-terminals regulate the channel Automating this step is expected to further improve the efficiency. Furthermore, our system anchored activities [31,34]. the KcsA channel proteins to the gel at a protein terminal; however, the N- and C-terminals regulate the channel activities [31,34]. 3.4. Channel Inorporation through Vesicle Fusion Physiological3.4. Channel Inorporation channel through proteins Vesicle were Fusion also measured by incorporating them into bilayers through vesicle fusion.Physiological Figure 6channel shows proteins the results were ofalso single-channel measured by incorporating recordings them of the into BK bilayers channel, through which is a voltage-vesicle and fusion. calcium-dependent Figure 6 shows the K+ resultschannel of single-channel purified from recordings pig uterus. of the Prior BK to channel, the bilayer which formation, is a a hydrogelvoltage- bead and calcium-dependent immobilized at the K+glass channel pipette purified tip from was dippedpig uterus. into Prior a membrane to the bilayer vesicle formation, suspension that containeda hydrogel BKbead channels immobilized in the at vesicularthe glass pipette membrane. tip was Immediatelydipped into a membrane after contact vesicle between suspension the gel and lipid–waterthat contained interface, BK thechannels current in the began vesicular to fluctuate, membrane. showing Immediately that BK after channels contact were between inserted the gel into the and lipid–water interface, the current began to fluctuate, showing that BK channels were inserted bilayer by vesicle fusion. It is likely that the incorporation rate was high compared with conventional into the bilayer by vesicle fusion. It is likely that the incorporation rate was high compared with bilayerconventional methods, bilayer because methods, the space because between the space the gel between and the the lipid gel and was the narrow, lipid was meaning narrow, that meaning the vesicle concentrationthat the vesicle was keptconcentration high. In Figurewas kept6A, high. we showIn Figure the 6A, current we show traces the recorded current traces in a solution recorded containing in a 250 mMsolution KCl, containing 5 mM Tris-Cl, 250 mM pH KCl, 7.4. 5 mM The Tris-Cl, bilayer pH was 7.4. held The bilayer at the indicatedwas held at voltages.the indicated Figure voltages.6B shows histogramsFigure 6B of shows the current histograms amplitudes of the current at membrane amplitudes potentials at membrane of +potentials100 mV of and +100 100mV mV.and − The100 open − probabilitymV. The changed open probability from >0.9 changed at +100 from mV >0.9 to < 0.1at +100 at 100mV mV.to <0.1 These at −100 results mV. are These consistent results are with the − typicalconsistent voltage dependencywith the typical of the voltage BK channel dependency [22]. Theof single-channelthe BK channel conductance [22]. The single-channel of the BK channel was determinedconductance toof bethe 230 BK pS, channel agreeing was well determined with the to values be 230 determined pS, agreeing by conventionalwell with the methodsvalues [35]. determined by conventional methods [35].

Figure 6. Single-channel current recordings of the voltage- and calcium-activated large conductance10 potassium channel (BK channel). (A) Current traces taken at indicated membrane voltages. (B) Current amplitude histograms at +100 mV and at 100 mV. − Micromachines 2020, 11, 1070 11 of 15

3.5. Simultaneous Measurements of Multiple Channel Types Bilayers were promptly made by pushing out the aqueous solution from a fine tube, such that the solution contacted a gel bead. The effect of this strategy is to miniaturize the apparatus. Above, we showed that we could measure the properties of various types of channels by using bilayers formed on a gel bead. Although our technique makes the recordings more efficient than the conventional bilayer technique, we should develop a simultaneous parallel recording technique to further increase the measurement efficiency. However, because our apparatus contains a bulky manipulator, it is not suitable for the parallel recordings of many channels. Therefore, we modified the bilayer formation technique to miniaturize the apparatus. Figure7A shows a gramicidin current trace recorded with a bilayer made on the surfaces of gel beads by moving the interface between the aqueous and lipid solutions. As illustrated in Figure2, the aqueous solution was extruded so as to come into contact with the gel bead, thus forming a bilayer at the interface. Immediately after the contact, the current began to fluctuate, showing bilayer formation and the insertion of gramicidin into it. The measured properties were typical of a gramicidin channel. The bilayer was durable against the membrane voltage and stable at 200 mV. Micromachines 2020, 11, x ± 12 of 15

Figure 7. (A) Gramicidin currents recorded at ±200 mV. (B) Simultaneous recordings of gramicidin Figure 7. (A) Gramicidin currents recorded at 200 mV. (B) Simultaneous recordings of gramicidin and alamethicin channels. The inset illustrates the± recording apparatus. and alamethicin channels. The inset illustrates the recording apparatus. Next, we used this modified bilayer formation technology to perform simultaneous recordings of different types of channels. The current recordings shown in Figure 7B are of simultaneously recorded gramicidin and alamethicin. We had previously reported simultaneous recordings of gramicidin channels [17]. In that experimental system, there was a single chamber for the aqueous recording solution, therefore the recording solutions on one side of all of the bilayers were electrically shunted. In contrast, in the present study, each bilayer was made in a different recoding solution, meaning that we could simultaneously measure different types of channels in different environments.

4. Conclusions By improving our previously developed channel recording techniques, here we developed an efficient method for measuring ion channel activities. We measured the natural properties of various types of channels incorporated into gel-supported bilayers. We were able to simultaneously measure different types of channels in different environments. This method allows miniaturization of a recording system. In the future, by making this system multi-channel, we may obtain a high- throughput device for channel current measurement. 12 Micromachines 2020, 11, 1070 12 of 15

Next, we used this modified bilayer formation technology to perform simultaneous recordings of different types of channels. The current recordings shown in Figure7B are of simultaneously recorded gramicidin and alamethicin. We had previously reported simultaneous recordings of gramicidin channels [17]. In that experimental system, there was a single chamber for the aqueous recording solution, therefore the recording solutions on one side of all of the bilayers were electrically shunted. In contrast, in the present study, each bilayer was made in a different recoding solution, meaning that we could simultaneously measure different types of channels in different environments.

4. Conclusions By improving our previously developed channel recording techniques, here we developed an efficient method for measuring ion channel activities. We measured the natural properties of various types of channels incorporated into gel-supported bilayers. We were able to simultaneously measure different types of channels in different environments. This method allows miniaturization of a recording system. In the future, by making this system multi-channel, we may obtain a high-throughput device for channel current measurement.

Author Contributions: Conceptualization, M.H. and T.I.; methodology, M.H. and T.I.; validation, M.H. and T.I.; formal analysis, T.I. and D.Y.; investigation, M.H., T.I., T.H., M.A., D.Y., S.M. and A.M.; writing—original draft preparation, T.I.; writing—A.M., M.H. and T.I.; supervision, T.I.; funding acquisition, M.H. and T.I. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by JSPS KAKENHI, grant numbers 15K13725 and 18K06157, MEXT Program for Building Regional Innovation Ecosystems. Conﬂicts of Interest: The authors declare no conﬂict of interest. Micromachines 2020, 11, x 13 of 15 Appendix A Appendix A

Figure A1. Experimental setup: (1) glass pipette; (2) bath electrode; (3) bath chamber, (4) coarse Figure A1. Experimental setup: (1) glass pipette; (2) bath electrode; (3) bath chamber, (4) coarse manipulator; (5) amplifier headstage. manipulator; (5) ampliﬁer headstage.

Figure A2. Procedure for the bilayer formation. (1) A gel bead is fixed at the tip of the pipette. (2) The bead is lifted up into the air. (3) The lipid solution is layered onto the recording solution. (4) The bead is plunged into the recording solution through the lipid solution layer.

Figure A3. Schematic of the experimental setup. A polyethylene tube and a glass pipette are connected to a syringe to exert pressure.

13 Micromachines 2020, 11, x 13 of 15 Micromachines 2020, 11, x 13 of 15

AppendixAppendix A A

FigureFigure A1. A1. Experimental Experimental setup: setup: (1) glass(1) glass pipette; pipette; (2) bath(2) bath electrode; electrode; (3) bath(3) bathchamber, chamber, (4) coarse (4) coarse Micromachines 2020, 11, 1070 13 of 15 manipulator;manipulator; (5) amplifier(5) amplifier headstage. headstage.

FigureFigure A2. A2. A2.Procedure ProcedureProcedure for thefor for thebilayer the bilayer bilayer formation. formation. formation. (1) A (1) gel A (1) be gelad A be gelisad fixed beadis fixed at isthe ﬁxedat tip the of attip the theof pipette. the tip pipette. of (2) the The pipette.(2) The bead is lifted up into the air. (3) The lipid solution is layered onto the recording solution. (4) The bead bead(2) The is lifted bead up is liftedinto the up air. into (3) the The air. lipid (3) solution The lipid is layered solution onto is layered the recording onto the solution. recording (4) The solution. bead is plunged into the recording solution through the lipid solution layer. is(4) plunged The bead into is plungedthe recording into the solution recording through solution the lipid through solution the lipid layer. solution layer.

Figure A3. Schematic of the experimental setup. A polyethylene tube and a glass pipette are connected to aFigure syringe A3. to SchematicexertSchematic pressure. of of the the experimental experimental setup. setup. A A polyet polyethylenehylene tube tube and and a a glas glasss pipette pipette are are connected connected to a syringe to exert pressure.

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