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MOLECULAR AND GENOMIC |
1 Division of Biophysics and Neurobiology, Department of Molecular Physiology, National Institute for Physiological Sciences, Aichi 444-8585, Japan
2 COE Program for Brain Integration and its Disorders, Tokyo Medical and Dental University Graduate School and Faculty of Medicine, Tokyo 113-8519, Japan
3 SORST, Japan Science and Technology Corporation, Saitama 332-0012, Japan
Phosphoinositides (PIPns) are known to regulate the activity of some ion channels. Here we determined that ATP-gated P2X2 channels also are regulated by PIPns, and investigated the structural background and the unique features of this regulation. We initially used two-electrode voltage clamp to analyse the electrophysiological properties of P2X2 channels expressed in Xenopus oocytes, and observed that preincubation with wortmannin or LY294002, two PI3K inhibitors, accelerated channel desensitization. K365Q or K369Q mutation of the conserved, positively charged, amino acid residues in the proximal region of the cytoplasmic C-terminal domain also accelerated desensitization, whereas a K365R or K369R mutation did not. We observed that the permeability of the channel to N-methyl-D-glucamine (NMDG) transiently increased and then decreased after ATP application, and that the speed of the decrease was accelerated by K365Q or K369Q mutation or PI3K inhibition. Using GST-tagged recombinant proteins spanning the proximal C-terminal region, we then analysed their binding of the P2X2 cytoplasmic domain to anionic lipids using PIPns-coated nitrocellulose membranes. We found that the recombinant proteins that included the positively charged region bound to PIPs and PIP2s, and that this binding was eliminated by the K365Q and K369Q mutations. We also used a fluorescence assay to confirm that fusion proteins comprising the proximal C-terminal region of P2X2 with EGFP expressed in COS-7 cells closely associated with the membrane. Taken together, these results show that membrane-bound PIPns play a key role in maintaining channel activity and regulating pore dilation through electrostatic interaction with the proximal region of the P2X2 cytoplasmic C-terminal domain.
(Received 9 June 2006;
accepted after revision 14 July 2006;
first published online 20 July 2006)
Corresponding authors Y. Fujiwara and Y. Kubo: Division of Biophysics and Neurobiology, Department of Molecular Physiology, National Institute for Physiological Sciences, Aichi 444-8585, Japan. Email: yuichiro{at}nips.ac.jp or ykubo{at}nips.ac.jp
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