Developing an E. coli-expression-function platform for study of 3-D structure-function relationships of channel molecules

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To elucidate structure-function relationships of ion channels a novel experimental platform is developed. So far only ion channels originated from prokaryotes were successfully crystallized. To study such targets more effectively, electrophysiological recording system for ion channels from prokaryotes is critically important. Based on Yabe's method for current recordings from giant E. coli, channel recording techniques were explored. Many technical problems arose for recording whole-cell current from giant bacteria. These problems have been mostly solved, although a few problems still remain.KcsA channel was expressed and recorded by inside-out excised patch methods from giant E. coli. Reconstituted KcsA channel into liposome was also recorded by a modified tip-dip method. On the other hand, whole-cell current of KcsA channel could not be observed from giant bacteria. The discrepancy between successful single channel recordings and unsuccessful whole-cell recordings poses fundamental issue on functional role of ion channels on bacteria.