We have been investigating the relationship between functions and structures of ion channels, especially inward rectifiers, by electrophysiological, biochemical and molecular biological approaches.
Patch clamp is a technique to measure the opening and closing of ion channels on the cell membrane with a tight seal between microelectrodes and membrane surface (Fig. 1). This technique is useful to identify not only the whole cell activity, but also a single channel function.
If a gene of the targe channel is isolated, we use Xenopus oocytes to see its activity. The frog egg expresses the ion channel, when it is injected its cRNA. We can manipulate the gene, so that this system enables us to address the ion channel function with genetically modified cRNAs.
Ion channels distribute at various specilaized membrane organization to help the cell to communicate with outer environment. We used to generate specific antibodies against ion channels to investigate subcellular localization of the channels. Fig. 3 shows that Kir4.1 localization in gastric mucosa. Not only with confocal microscopy, but also electron microscopy are necessary to investigate the ion channel function in detail (Fig. 4).
In some cases, the cell localization of the ion channel is regulated by the association with the other proteins. Biochemical approach and yeast two-hybrid system are used to investigate this point. To learn the molecule function at an atomic level, a modeling of channel structure and a wet structural biology technique are also available in our laboratory.
