Identification and characterization of the molecular complex formed by the P2X<sub>2</sub> receptor subunit and the adapter protein Fe65 in rat brain

Abstract

Synaptic transmission in the nervous system is achieved through the release of neurotransmitters from presynaptic terminals, resulting in the activation of neurotransmitter receptors at postsynaptic membrane. ATP is a neurotransmitter that mediates fast synaptic transmission in the central and peripheral nervous system, by activating a family of ATP-gated ionotropic receptors called P2X receptors. Mammalian P2X receptors are formed by seven subunits (P2X1 P2X7) of which mainly P2X2, P2X4 and P2X6 subunits are expressed in the brain. By using postembedding immunogold labeling combined with electron microscopy, the three subunits have been localized at the peripheral portion of the excitatory postsynaptic membrane. This precise localization of P2X subunits at excitatory synapses suggests an interaction with intracellular regulatory or anchoring proteins, as described for other synaptic receptors. However, interaction partners of P2X receptor subunits in the synapse have not been identified so far.In the present work, that question was answered by performing a yeast two-hybrid (Y2H) screening of a rat brain cDNA library employing the C-terminal domain of the P2X2 subunit as a bait. This approach allowed us to isolate the protein Fe65 as the main interacting partner of neuronal P2X2 receptor subunits. Characteristics of the interaction were confirmed in vitro by complementary Y2H assays and GST-pulldown experiments. Other members of the P2X family, as P2X4 and P2X7 were not able to interact with Fe65, revealing the specificity of the interaction. An interaction was found for P2X2 and the Fe65-related protein Fe65-like1, but was not observed between Fe65 and the naturally occurring P2X2 splice variant P2X2(b), indicating that alternative splicing may regulate complex assembly. Deletions and point mutations on both interacting partners helped to identify that the interaction is driven by the WW domain of Fe65 which specifically recognizes the proline rich sequence PPPP at the C-terminus of the P2X2 receptor.Two antibodies against Fe65 were generated, characterized and employed to assay the subcellular localization of this protein by immunogold-labeling electron microscopy. Labeling for Fe65 was found at the pre- and postsynaptic specialization of CA1 hippocampal pyramidal cell/Schaffer collateral synapses. By double immunogold labeling, the co-localization of Fe65 with P2X2 subunits at the postsynaptic specialization of excitatory synapses was observed, suggesting that the interaction occurs in vivo. As expected by the overlapping distribution of P2X2 and Fe65, both proteins could be co-immunoprecipitated from brain membrane extracts. This experiment demonstrates that both proteins are present in the same molecular complex providing more evidence on the occurrence in vivo of such association. The assembly with Fe65 was found to regulate certain functional properties of P2X2 receptors as demonstrated by electrophysiology recordings on HEK cells and Xenopus laevis oocyte heterologously expressing both proteins. While Fe65-bound P2X2 receptors showed comparable basic pharmacological properties, as current amplitudes, kinetics and ATP EC50 values, permeability changes were affected. Thus, the time- and activation-dependent change in ionic selectivity of P2X2 receptors was inhibited by coexpression with Fe65. We propose that Fe65 tethers the C-terminus of the receptor and impairs its ability permeate bulky organic cations, suggesting a novel mechanism for intracellular proteins in regulating receptor function and thus ATP-mediated synaptic transmission.