Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PORTFOLIO  

Artificial lipid bilayer single-channel recording technique has been employed to determine the biophysical and pharmacological properties of various ion channels. However, its measurement efficiency is very low, as it requires two time-consuming processes: preparation of lipid bilayer membranes and incorporation of ion channels into the membranes. In order to address these problems, we previously developed a technique based on hydrophilically modified gold probes on which are immobilized ion channels that can be promptly incorporated into the bilayer membrane at the same time as the membrane is formed on the probes' hydrophilic area. Here, we improved further this technique by optimizing the gold probe and developed an automated channel current measurement system. We found that use of probes with rounded tips enhanced the efficiency of channel current measurements, and introducing a hydrophobic area on the probe surface, beside the hydrophilic one, further increased measurement efficiency by boosting membrane stability. Moreover, we developed an automated measurement system using the optimized probes; it enabled us to automatically measure channel currents and analyze the effects of a blocker on channel activity. Our study will contribute to the development of high-throughput devices to identify drug candidates affecting ion channel activity.

DOI： 10.1038/s41598-021-97237-z

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Over the years, fluorescence microscopy has evolved and has become a necessary element of life science studies. Microscopy has elucidated biological processes in live cells and organisms, and also enabled tracking of biomolecules in real time. Development of highly sensitive photodetectors and light sources, in addition to the evolution of various illumination methods and fluorophores, has helped microscopy acquire single-molecule fluorescence sensitivity, enabling single-molecule fluorescence imaging and detection. Low-light photodetectors used in microscopy are classified into two categories: point photodetectors and wide-field photodetectors. Although point photodetectors, notably photomultiplier tubes (PMTs), have been commonly used in laser scanning microscopy (LSM) with a confocal illumination setup, wide-field photodetectors, such as electron-multiplying charge-coupled devices (EMCCDs) and scientific complementary metal-oxide-semiconductor (sCMOS) cameras have been used in fluorescence imaging. This review focuses on the former low-light point photodetectors and presents their fluorescence microscopy applications and recent progress. These photodetectors include conventional PMTs, single photon avalanche diodes (SPADs), hybrid photodetectors (HPDs), in addition to newly emerging photodetectors, such as silicon photomultipliers (SiPMs) (also known as multi-pixel photon counters (MPPCs)) and superconducting nanowire single photon detectors (SSPDs). In particular, this review shows distinctive features of HPD and application of HPD to wide-field single-molecule fluorescence detection.

DOI： 10.3390/app11062773

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI  

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.

DOI： 10.3390/mi11121070

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Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal toxin with an aerolysin-type architecture, and it is expected to be used as a novel bioinsecticide. Cry46Ab acts as a functional pore-forming toxin, and characteristics of the resulting channel pores, including ion selectivity, have been analyzed. However, the relationship between channel-pore ion selectivity and insecticidal activity remains to be elucidated. To clarify the effects of charged amino acid residues on the ion permeability of channel-pores and the resulting insecticidal activity, in the present study, we constructed Cry46Ab mutants in which a charged amino acid residue within a putative transmembrane β-hairpin region was replaced with an oppositely charged residue. Bioassays using Culex pipiens mosquito larvae revealed that the mosquitocidal activity was altered by the mutation. A K155E Cry46Ab mutant exhibited toxicity apparently higher than that of wild-type Cry46Ab, but the E159K and E163K mutants exhibited decreased toxicity. Ions selectivity measurements demonstrated that the channel pores formed by both wild-type and mutant Cry46Abs were cation selective, and their cation preference was also similar. However, the degree of cation selectivity was apparently higher in channel pores formed by the K155E mutant, and reduced selectivity was observed with the E159K and E163K mutants. Our data suggest that channel-pore cation selectivity is a major determinant of Cry46Ab mosquitocidal activity and that cation selectivity can be controlled via mutagenesis targeting the transmembrane β-hairpin region. KEY POINTS: • Cry46Ab mutants were constructed by targeting the putative transmembrane β-hairpin region. • Charged residues within the β-hairpin control the flux of ions through channel pores. • Channel-pore cation selectivity is correlated with insecticidal activity.

DOI： 10.1007/s00253-020-10893-5

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PTEN, a 3-phosphatase of phosphoinositide, regulates asymmetric PI(3,4,5)P3 signaling for the anterior-posterior polarization and migration of motile cells. PTEN acts through posterior localization on the plasma membrane, but the mechanism for this accumulation is poorly understood. Here we developed an in vitro single-molecule imaging assay with various lipid compositions and use it to demonstrate that the enzymatic product, PI(4,5)P2, stabilizes PTEN's membrane-binding. The dissociation kinetics and lateral mobility of PTEN depended on the PI(4,5)P2 density on artificial lipid bilayers. The basic residues of PTEN were responsible for electrostatic interactions with anionic PI(4,5)P2 and thus the PI(4,5)P2-dependent stabilization. Single-molecule imaging in living Dictyostelium cells revealed that these interactions were indispensable for the stabilization in vivo, which enabled efficient cell migration by accumulating PTEN posteriorly to restrict PI(3,4,5)P3 distribution to the anterior. These results suggest that PI(4,5)P2-mediated positive feedback and PTEN-induced PI(4,5)P2 clustering may be important for anterior-posterior polarization.

DOI： 10.1038/s42003-020-0818-3

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Photoactivated adenylyl cyclase (PAC) is a unique protein that, upon blue light exposure, catalyzes cAMP production. The crystal structures of two PACs, from Oscillatoria acuminata (OaPAC) and Beggiatoa sp. (bPAC), have been solved, and they show a high degree of similarity. However, the photoactivity of OaPAC is much lower than that of bPAC, and the regulatory mechanism of PAC photoactivity, which induces the difference in activity between OaPAC and bPAC, has not yet been clarified. Here, we investigated the role of the C-terminal region in OaPAC, the length of which is the only notable difference from bPAC. We found that the photoactivity of OaPAC was inversely proportional to the C-terminal length. However, the deletion of more than nine amino acids did not further increase the activity, indicating that the nine amino acids at the C-terminal critically affect the photoactivity. Besides, absorption spectral features of light-sensing domains (BLUF domains) of the C-terminal deletion mutants showed similar light-dependent spectral shifts as in WT, indicating that the C-terminal region influences the activity without interacting with the BLUF domain. The study characterizes new PAC mutants with modified photoactivities, which could be useful as optogenetics tools.

DOI： 10.1038/s41598-019-56721-3

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Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal toxin with aerolysin-type architecture, and has been shown that co-administration of Cry46Ab with other mosquitocidal Cry toxins results in synergistic toxicity against Culex pipiens Coquillett (Diptera: Culicidae) mosquito larvae. Cry46Ab, therefore, is expected to find use in improving the insecticidal activity of B. thuringiensis-based bioinsecticides. In the present study, the mode of action of Cry46Ab was explored by single-channel measurements of Cry46Ab channel-pores. The single-channel conductances of channel-pores formed in planar lipid bilayers by Cry46Ab were determined to be 31.8 +/- 2.7 pS in 150 mM NaCl and 24.2 +/- 0.7 pS in 150 mM CaCl2. Ion-selectivity measurements revealed that the channel-pores formed by Cry46Ab were cation selective. The permeability ratio of K+ to Cl-was approximately 4, and the preferences for cations were K+ > Na+, K+ > Ca2+, and Ca2+ > Na+. A calcein release assay using liposomes suggested that Cry46Ab influences the integrity of membrane vesicles. Formation of cation-selective channel-pores has been observed with other insecticidal Cry toxins that have structures distinct from those of Cry46Ab; the capability of forming such pores may be a property required of insecticidal toxins.

DOI： 10.1007/s13355-019-00635-z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

KcsA is a proton-activated K+ channel that is regulated at two gates: an activation gate located in the inner entrance of the pore and an inactivation gate at the selectivity filter. Previously, we revealed that the cytoplasmic domain (CPD) of KcsA senses proton and that electrostatic changes of the CPD influences the opening and closing of the activation gate. However, our previous studies did not reveal the effect of CPD on the inactivation gate because we used a non-inactivating mutant (E71A). In the present study, we used mutants that did not harbor the E71A mutation, and showed that the electrostatic state of the CPD influences the inactivation gate. Three novel CPD mutants were generated in which some negatively charged amino acids were replaced with neutral amino acids. These CPD mutants conducted K+, but showed various inactivation properties. Mutants carrying the D149N mutation showed high open probability and slow inactivation, whereas those without the D149N mutation showed low open probability and fast inactivation, similar to wild-type KcsA. In addition, mutants with D149N showed poor K+ selectivity, and permitted Na+ to flow. These results indicated that electrostatic changes in the CPD by D149N mutation triggered the loss of fast inactivation and changes in the conformation of selectivity filter. Additionally, the loss of fast inactivation induced by D149N was reversed by R153A mutation, suggesting that not only the electrostatic state of D149, but also that of R153 affects inactivation.

DOI： 10.1016/j.bbamem.2018.07.011

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The artificial bilayer single channel recording technique is commonly used to observe the detailed physiological properties of various ion channel proteins. It permits easy control of the solution and membrane lipid composition, and is also compatible with pharmacological screening devices. However, its use is limited due to low measurement efficiency. Here, we developed a novel artificial bilayer single channel recording technique in which solubilized ion channel proteins immobilized on a gold nano-electrode are directly incorporated into a lipid bilayer at the same time as the bilayer is formed at the tip of it on coming in contact with an aqueous-oil interface. Using this technique, we measured the single channel currents of several types of channels including KcsA, MthK, hBK and P2X4. This technique requires only one action to simultaneously form the bilayers and reconstitute the channels into the membranes. This simplicity greatly increases the measurement efficiency and allows the technique to potentially be combined with high-throughput screening devices.

DOI： 10.1039/c7nr08098k

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

A Cry46Ab toxin derived from Bacillus thuringiensis strain TK-E6 shows mosquitocidal activity against Culex pipiens pallens Coquillett (Diptera: Culicidae) larvae as well as preferential cytotoxicity against human cancer cells. In B. thuringiensis cells, Cry46Ab is produced and accumulates as a protein crystal that is processed into the active 29-kDa toxin upon solubilization in the alkaline environment of the insect midgut. The Cry46Ab protoxin is 30 kDa, and is therefore thought to require an accessory protein such as P20 and/or ORF2 for efficient crystal formation. In the present study, the potency of the 4AaCter-tag was investigated for the production of alkali-soluble inclusion bodies of recombinant Cry46Ab in Escherichia coli. The 4AaCter-tag is a polypeptide derived from the C-terminal region of the B. thuringiensis Cry4Aa toxin and facilitates the formation of alkali-soluble protein inclusion bodies in E. coli. Fusion with the 4AaCter-tag enhanced both Cry46Ab production and the formation of Cry46Ab inclusion bodies. In addition, upon optimization of protein expression procedures, the Cry46Ab-4AaCter inclusion bodies showed mosquitocidal activity and stability in aqueous environments comparable to Cry46Ab without the 4AaCter-tag. Our study suggests that use of the 4AaCter-tag is a straightforward approach for preparing formulations of smaller-sized Cry toxins such as Cry46Ab in E. coli.

DOI： 10.1007/s13355-017-0529-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Cry46Ab is a Cry toxin derived from Bacillus thuringiensis TK-E6. Cry46Ab is not significantly homologous to other mosquitocidal Cry or Cyt toxins and is classified as an aerolysin-type pore-forming toxin based on structural similarity. In this study, the potency of Cry46Ab was assessed for its potential application to mosquito control. A synthetic Cry46Ab gene, cry46Ab-S1, was designed to produce recombinant Cry46Ab as a glutathione-S-transferase fusion in Escherichia coli. Recombinant Cry46Ab showed apparent toxicity to Culex pipiens larvae, with a 50% lethal dose of 1.02 μg/ml. In an artificial lipid bilayer, Cry46Ab activated by trypsin caused typical current transitions between open and closed states, suggesting it functions as a pore-forming toxin similar to other Cry and Cyt toxins. The single-channel conductance was 103.3 ± 4.1 pS in 150 mM KCl. Co-administration of recombinant Cry46Ab with other mosquitocidal Cry toxins, especially the combination of Cry4Aa and Cry46Ab, resulted in significant synergistic toxicity against C. pipiens larvae. Co-administration of multiple toxins exhibiting different modes of action is believed to prevent the onset of resistance in insects. Our data, taken in consideration with the differences in its structure, suggest that Cry46Ab could be useful in not only reducing resistance levels but also improving the insecticidal activity of Bt-based bio-insecticides.

DOI： 10.1016/j.ibmb.2017.06.015

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A 2,175-bp modified gene (cry11Ba-S1) encoding Cry11Ba from Bacillus thuringiensis subsp. jegathesan was designed and the recombinant protein was expressed as a fusion protein with glutathione S-transferase in Escherichia coli. The recombinant Cry11Ba was highly toxic against Culex pipiens mosquito larvae, being nine and 17 times more toxic than mosquitocidal Cry4Aa and Cry11Aa from Bacillus thuringiensis subsp. israelensis, respectively. Interestingly, a further increase in the toxicity of the recombinant Cry11Ba was achieved by mixing with Cry4Aa, but not with Cry11Aa. These findings suggested that Cry11Ba worked synergistically with Cry4Aa, but not with Cry11Aa, in exhibiting toxicity against C. pipiens larvae. On the other hand, the amount of Cry toxin bound to brush border membrane vesicles (BBMVs) did not significantly change between individual toxins and the toxin mixtures, suggesting that the increase in toxins binding to BBMVs was not a reason for the observed synergistic effect. It is generally accepted that synergism of toxins is a potentially powerful tool for enhancing insecticidal activity and managing Cry toxin resistance in mosquitoes. The mixture of Cry4Aa and Cry11Ba in order to increase toxicity would be very valuable in terms of mosquito control.

DOI： 10.1007/s13355-016-0454-z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Chaperoning functions of liposomes were investigated using cell-free membrane protein synthesis. KcsA potassium channel-reconstituted liposomes were prepared directly using cell-free protein synthesis. In the absence of liposomes, all synthesized KcsA protein aggregated. In the presence of liposomes, however, synthesized KcsA spontaneously integrated into the liposome membrane. The KscA-reconstituted liposomes were transferred to the planar bilayer across a small hole in a thin plastic sheet and the channel function of KcsA was examined. The original electrophysiological activities, such as voltage- and pH-dependence, were observed. These results suggested that in cell-free membrane protein synthesis, liposomes act as chaperones, preventing aggregation and assisting in folding and tetrameric formation, thereby allowing full channel activity.

DOI： 10.1039/c5bm00285k

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC ANALYTICAL CHEMISTRY  

The artificial bilayer single-channel recording technique is commonly used to observe detailed pharmacological properties of various ion channel proteins. It permits easy control of the solution and membrane lipid composition, and is also compatible with pharmacological screening devices. However, its use is limited due to low measurement efficiency. Here, we develop a novel artificial bilayer single-channel recording technique in which bilayers are made and channels are reconstituted into the membranes by contacting a gold electrode to the lipid-solution interface. Using this technique, we measured the single-channel currents of two channel-forming peptides, gramicidin and alamethicin, and a channel-forming protein, α-hemolysin. This technique requires only one action, allowing the technique to potentially be combined with high-throughput screening devices.

DOI： 10.2116/analsci.32.1353

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The KcsA channel is a proton-activated potassium channel. We have previously shown that the cytoplasmic domain (CPD) acts as a pH-sensor, and the charged states of certain negatively charged amino acids in the CPD play an important role in regulating the pH-dependent gating. Here, we demonstrate the KcsA channel is constitutively open independent of pH upon mutating E146 to a neutrally charged amino acid. In addition, we found that rearrangement of the CPD following this mutation was not large. Our results indicate that minimal rearrangement of the CPD, particularly around E146, is sufficient for opening of the KcsA channel.

DOI： 10.1016/j.bbrc.2014.07.032

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.54.S285_6

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Chloroplasts require protein translocons at the outer and inner envelope membranes, termed TOC and TIC, respectively, to import thousands of cytoplasmically synthesized preproteins. However, the molecular identity of the TIC translocon remains controversial. Tic20 forms a 1-megadalton complex at the inner membrane and directly interacts with translocating preproteins. We purified the 1-megadalton complex from Arabidopsis, comprising Tic20 and three other essential components, one of which is encoded by the enigmatic open reading frame ycf1 in the chloroplast genome. All four components, together with well-known TOC components, were found stoichiometrically associated with different translocating preproteins. When reconstituted into planar lipid bilayers, the purified complex formed a preprotein-sensitive channel. Thus, this complex constitutes a general TIC translocon.

DOI： 10.1126/science.1229262

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DOI： 10.1016/j.bpj.2012.11.735

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Although ion channels are attractive targets for drug discovery, the systematic screening of ion channel-targeted drugs remains challenging. To facilitate automated single ion-channel recordings for the analysis of drug interactions with the intra- and extracellular domain, we have developed a parallel recording methodology using artificial cell membranes. The use of stable lipid bilayer formation in droplet chamber arrays facilitated automated, parallel, single-channel recording from reconstituted native and mutated ion channels. Using this system, several types of ion channels, including mutated forms, were characterised by determining the protein orientation. In addition, we provide evidence that both intra- and extracellular amyloid-beta fragments directly inhibit the channel open probability of the hBK channel. This automated methodology provides a high-throughput drug screening system for the targeting of ion channels and a data-intensive analysis technique for studying ion channel gating mechanisms.

DOI： 10.1038/srep01995

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This paper describes an investigation of drug effects for ion channels with recognizing the intra/extracellular directions. We have previously reported the ion channel recordings with bilayer lipid membranes (BLMs) which formed on a parylene micropore using droplets contact method. However, the low reconstitution probability of ion channels into BLMs has been a major issue. In this study, BLMs area and position are regulated with changing the numbers and positions of the parylene micropores. As a result, the simultaneous single channel recordings of K+ channel expressed in nerve system with BLM array were able to be achieved at the high probability. © 2013 IEEE.

DOI： 10.1109/MEMSYS.2013.6474175

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We have developed a quantitative immunoassay chip targeting point-of-care testing. To implement a lateral flow immunoassay, a glass fiber sheet was chosen as the material for the microfluidic channel in which the negative charge on the fiber surfaces efficiently generates the electroosmotic flow (EOF). The EOF, in turn, allows controllable bound/free separation of antigen/antibody interactions on the chip and enables precise determination of the antigen concentration. In addition, the defined size of the porous matrix was suitable for the filtration of undesired large particles. We confirmed the linear relationship between the concentration of analyte and the resulting fluorescence intensity from the immunoassay of two model analytes, C-reactive protein (CRP) and insulin, demonstrating that analyte concentration was quantitatively determined within the developed chip in 20 min. The limits of detection were 8.5 ng mL(-1) and 17 ng mL(-1) for CRP and insulin, respectively.

DOI： 10.1039/c2lc40994a

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.52.289

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This paper describes a quantitative immunoassay chip harnessing EOF (electroosmotic flow) in glass fiber sheet. The chip consists of a glass fiber sheet-based channel covered with PMMA (polymethylmethacrylate) frames and antibody-immobilized microbeads at the observation spot (Fig. 1). Applying EOF, another labeled-antibody and rinsing buffer sequentially migrate through the antigen-antibody complex on the beads and stain the beads depending on the sample (antigen) concentration. We demonstrated a quantitative assay of insulin and succeeded in determining the concentration in 20 min. The developed chip would provide a rapid, low cost and contamination-free assay system.

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The KcsA channel is a representative potassium channel that is activated by changes in pH. Previous studies suggested that the region that senses pH is entirely within its transmembrane segments. However, we recently revealed that the cytoplasmic domain also has an important role, because its conformation was observed to change dramatically in response to pH changes. Here, to investigate the effects of the cytoplasmic domain on pH-dependent gating, we made a chimera mutant channel consisting of the cytoplasmic domain of the KcsA channel and the transmembrane region of the MthK channel. The chimera showed a pH dependency similar to that of KcsA, indicating that the cytoplasmic domain can act as a pH sensor. To identify how this region detects pH, we substituted certain cytoplasmic domain amino acids that are normally negatively charged at pH 7 for neutral ones in the KcsA channels. These mutants opened independently of pH, suggesting that electrostatic charges have a major role in the cytoplasmic domain's ability to sense and respond to pH.

DOI： 10.1016/j.bpj.2011.09.024

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

Ion channels are membrane proteins that regulate cell functions by controlling the ion permeability of cell membranes. An ion channel contains an ion-selective pore that permeates ions and a sensor that senses a specific stimulus such as ligand binding to regulate the permeability. The detailed molecular mechanisms of this regulation, or gating, are unknown. Gating is thought to occur from conformational changes in the sensor domain in response to the stimulus, which results in opening the gate to permit ion conduction. Using an atomic force microscope and artificial bilayer system, a mechanical stimulus is applied to a potassium channel, and its gating is monitored in real time. The channel-open probability increases greatly when pushing the cytoplasmic domain toward the membrane. This result shows that a mechanical stimulus at the cytoplasmic domain causes changes in the gating and is the first to show direct evidence of coupling between conformational changes in the cytoplasmic domain and channel gating. This novel technology has the potential to be a powerful tool for investigating the activation dynamics in channel proteins.

DOI： 10.1002/smll.201002337

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Amphotericin B (AmB) is a widely used antifungal antibiotic with high specificity for fungi. We previously synthesized several covalently conjugated AmB dimers to clarify the AmB channel structure. Among these dimers, that with an aminoalkyl linker was found to exhibit potent hemolytic activity. We continue this work by investigating the channel activity of the dimer, finding that all channels comprised of AmB dimers show rectification. The direction of the dimer channel in the membrane depended on the electric potential at which the dimer channel was formed. On the other hand, only about half the monomer channels showed rectification. In addition, these channels were easily switched from a rectified to a nonrectified state following voltage stimulation, indicating instability. We propose a model to describe the AmB channel structure that explains why AmB dimer channels necessarily show rectification.

DOI： 10.1007/s00232-011-9354-x

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DOI： 10.2142/biophys.51.S105_1

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DOI： 10.2142/biophys.51.S21_1

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Transport from the endoplasmic reticulum to the Golgi body is ensured by a protein complex called COPII. Because the COPII vesicles are covered by the COPII coat protein - which consists of the low molecular weight GTPase Sar1p, Sec23/24p, and Sec13/31p, the transported proteins are selectively incorporated into the COPII vesicles by binding directly to the COPII coat. In this study, we reconstituted the formation of COPII vesicles on artificial planar lipid bilayer membranes, and visualized the dynamics of fluorescent-labeled transported proteins at a single molecular level, using a Total Internal Reflection Fluorescence Microscope (TIRFM). Then, the clusters of cargo molecules were observed by addition of Sec13/31p, revealing that the cargo molecules were concentrated inside the clusters. In addition, it has been revealed that the non-cargo molecules were excluded from the clusters. In this communication, we discuss the dynamics of cargo molecule in the process of COPII vesicle formation. © Springer Science+Business Media B.V. 2011.

DOI： 10.1007/978-90-481-9864-1_8

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DOI： 10.1002/cphc.201000560

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Single molecule imaging of working ion-channels is much more difficult than that of water-soluble proteins because of the fragile nature of membranes and lateral diffusion of particles in the membranes, which does not allow fluorescent contamination for optical single channel recording. In this report, we reconstituted maxi-potassium channels from porcine uterine smooth muscle into artificial planar bilayers formed on poly(ethylene glycol) (PEG) modified glass and performed simultaneous optical and electrical recording of the single channels. The channels were immobilized in the membranes by anchoring to PEG molecules on the glass. The technique developed in this study should pave the way for single molecule pharmacology of ion-channels.

DOI： 10.1021/la9045594

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A change of cytosolic pH 7 to 4 opens the bacterial potassium channel KcsA. However, the overall gating mechanism leading to channel opening, especially the contribution of the cytoplasmic domain, remains unsolved. Here we report that deletion of the cytoplasmic domain resulted in changes in channel conductance and gating behavior at pH 4 without channel opening at pH 7. To probe for rearrangements in the cytoplasmic domain during channel opening, amino acid residues were substituted with cysteines and labeled with a fluorophore (tetramethylrhodamine maleimide) that exhibits increased fluorescence intensity upon transfer from a hydrophilic to hydrophobic environment. In all cases channel open probability (P(o)) was approximately 1 at pH 4 and approximately 0 at pH 7. Major increases in fluorescence intensity were observed for tetramethylrhodamine maleimide-labeled residues in the cytoplasmic domain as pH changed from 7 to 4, which suggests the fluorophores shifted from a hydrophilic to hydrophobic environment. Dipicrylamide, a lipid soluble quencher, reduced the fluorescence intensities of labeled residues in the cytosolic domain at pH 4. These results reveal that a decrease in pH introduces major conformational rearrangements associated with channel opening in the KcsA cytoplasmic domain.

DOI： 10.1074/jbc.M109.084368

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Single channel currents of lysenin were measured using artificial lipid bilayers formed on a glass micropipette tip. The single channel conductance for KCl, NaCl, CaCl(2), and Trimethylammonium-Cl were 474 ± 87, 537 ± 66, 210 ± 14, and 274 ± 10 pS, respectively, while the permeability ratio P(Na)/P(Cl) was 5.8. By adding poly(deoxy adenine) or poly(L-lysine) to one side of the bilayer, channel currents were influenced when membrane voltages were applied to pass the charged molecules through the channel pores. Current inhibition process was concentration-dependent with applied DNA. As the current fluctuations of α-hemolysin channels is often cited as the detector in a molecular sensor, these results suggest that by monitoring channel current changes, the lysenin channel has possibilities to detect interactions between it and certain biomolecules by its current fluctuations.

DOI： 10.2183/pjab.86.920

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DOI： 10.2142/biophys.50.S139_4

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DOI： 10.2142/biophys.50.S187_4

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DOI： 10.1002/anie.200904909

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DOI： 10.2142/biophys.50.S186_5

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.50.S186_4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Selective protein export from the endoplasmic reticulum is mediated by COPII vesicles. Here, we investigated the dynamics of fluorescently labelled cargo and non-cargo proteins during COPII vesicle formation using single-molecule microscopy combined with an artificial planar lipid bilayer. Single-molecule analysis showed that the Sar1p-Sec23/24p-cargo complex, but not the Sar1p-Sec23/24p complex, undergoes partial dimerization before Sec13/31p recruitment. On addition of a complete COPII mixture, cargo molecules start to assemble into fluorescent spots and clusters followed by vesicle release from the planar membrane. We show that continuous GTPase cycles of Sar1p facilitate cargo concentration into COPII vesicle buds, and at the same time, non-cargo proteins are excluded from cargo clusters. We propose that the minimal set of COPII components is required not only to concentrate cargo molecules, but also to mediate exclusion of non-cargo proteins from the COPII vesicles. The EMBO Journal (2009) 28, 3279-3289. doi: 10.1038/emboj.2009.269; Published online 17 September 2009

DOI： 10.1038/emboj.2009.269

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Current ion channel current measurement techniques are cumbersome, as they require many steps and much time. This is especially true when reconstituting channels into liposomes and incorporating them into lipid bilayers. Here, we report a novel method that measures ion channel current more efficiently than current methods. We applied our method to KcsA and MthK channels by binding them to cobalt affinity gel beads with histidine tags and then forming a lipid bilayer membrane on the bead. This allowed channels to incorporate into the bilayer and channel currents to be measured quickly and easily. The efficiency was such that currents could be recorded with extremely low amounts of protein. In addition, the channel direction could be determined by the histidine tag. This method has the potential to be applied to various channel proteins and channel research in general.

DOI： 10.1021/ac900286z

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.49.S92_4

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.49.S135_1

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Language：English   Publisher：SPRINGER TOKYO  

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.49.S135_2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Single-channel recording using artificial lipid bilayers is along with the patch-clamp technique a very powerful tool to physiologically and pharmacologically study ion channels. It is particularly advantageous in studying channels that are technically difficult to access with a patch pipet. However, the fragility of the bilayers and the difficulty to incorporate ion channels into them significantly compromises measurement efficiency. We have developed a novel method for forming artificial lipid bilayers on a hydrogel surface that significantly improves the measurement efficiency. Bilayers formed almost instantly (<1 s) and were able to incorporate various types of ion channel proteins within a short time (<30 s) enabling multichannel measurements. These results indicate that this method can potentially be applied to developing high-throughput screening devices for drug design.

DOI： 10.1021/ac801224a

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

The organelles within secretory and endocytotic pathways in mammalian cells have acidified lumens, and regulation of their acidic pH is critical for the trafficking, processing and glycosylation of cargo proteins and lipids, as well as the morphological integrity of the organelles. How organelle lumen acidification is regulated, and how luminal pH elevation disturbs these fundamental cellular processes, is largely unknown. Here, we describe a novel molecule involved in Golgi acidification. First, mutant cells defective in Golgi acidification were established that exhibited delayed protein transport, impaired glycosylation and Golgi disorganization. Using expression cloning, a novel Golgi-resident multi-transmembrane protein, named Golgi pH regulator (GPHR), was identified as being responsible for the mutant cells. After reconstitution in planar lipid bilayers, GPHR exhibited a voltage-dependent anion-channel activity that may function in counterion conductance. Thus, GPHR modulates Golgi functions through regulation of acidification.

DOI： 10.1038/ncb1773

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

We synthesized a novel fluorescent analogue of acetylcholine, Cy3-3-acylcholine. The molecular weight of the products agreed with structural predictions. Discrete intensity changes of fluorescent spots due to a single ligand binding/unbinding to nAChR were visualized by TIRF microscopy. The agonist effect of the Cy3-3-acylcholine on nicotinic acetylcholine receptor (nAChR) was confirmed electrophysiologically. This newly synthesized fluorescent analogue will enable us to conduct more elaborate studies on single channel interaction processes between nAChR and ligands.

DOI： 10.1016/j.bmcl.2007.12.002

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.S159_5

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.S157_6

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.S159_1

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.S161_7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Surface Science Society of Japan  

We have developed a practical method to produce durable artificial lipid bilayers using a hydrogel-hydrogel interface for ion channel measurements. Bilayers were formed by forcing a hydrogel-bead into contact with the hydrogel layer (hydrogel plate) in a lipid solution. The immediate formation of a bilayer was observed (<1 s). This allows channel recordings to be repeated more easily and quickly as compared to conventional methods. Currents of various types of channel such as gramicidin, hemolysin and BK-channel have been recorded. Our channel property results mirrored those of other techniques and were reproducible. Hydrogel solutions containing gramicidin were extremely stable and could be used months after preparation for bilayer experiments. [DOI: 10.1380/ejssnt.2008.130]

DOI： 10.1380/ejssnt.2008.130

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.47.S187_1

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.47.S186_3

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.47.S186_2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SURFACE SCI SOC JAPAN  

In this report, we show an improved method for the simultaneous measurement of optical and electrical properties of single-channel proteins for analysis of the gating mechanism. Large-conductance Ca2+-activated potassium (BK) channels were isolated from porcine uterine smooth muscle and labeled with Cy5 via antibody against the N-terminal. These Cy5-labeled BK channels were incorporated into lipid bilayer membranes followed by single channel current measurements. Cy5-labeled BK channels possessed Ca2+ and voltage sensitivity. The orientation of the vesicles was determined to be outside-out. Charybdotoxin applied from the cis side blocked the channel current. For stable observations of ligand and channel binding, BK channel immobilization was also examined. The lateral diffusion coefficient of BK channels decreased over 200 fold in 1 mu M annexin V while the open probability did not change. This study is a significant advancement in simultaneous measurements of ligand binding and current change at the single channel level.

DOI： 10.1380/ejssnt.2007.1

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.47.S186_4

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Lysenin, a hemolytic protein derived from the body fluid of earthworm, was incorporated into artificial bilayer membranes. Upon insertion, it formed a voltage-dependent large conductance channel in asolectin bilayers in a sphingomyelin-dependent manner. The channel had low ion-selectivity. Single-channel conductance was calculated as approximately 550 pS in 100 mM KCl. The channel in asolectin bilayers closed when the membrane was held at a positive potential. In contrast, the channel showed no voltage dependency in membranes made of pure phosphatidylcholine and sphingomyelin, suggesting some lipid contents included in the asolectin membranes affected channel gating.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The effects of annexin A5 on the lateral diffusion of single-molecule lipids and single-molecule proteins were studied in an artificial lipid bilayer membrane. Annexin A5 is a member of the annexin superfamily, which binds preferentially to anionic phospholipids in a Ca2+-dependent manner. In this report, we were able to directly monitor single BODIPY 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DHPE) and ryanodine receptor type 2 (RyR2) labeled with Cy5 molecules in lipid bilayers containing phosphatidylserine (PS) by using fluorescence microscopy. The diffusion coefficients were calculated at various annexin A5 concentrations. The diffusion coefficients of BODIPY-DHPE and Cy5-RyR2 in the absence of annexin A5 were 4.81 x 10(-8) cm(2)/s and 2.13 x 10(-8) cm(2)/s, respectively. In the presence of 1 microM annexin A5, the diffusion coefficients of BODIPY-DHPE and Cy5-RyR2 were 2.2 x 10(-10) cm(2)/s and 9.5 x 10(-11) cm(2)/s, respectively. Overall, 1 microM of annexin A5 was sufficient to induce a 200-fold decrease in the lateral diffusion coefficient. Additionally, we performed electrophysiological examinations and determined that annexin A5 has little effect on the function of RyR2. This means that annexin A5 can be used to immobilize RyR2 in a lipid bilayer when imaging and analyzing RyR2.

DOI： 10.1021/la0535025

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.46.S236_4

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.46.S239_4

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Many proposals have been made regarding the development of biosensors using single-channel recording with an artificial planar bilayer. The fragile nature of bilayer membranes is the major difficulty for the application of the artificial bilayer technique to the development of biosensors. We have developed an apparatus that promptly forms artificial bilayers. This technique is more efficient than other techniques for forming artificial bilayers. Bilayer membranes could be formed within 10s requiring 1 microl of analyte solution to record single-channel currents using our apparatus. A bilayer was formed by pressing the membrane on an agarose layer with hydraulic pressure. With this novel apparatus, we have recorded single-channel currents of various types of channels such as the BK-channel, the nicotinic receptor channel and the ryanodine receptor channel. The properties of the channels determined with this novel technique agreed well with those determined with conventional techniques.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Surface Science Society Japan  

The purpose of this study is to develop an apparatus for simultaneous measurement of electrical and spectroscopic parameters of single ion-channels. We have combined the single channel recording apparatus with an artificial lipid bilayer and a fluorescence microscope designed to detect single fluorescent molecules. The artificial membranes were formed on a PEG (polyethylene glycol)-coated glass and observed with an objective-type total internal reflection fluorescence (TIRF) microscope. The lateral motion of a single lipid molecule (beta-BODIPY 530/550 HPC) was recorded. The lateral diffusion constant of the lipid molecule was calculated from the trajectories of single molecules as D = 8.0 +/- 4.0 x 10(-8) cm(2)/s. Ionic channels were incorporated into the membrane and single-channel current fluctuations were recorded. These data show the possibility of the present technique for simultaneous measurement of electrical and spectroscopic parameters of single-channel activities.

DOI： 10.1380/ejssnt.2005.70

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SURFACE SCI SOC JAPAN  

The effects of annexin 5 on the lateral diffusion of single molecule lipids and single molecule proteins were studied in a lipid bilayer membrane. Single beta-BODIPY HPC and ryanodine receptor type 2 (RyR2) labeled with Cy5 molecules were monitored by fluorescence microscopy. The diffusion coefficient was calculated in the presence and absence of annexin 5. The diffusion coefficients of beta-BODIPY HPC and RyR2 were 11 x 10(-8) cm(2)/s and 2.7x10(-8) cm(2)/s in the absence of annexin 5, respectively. The diffusion coefficients of beta-BODIPY HPC and RyR2 in the presence of 1 mu M annexin 5 were 2.4 x 10(-10) cm(2)/s and 2.6 x 10(-10) cm(2)/s, respectively. Overall, 1 mu M annexin 5 decreases the lateral diffusion coefficient 100-500 fold. Regarding RyR2, annexin 5 has little effect on function and can be used to immobilize RyR2 in a lipid bilayer system.

DOI： 10.1380/ejssnt.2005.213

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SURFACE SCI SOC JAPAN  

In order to detect and visualize the electrostatic features of biological macromolecules in a non-contact mode, we have refined the technique of scanning probe microscopy. The forces in the sub-piconewton range between the probe stylus and the sample surfaces have been measured with a gap distance controlled with nanometer accuracy. Images of the electrostatic surface forces of myosin filaments were detected in pure water using positively charged whiskers as cantilever probe tips. The images were consistent with the structure of myosin filaments that have a bipolar spindle shape; they were charged with a great number of negative charges in the central bare zone compared with the rest of the filament. Thus, in this non-contact mode, the electrostatic features of the protein surface rather than the surface topography were measured. This method has been further extended to measure forces exerted between protein molecules. Long-range interaction between kinesin and microtubules has been examined. It is likely that long-range attractive forces, in the order of several nanometers, exist between kinesin and microtubules.

DOI： 10.1380/ejssnt.2005.46

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.45.S69_2

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Amphotericin B-sterol conjugates were synthesized and examined for their membrane permeabilizing activity. Ergosterol and cholesterol, each connected with amphotericin B via an ethylenecarbamate or hexamethylenecarbamate linker, were examined by K(+) flux assays using liposomes and by single-channel recording across phospholipid membrane. Among four conjugates tested, AmB-ergosterol bearing an ethylenecarbamate linker exhibited the most powerful activity, which substantially exceeded that of the cholesterol homolog. Single-channel recording clearly exhibited that the ergosterol conjugate elicited channel current with the conductance of 28 pS, which was comparable with those by AmB, and revealed a higher channel open probability than the cholesterol conjugate. These results imply that direct interaction between amphotericin B and ergosterol is reproduced by their conjugate, which may serve as a model compound for understanding the drug's selective toxicity.

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S87_2

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.43.S177_3

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Language：English   Publishing type：Research paper (scientific journal)  

In recent years, the single-molecule imaging technique has proven to be a valuable tool in solving many basic problems in biophysics. The technique used to measure single-molecule functions was initially developed to study electrophysiological properties of channel proteins. However, the technology to visualize single channels at work has not received as much attention. In this study, we have for the first time, simultaneously measured the optical and electrical properties of single-channel proteins. The large conductance calcium-activated potassium channel (BK-channel) labeled with fluorescent dye molecules was incorporated into a planar bilayer membrane and the fluorescent image captured with a total internal reflection fluorescence microscope simultaneously with single-channel current recording. This innovative technology will greatly advance the study of channel proteins as well as signal transduction processes that involve ion permeation processes.

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Language：English   Publisher：PHYSIOLOGICAL SOCIETY OF JAPAN  

DOI： 10.2170/jjphysiol.49.19

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：CELL PRESS  

DOI： 10.1016/j.bpj.2014.11.309

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER SCIENCE BV  

DOI： 10.1016/j.bbabio.2010.04.153

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Language：English   Publisher：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.46.S278_4

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.45.S69_3

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Language：English   Presentation type：Oral presentation (general)  

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