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Direct Modulation of P2X₁ Receptor-Channels by the Lipid Phosphatidylinositol 4,5-Bisphosphate

Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada (L.-P.B., A.R.A., X.T., E.H., D.B., P.S.); and Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York University, New York, New York (Q.Z., D.E.L.)

The P2X₁ receptor-channels activated by extracellular ATP contribute to the neurogenic component of smooth muscle contraction in vascular beds and genitourinary tracts of rodents and humans. In the present study, we investigated the interactions of plasma membrane phosphoinositides with P2X₁ ATP receptors and their physiological consequences. In an isolated rat mesenteric artery preparation, we observed a strong inhibition of P2X₁-mediated constrictive responses by depletion of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] with the phosphatidylinositol 4-kinase inhibitor wortmannin. Using the Xenopus laevis oocyte expression system, we provided electrophysiological evidence that lowering PI(4,5)P₂ levels with wortmannin significantly decreases P2X₁ current amplitude and recovery. Previously reported modulation of recovery of desensitized P2X₁ currents by phospholipase C-coupled 5-hydroxytryptamine_2A metabotropic receptors was also found to be wortmannin-sensitive. Treatment with wortmannin alters the kinetics of P2X₁ activation and inactivation without changing its sensitivity to ATP. The functional impact of wortmannin on P2X₁ currents could be reversed by addition of intracellular PI(4,5)P₂, but not phosphatidylinositol 3,4,5-trisphosphate, and direct application of PI(4,5)P₂ to excised inside-out macropatches rescued P2X₁ currents from rundown. We showed that the proximal region of the intracellular C terminus of P2X₁ subunit directly binds to PI(4,5)P₂ and other anionic phospholipids, and we identified the basic residue Lys₃₆₄ as a critical determinant for phospholipid binding and sensitivity to wortmannin. Overall, these results indicate that PI(4,5)P₂ plays a key role in the expression of full native and heterologous P2X₁ function by regulating the amplitude, recovery, and kinetics of ionotropic ATP responses through direct receptor-lipid interactions.

Received for publication March 5, 2008.

Accepted for publication June 3, 2008.

Address correspondence to: Dr. Philippe Séguéla, Montreal Neurological Institute, 3801 University, Suite 778, Montreal, QC, Canada H3A 2B4. E-mail: philippe.seguela{at}mcgill.ca

This article has been cited by other articles:

				L.-P. Bernier, A. R. Ase, S. Chevallier, D. Blais, Q. Zhao, E. Boue-Grabot, D. Logothetis, and P. Seguela Phosphoinositides Regulate P2X4 ATP-Gated Channels through Direct Interactions J. Neurosci., November 26, 2008; 28(48): 12938 - 12945. [Abstract] [Full Text] [PDF]