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J. Biol. Chem., Vol. 281, Issue 43, 32649-32659, October 27, 2006

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Participation of the Lys³¹³-Ile³³³ Sequence of the Purinergic P2X₄ Receptor in Agonist Binding and Transduction of Signals to the Channel Gate^*

Zonghe Yan, Zhaodong Liang, Tomas Obsil, and Stanko S. Stojilkovic¹

From the Section on Cellular Signaling, Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892-4510 and the Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, 128 43 Prague, Czech Republic

To study the roles of the Lys³¹³-Ile³³³ ectodomain sequence of the rat P2X₄ receptor in ATP binding and transduction of signals to the channel gate, the conserved Lys³¹³, Tyr³¹⁵, Gly³¹⁶, Ike³¹⁷, Arg³¹⁸, Asp³²⁰, Val³²³, Lys³²⁹, Phe³³⁰, and Ile³³³ residues were mutated. Current recordings were done on lifted cells and ATP was applied using an ultrafast solution-switching system. The rates of wild type channel opening and closing in the presence of ATP, but not the rate of washout-induced closing, were dependent on agonist concentration. All mutants other than I317A were expressed in the plasma membrane at comparable levels. The majority of mutants showed significant changes in the peak amplitude of responses and the EC₅₀ values for ATP. When stimulated with the supramaximal (1.4 mM) ATP concentration, mutants also differed in the kinetics of their activation, deactivation, and/or desensitization. The results suggest a critical role of the Lys³¹³ residue in receptor function other than coordination of the phosphate group of ATP and possible contribution of the Tyr³¹⁵ residue to the agonist binding module. The pattern of changes of receptor function by mutation of other residues was consistent with the operation of the Gly³¹⁶-Ile³³³ sequence as a signal transduction module between the ligand binding domain and the channel gate in the second transmembrane domain.

Received for publication, November 30, 2005 , and in revised form, August 31, 2006.

^* This work was supported by the Intramural Research Program of the NICHD, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S5.

¹ To whom correspondence should be addressed: ERRB, NICHD, Bldg. 49, Rm. 6A-36, 49 Convent Dr., Bethesda, MD 20892-4510. Tel.: 301-496-1638; Fax: 301-594-7031; E-mail: stankos{at}helix.nih.gov.

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