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

In this paper, we report the development of a device that improves the conventional artificial lipid bilayer method and can measure channel currents more efficiently. Ion channel proteins are an attractive research target in biophysics, because their functions can be measured at the single-molecule level with high time resolution. In addition, they have attracted attention as targets for drug discovery because of their crucial roles in vivo. Although electrophysiological methods are powerful tools for studying channel proteins, they suffer from low measurement efficiency and require considerable skill. In our previous paper, we reported that by immobilizing channel proteins on agarose gel beads and forming an artificial lipid bilayer on the bead surface, we simultaneously solved two problems that had been hindering the efficiency of the artificial bilayer method: the time-consuming formation of artificial lipid bilayers and the time-consuming incorporation of channels into artificial bilayers. Previous studies have utilized crosslinked hard beads; however, here we show that channel current measurement can be achieved more simply and efficiently using non-crosslinked soft beads. In this study, we detailed the process of immobilizing channel proteins on the surface of non-crosslinked beads through chemical modification, allowing us to measure their channel activity. This method enables current measurements without the need for stringent bead size selection or high negative pressure.

DOI： 10.1007/s44211-025-00792-y

PubMed

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Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

We constructed a library of Mpp46Ab mutants, in which S153 within the transmembrane β-hairpin was randomly replaced by other amino acids. Mutagenesis and subsequent primary screening yielded 10 different Mpp46Ab mutants in addition to the wild type. Remarkably, S153 was replaced with a more hydrophobic amino acid in most of the mutants, and the S153I mutant in particular exhibited significantly increased toxicity. Electrophysiologic analysis using artificial lipid bilayers revealed that the single-channel conductance and PK/PCl permeability ratio were significantly increased for S153I pores. This suggests that the formation of highly ion-permeable and highly cation-selective toxin pores increases the influx of cations and water into cells, thereby facilitating osmotic shock. In addition, the S153F, S153L, and S153I mutants exhibited significantly reduced synergistic toxicity with Cry4Aa. Electrophysiologic analysis showed that the S153F, S153L, and S153I mutants form toxin pores with a significantly reduced PK/PNa permeability ratio and a significantly increased PK/PCa permeability ratio compared to wild-type pores. Thus, our results suggest that pore formation is central to the insecticidal activity of Mpp46Ab and that the ion permeability of toxin pores is a potential indicator correlated with both toxicity and synergistic toxicity with other toxins.

DOI： 10.3390/biology14050489

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

The human BK channel (hBK) is an essential membrane protein that regulates various biological functions, and its dysfunction leads to serious diseases. Understanding the biophysical properties of hBK channels is crucial for drug development. Artificial lipid bilayer recording is used to measure biophysical properties at the single-channel level. However, this technique is time-consuming and complicated; thus, its measurement efficiency is very low. Previously, we developed a novel technique to improve the measurement efficiency by rapidly forming lipid bilayer membranes and incorporating ion channels into the membrane using a hydrophilically modified gold probe. To further improve our technique for application to the hBK channel, we combined it using the gold probe with a liposome fusion method. Using a probe on which liposomes containing hBK channels were immobilized, the channels were efficiently incorporated into the lipid bilayer membrane, and the measured channel currents showed the current characteristics of the hBK channel. This technique will be useful for the efficient measurements of the channel properties of hBK and other biologically important channels.

DOI： 10.1007/s44211-024-00707-3

PubMed

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Publishing type：Research paper (scientific journal)   Publisher：Wiley  

Electrophysiologic analysis using artificial lipid bilayers is useful for studying the formation of pores by insecticidal proteins, especially the ion permeability of toxin pores. However, such studies are time‐consuming and require special skills, particularly regarding the construction of lipid bilayers and promoting toxin pore formation. To facilitate the analysis of toxin pore formation in the present study, we evaluated the usefulness of agarose gel‐supported lipid bilayers for electrophysiologic measurements using two structurally different mosquito‐larvicidal proteins, Mpp46Ab and Cry4Aa. The agarose gel‐supported lipid bilayers enabled the measurement of channel currents through pores made by both toxins and, notably, the lipid bilayers could be easily reconstructed even after disruption of the lipid bilayer. Using this system, measurements could be repeated at least five times using the same apparatus and toxins. We also investigated the effect of the lipid bilayer component on toxin pore formation and found that the incorporation of both cholesterol and sphingomyelin into the lipid bilayer facilitates the formation of pores by both Mpp46Ab and Cry4Aa. Both cholesterol and sphingomyelin are major components of lipid raft microdomains, suggesting that, in addition to recruiting toxin receptors, raft microdomains play a key role in membrane insertion and pore formation by insecticidal proteins.

DOI： 10.1111/febs.70070

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

The human BK channel (hBK) is an essential membrane protein that regulates various biological functions, and its dysfunction leads to serious diseases. Understanding the biophysical properties of hBK channels is crucial for drug development. Artificial lipid bilayer recording is used to measure biophysical properties at the single-channel level. However, this technique is time-consuming and complicated; thus, its measurement efficiency is very low. Previously, we developed a novel technique to improve the measurement efficiency by rapidly forming lipid bilayer membranes and incorporating ion channels into the membrane using a hydrophilically modified gold probe. To further improve our technique for application to the hBK channel, we combined it using the gold probe with a liposome fusion method. Using a probe on which liposomes containing hBK channels were immobilized, the channels were efficiently incorporated into the lipid bilayer membrane, and the measured channel currents showed the current characteristics of the hBK channel. This technique will be useful for the efficient measurements of the channel properties of hBK and other biologically important channels.

Graphical abstract

DOI： 10.1007/s44211-024-00707-3

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Other Link： https://link.springer.com/article/10.1007/s44211-024-00707-3/fulltext.html