Abstract
Activity regulation by phosphoinositides (PIPns) are known for various ion channels. In this study we aimed to examine whether the ATP-gated P2X2 receptor channel is also regulated by PIPns or not, and analyzed the electrophysiological properties of P2X2 wild type expressed in Xenopus oocytes under two-electrode voltage clamp. We observed that the preincubation in wortmannin or LY294002, PI3K inhibitors, accelerated the channel desensitization, while the preincubation in PAO, a PI4K inhibitor, decelerated it. As all PIPns are anionic lipids, we focused as the structural determinant on the proximal region of the C-terminus cytoplasmic domain where positively charged amino acids are clustered. We analyzed properties of several mutant P2X2 channels, and observed that K360Q, K365Q, K365N, K369Q, R371Q or K374Q mutation accelerated the desensitization, while K365R did not. These results suggest that these positively charged amino acid residues play critical roles in preventing the channel desensitization. As a next step we purified a GST-tagged recombinant protein from L353 to S377 expressed in E. coli., and analyzed their binding to anionic lipids using a PIPns-coated nitrocellulose membrane. We observed that the recombinant protein including the positively charged region was bound to PIPs and PIP2s, most strongly to PI(3,5)P2. Taken together, we speculate that an electrostatic binding of PI(3,5)P2 to the proximal C-terminus cytoplasmic domain plays a critical role in maintaing the channel activity. [Jpn J Physiol 55 Suppl:S127 (2005)]
