start-ver=1.4 cd-journal=joma no-vol=81 cd-vols= no-issue=3 article-no= start-page=80 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2024 dt-pub=20240128 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Mutational analysis of the transmembrane 4-helix of Bacillus thuringiensis mosquito-larvicidal Cry4Aa toxin en-subtitle= kn-subtitle= en-abstract= kn-abstract=Cry4Aa, produced by Bacillus thuringiensis subsp. israelensis, exhibits specific toxicity to larvae of medically important mosquito genera. Cry4Aa functions as a pore-forming toxin, and a helical hairpin (4-loop-5) of domain I is believed to be the transmembrane domain that forms toxin pores. Pore formation is considered to be a central mode of Cry4Aa action, but the relationship between pore formation and toxicity is poorly understood. In the present study, we constructed Cry4Aa mutants in which each polar amino acid residues within the transmembrane 4 helix was replaced with glutamic acid. Bioassays using Culex pipiens mosquito larvae and subsequent ion permeability measurements using symmetric KCl solution revealed an apparent correlation between toxicity and toxin pore conductance for most of the Cry4Aa mutants. In contrast, the Cry4Aa mutant H178E was a clear exception, almost losing its toxicity but still exhibiting a moderately high conductivity of about 60% of the wild-type. Furthermore, the conductance of the pore formed by the N190E mutant (about 50% of the wild-type) was close to that of H178E, but the toxicity was significantly higher than that of H178E. Ion selectivity measurements using asymmetric KCl solution revealed a significant decrease in cation selectivity of toxin pores formed by H178E compared to N190E. Our data suggest that the toxicity of Cry4Aa is primarily pore related. The formation of toxin pores that are highly ion-permeable and also highly cation-selective may enhance the influx of cations and water into the target cell, thereby facilitating the eventual death of mosquito larvae. en-copyright= kn-copyright= en-aut-name=TakahashiHirokazu en-aut-sei=Takahashi en-aut-mei=Hirokazu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=AsakuraMami en-aut-sei=Asakura en-aut-mei=Mami kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=IdeToru en-aut-sei=Ide en-aut-mei=Toru kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=HayakawaTohru en-aut-sei=Hayakawa en-aut-mei=Tohru kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= affil-num=1 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=2 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=3 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=4 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= END start-ver=1.4 cd-journal=joma no-vol=12 cd-vols= no-issue=12 article-no= start-page=1481 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2023 dt-pub=20231201 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Random Mutational Analysis Targeting Residue K155 within the Transmembrane -Hairpin of the Mosquitocidal Mpp46Ab Toxin en-subtitle= kn-subtitle= en-abstract= kn-abstract=Mpp46Ab is a mosquito-larvicidal pore-forming toxin derived from Bacillus thuringiensis TK-E6. Pore formation is believed to be a central mode of Mpp46Ab action, and the cation selectivity of the channel pores, in particular, is closely related to its mosquito-larvicidal activity. In the present study, we constructed a mutant library in which residue K155 within the transmembrane -hairpin was randomly replaced with other amino acid residues. Upon mutagenesis and following primary screening using Culex pipiens mosquito larvae, we obtained 15 mutants in addition to the wild-type toxin. Bioassays using purified proteins revealed that two mutants, K155E and K155I, exhibited toxicity significantly higher than that of the wild-type toxin. Although increased cation selectivity was previously reported for K155E channel pores, we demonstrated in the present study that the cation selectivity of K155I channel pores was also significantly increased. Considering the characteristics of the amino acids, the charge of residue 155 may not directly affect the cation selectivity of Mpp46Ab channel pores. Replacement of K155 with glutamic acid or isoleucine may induce a similar conformational change in the region associated with the ion selectivity of the Mpp46Ab channel pores. Mutagenesis targeting the transmembrane -hairpin may be an effective strategy for enhancing the ion permeability of the channel pores and the resulting mosquito-larvicidal activity of Mpp46Ab. en-copyright= kn-copyright= en-aut-name=MiyazakiMidoka en-aut-sei=Miyazaki en-aut-mei=Midoka kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=AsakuraMami en-aut-sei=Asakura en-aut-mei=Mami kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=IdeToru en-aut-sei=Ide en-aut-mei=Toru kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=HayakawaTohru en-aut-sei=Hayakawa en-aut-mei=Tohru kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= affil-num=1 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=2 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=3 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=4 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= en-keyword=mosquito-larvicidal toxin kn-keyword=mosquito-larvicidal toxin en-keyword=Bacillus thuringiensis TK-E6 kn-keyword=Bacillus thuringiensis TK-E6 en-keyword=Culex pipiens mosquito larvae kn-keyword=Culex pipiens mosquito larvae en-keyword=site-directed mutagenesis kn-keyword=site-directed mutagenesis en-keyword=electrophysiologic analysis kn-keyword=electrophysiologic analysis END start-ver=1.4 cd-journal=joma no-vol=57 cd-vols= no-issue=1 article-no= start-page=63 end-page=70 dt-received= dt-revised= dt-accepted= dt-pub-year=2021 dt-pub=20211023 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Characteristics of channel pores formed by Bacillus thuringiensis mosquito-larvicidal Cry4Aa toxin en-subtitle= kn-subtitle= en-abstract= kn-abstract=Cry4Aa toxin produced by Bacillus thuringiensis subsp. israelensis exhibits specific toxicity to larvae of medically-important mosquito genera. In the present study, we analyzed the characteristics of channel-pores formed by recombinant Cry4Aa in a solvent-free planar lipid bilayer. Stable channel-currents were observed in electrophysiologic measurements, and the single-channel conductance was 187 } 10 pS in symmetrical buffer containing 150 mM KCl. The channel-pores formed by Cry4Aa were cation selective, with an estimated PK/PCl permeability ratio of 4.9. In addition, Cry4Aa channel-pores exhibited apparent cation preference in the order Na+ > K+, Na+ > Ca2+, and K+ > Ca2+. Although the effect was limited, the cation preference of Cry4Aa channel-pores seemed to be correlated with toxicity. Culex pipiens mosquito larvae reared in NaCl solution exhibited greater sensitivity to Cry4Aa, particularly early period after exposure. The presence of cations that preferentially translocate through Cry4Aa channel-pores may facilitate excessive influx of water into the midgut cells, leading to colloid-osmotic lysis. Whereas CaCl2 had some effect on the mosquito-larvicidal activity of Cry4Aa, KCl had no effect. The effect of some cations may be mitigated by the variety of ion channels present on the midgut cell membrane. en-copyright= kn-copyright= en-aut-name=ShiraishiYuri en-aut-sei=Shiraishi en-aut-mei=Yuri kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=ShiozakiTomoya en-aut-sei=Shiozaki en-aut-mei=Tomoya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=AsakuraMami en-aut-sei=Asakura en-aut-mei=Mami kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=IdeToru en-aut-sei=Ide en-aut-mei=Toru kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=HayakawaTohru en-aut-sei=Hayakawa en-aut-mei=Tohru kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= affil-num=1 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=2 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=3 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=4 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=5 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= en-keyword=Bacillus thuringiensis Cry4Aa toxin kn-keyword=Bacillus thuringiensis Cry4Aa toxin en-keyword=Culex pipiens mosquito larvae kn-keyword=Culex pipiens mosquito larvae en-keyword=electrophysiologic analysis of channel-pores kn-keyword=electrophysiologic analysis of channel-pores END start-ver=1.4 cd-journal=joma no-vol=11 cd-vols= no-issue=12 article-no= start-page=1070 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2020 dt-pub=20201201 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=A Lipid Bilayer Formed on a Hydrogel Bead for Single Ion Channel Recordings en-subtitle= kn-subtitle= en-abstract= kn-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, alpha-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. en-copyright= kn-copyright= en-aut-name=HiranoMinako en-aut-sei=Hirano en-aut-mei=Minako kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=YamamotoDaiki en-aut-sei=Yamamoto en-aut-mei=Daiki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=AsakuraMami en-aut-sei=Asakura en-aut-mei=Mami kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=HayakawaTohru en-aut-sei=Hayakawa en-aut-mei=Tohru kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=MiseShintaro en-aut-sei=Mise en-aut-mei=Shintaro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=MatsumotoAkinobu en-aut-sei=Matsumoto en-aut-mei=Akinobu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=IdeToru en-aut-sei=Ide en-aut-mei=Toru kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= affil-num=1 en-affil=Bio Photonics Laboratory, The Graduate School for the Creation of New Photonics Industries kn-affil= affil-num=2 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=3 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=4 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=5 en-affil=Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University kn-affil= affil-num=6 en-affil=Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University kn-affil= affil-num=7 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= en-keyword=ion channel kn-keyword=ion channel en-keyword=lipid bilayer kn-keyword=lipid bilayer en-keyword=single-channel recording kn-keyword=single-channel recording END start-ver=1.4 cd-journal=joma no-vol=104 cd-vols= no-issue= article-no= start-page=8789 end-page=8799 dt-received= dt-revised= dt-accepted= dt-pub-year=2020 dt-pub=20200911 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Channel-pore cation selectivity is a major determinant of Bacillus thuringiensis Cry46Ab mosquitocidal activity en-subtitle= kn-subtitle= en-abstract= kn-abstract=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 -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 -hairpin region. en-copyright= kn-copyright= en-aut-name=HayakawaTohru en-aut-sei=Hayakawa en-aut-mei=Tohru kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=MiyazakiMidoka en-aut-sei=Miyazaki en-aut-mei=Midoka kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=HaradaSyoya en-aut-sei=Harada en-aut-mei=Syoya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=AsakuraMami en-aut-sei=Asakura en-aut-mei=Mami kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=IdeToru en-aut-sei=Ide en-aut-mei=Toru kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= affil-num=1 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=2 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=3 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=4 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=5 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= en-keyword=Bacillus thuringiensis TK-E6 kn-keyword=Bacillus thuringiensis TK-E6 en-keyword=Cry46Ab toxin kn-keyword=Cry46Ab toxin en-keyword=Culex pipiens mosquito larvae kn-keyword=Culex pipiens mosquito larvae en-keyword=Site-directed mutagenesis kn-keyword=Site-directed mutagenesis en-keyword=Electrophysiologic analysis kn-keyword=Electrophysiologic analysis END start-ver=1.4 cd-journal=joma no-vol=54 cd-vols= no-issue=4 article-no= start-page=389 end-page=398 dt-received= dt-revised= dt-accepted= dt-pub-year=2019 dt-pub=20190816 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Characterization of the channel-pores formed by Bacillus thuringiensis Cry46Ab toxin in planar lipid bilayers en-subtitle= kn-subtitle= en-abstract= kn-abstract= 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. en-copyright= kn-copyright= en-aut-name=SakakibaraAkira en-aut-sei=Sakakibara en-aut-mei=Akira kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=TakebeSo en-aut-sei=Takebe en-aut-mei=So kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=IdeToru en-aut-sei=Ide en-aut-mei=Toru kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=HayakawaTohru en-aut-sei=Hayakawa en-aut-mei=Tohru kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= affil-num=1 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=2 en-affil=Graduate School of Biology-Oriented Science and Technology, Kindai University kn-affil= affil-num=3 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= affil-num=4 en-affil=Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University kn-affil= en-keyword=Bacillus thuringiensis kn-keyword=Bacillus thuringiensis en-keyword=Cry46Ab toxin kn-keyword=Cry46Ab toxin en-keyword=Planar lipid bilayer kn-keyword=Planar lipid bilayer en-keyword=Single-channel analysis kn-keyword=Single-channel analysis en-keyword=Calcein release assay kn-keyword=Calcein release assay END