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J. Biol. Chem., Vol. 281, Issue 7, 4100-4108, February 17, 2006

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Fe65 Interacts with P2X₂ Subunits at Excitatory Synapses and Modulates Receptor Function^*

Marianela Masin, Daniel Kerschensteiner¹, Kerstin Dümke, Maria E. Rubio^¶, and Florentina Soto²

From the Department of Molecular Biology of Neuronal Signals, Max-Planck Institute for Experimental Medicine, D-37075 Göttingen, Germany, the Laboratory of Molecular Pharmacology, Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom, and the ^¶Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut 06269

Ionotropic receptors in the neuronal plasma membrane are organized in macromolecular complexes, which assure their proper localization and regulate signal transduction. P2X receptors, the ionotropic receptors activated by extracellular ATP, have been shown to influence synaptic transmission. Using a yeast two-hybrid approach with the P2X₂ subunit C-terminal domain as bait we isolated the -amyloid precursor protein-binding proteins Fe65 and Fe65-like 1 as the first identified proteins interacting with neuronal P2X receptors. We confirmed the direct interaction of Fe65 and the P2X₂ C-terminal domain by glutathione S-transferase pull-down experiments. No interaction was observed between Fe65 and the naturally occurring P2X₂ splice variant P2X_2(b), indicating that alternative splicing can regulate the receptor complex assembly. We generated two antibodies to Fe65 to determine its subcellular localization using postembedding immunogold labeling electron microscopy. We found labeling for Fe65 at the pre- and postsynaptic specialization of CA1 hippocampal pyramidal cell/Schaffer collateral synapses. By double immunogold labeling, we determined that Fe65 colocalizes with P2X₂ subunits at the postsynaptic specialization of excitatory synapses. Moreover, P2X₂ and Fe65 could be coimmunoprecipitated from brain membrane extracts, demonstrating that the interaction occurs in vivo. The assembly with Fe65 regulates the functional properties of P2X₂ receptors. Thus, the time- and activation-dependent change in ionic selectivity of P2X₂ receptors was inhibited by coexpression of Fe65, suggesting a novel role for Fe65 in regulating P2X receptor function and ATP-mediated synaptic transmission.

Received for publication, July 15, 2005 , and in revised form, November 10, 2005.

^* This work was supported by Deutsche Forschungsgemeinschaft Grant So-390/1–2 (to F. S.) and the University of Connecticut (to M. E. R.). 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.

¹ Present address: Dept. of Anatomy and Neurobiology, Washington University Medical School, 660 S. Euclid Ave., St. Louis, MO 63110.

² To whom correspondence should be addressed: Dept. of Pharmacological and Physiological Science, St. Louis University Medical School, 1402 S. Grand Blvd., St. Louis, MO 63104. Tel.: 314-977-6445; Fax: 314-977-6410; E-mail: sotof{at}slu.edu.

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