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J. Biol. Chem., Vol. 280, Issue 28, 25982-25993, July 15, 2005

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An Intersubunit Zinc Binding Site in Rat P2X₂ Receptors^*

Naomi Nagaya, Rachel K. Tittle, Nir Saar, Shlomo S. Dellal, and Richard I. Hume

From the Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109-1048

P2X receptors are ATP-gated ion channels made up of three similar or identical subunits. It is unknown whether ligand binding is intersubunit or intrasubunit, either for agonists or for allosteric modulators. Zinc binds to rat P2X₂ receptors and acts as an allosteric modulator, potentiating channel opening. To probe the location of this zinc binding site, P2X₂ receptors bearing mutations of the histidines at positions 120 and 213 were expressed in Xenopus oocytes. Studies of H120C and H213C mutants produced five lines of evidence consistent with the hypothesis that the residues in these positions bind zinc. Mixing of subunits containing the H120A or H213A mutation generated receptors that showed zinc potentiation, even though neither of these mutant receptors showed zinc potentiation on its own. Furthermore, expression of trimeric concatamers with His Ala mutations at some but not all six positions showed that zinc potentiation correlated with the number of intersubunit histidine pairs. These results indicate that zinc potentiation requires an interaction across a subunit interface. Expression of the H120C/H213C double mutant resulted in the formation of ectopic disulfide bonds that could be detected by changes in the physiological properties of the receptors after treatment with reducing and oxidizing agents. Immunoblot analysis of H120C/H213C protein separated under nonreducing conditions demonstrated that the ectopic bonds were between adjacent subunits. Taken together, these data indicate that His¹²⁰ and His²¹³ sit close to each other across the interface between subunits and are likely to be key components of the zinc binding site in P2X₂ receptors.

Received for publication, April 26, 2005 , and in revised form, May 13, 2005.

^* This work was supported by National Institutes of Health Grant R01-NS039196 (to R. I. H.). 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.

These two authors contributed equally to this work.

To whom correspondence should be addressed: Dept. of Molecular, Cellular, and Developmental Biology, University of Michigan, 830 N. University Ave., Ann Arbor, MI 48109-1048. E-mail: rhume{at}umich.edu.

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