Portions of these findings were presented at the Annual Meeting of the Research Society on Alcoholism in Washington, DC (Alcohol. Clin. Exp. Res. Vol. 32, Issue 6, p. 211A, 2008) and at the Purines Meeting in Copenhagen, Denmark (Purinergic Signaling, Vol. 4, Issue 5, p. 206, 2008).
A point mutation in the ectodomain-transmembrane 2 interface eliminates the inhibitory effects of ethanol in P2X4 receptors
Article first published online: 28 OCT 2009
© 2009 The Authors. Journal Compilation © 2009 International Society for Neurochemistry
Journal of Neurochemistry
Volume 112, Issue 1, pages 307–317, January 2010
How to Cite
Popova, M., Asatryan, L., Ostrovskaya, O., Wyatt, L. R., Li, K., Alkana, R. L. and Davies, D. L. (2010), A point mutation in the ectodomain-transmembrane 2 interface eliminates the inhibitory effects of ethanol in P2X4 receptors. Journal of Neurochemistry, 112: 307–317. doi: 10.1111/j.1471-4159.2009.06460.x
- Issue published online: 8 DEC 2009
- Article first published online: 28 OCT 2009
- Received June 9, 2009; revised manuscript received October 7, 2009; accepted October 21, 2009.
- ion channels;
- purinergic P2X4 receptors;
- sites of ethanol action;
- two-electrode voltage clamp;
- Xenopus oocytes
J. Neurochem. (2010) 112, 307–317.
ATP-gated P2X4 receptors (P2X4R) are abundantly expressed in the CNS. However, little is known about the molecular targets for ethanol action in P2X4Rs. The current investigation tested the hypothesis that the ectodomain-transmembrane (TM) interface contains residues that are important for the action of ethanol in P2X4Rs. Wild type (WT) and mutant P2X4R were expressed in Xenopus oocytes. ATP concentration–response curves and ethanol (10–200 mM)-induced changes in ATP EC10-gated currents were determined using two-electrode voltage clamp (−70 mV). Alanine substitution at the ectodomain-TM1 interface (positions 50–61) resulted in minimal changes in ethanol response. On the other hand, alanine substitution at the ectodomain-TM2 interface (positions 321–337) identified two key residues (D331 and M336) that significantly reduced ethanol inhibition of ATP-gated currents without causing marked changes in ATP Imax, EC50, or Hill’s slope. Other amino acid substitutions at positions 331 and 336 significantly altered or eliminated the modulatory effects of ethanol. Linear regression analyses revealed a significant relationship between hydropathy and polarity, but not molecular volume/molecular weight of the residues at these two positions. The results support the proposed hypothesis and represent an important step toward developing ethanol-insensitive receptors for investigating the role of P2X4Rs in mediating behavioral effects of ethanol.