Abstract
We previously observed that the channel pore properties such as rectification and ionic selectivity of ATP receptor P2X2 change depending on the expression level, suggesting dynamic structural rearrangements. However, the structure, even the stoichiometry, remains unknown. Towards the structure analysis, we aimed to purify recombinant P2X2 protein. We first made constructs of P2X2 tagged with FLAG at either N-or C-terminus, and confirmed that they have intact function when expressed in Xenopus oocytes. We then made them express in insect Sf9 cells using baculovirus expression system and solubilized the FLAG-tagged P2X2 proteins of membrane fraction in a solution containing 50 mM dodecyl-β-maltoside. They were purified by FLAG affinity gel and further by gel filtration chromatography. A single major band was successfully observed by silver staining and Western blotting of SDS-PAGE. By evaluating the molecular size of the purified protein treated by 25 mM glutaraldehyde which bridges the subunits, P2X2 protein was shown to be a trimer. The trimer band on SDS-PAGE shifted upwards by pretreating with 200 μM ATP, but the shift was not observed when ATP was applied after glutaraldehyde treatment. These results suggest that a sort of conformational changes were induced by ATP. Electron microscopic images of negative-stained proteins also supported that P2X2 is a trimeric protein. [Jpn J Physiol 55 Suppl:S126 (2005)]
