RESUMO
Patch clamp electrophysiology is the main technique to study mechanosensitive ion channels (MSCs), however, conventional patch clamping is laborious and success and output depends on the skills of the operator. Even though automated patch systems solve these problems for other ion channels, they could not be applied to MSCs. Here, we report on activation and single channel analysis of a bacterial mechanosensitive ion channel using an automated patch clamp system. With the automated system, we could patch not only giant unilamellar liposomes but also giant Escherichia coli (E. coli) spheroplasts. The tension sensitivity and channel kinetics data obtained in the automated system were in good agreement with that obtained from the conventional patch clamp. The findings will pave the way to high throughput fundamental and drug screening studies on mechanosensitive ion channels.
Assuntos
Automação Laboratorial/métodos , Proteínas de Escherichia coli/metabolismo , Canais Iônicos/metabolismo , Técnicas de Patch-Clamp/métodos , Escherichia coli/metabolismo , Esferoplastos/metabolismoRESUMO
The flavoenzyme L-galactono-gamma-lactone dehydrogenase (GALDH) catalyzes the terminal step of vitamin C biosynthesis in plants. Little is known about the catalytic mechanism of GALDH and related aldonolactone oxidoreductases. Here we identified an essential Glu-Arg pair in the active site of GALDH from Arabidopsis thaliana. Glu386 and Arg388 variants show high K(m) values for L-galactono-1,4-lactone and low turnover rates. Arg388 is crucial for the stabilization of the anionic form of the reduced FAD cofactor. Glu386 is involved in productive substrate binding. The E386D variant has lost its specificity for L-galactono-1,4-lactone and shows the highest catalytic efficiency with L-gulono-1,4-lactone.