The 5-HT3 receptors are serotonin-gated ion channels that physically couple with purinergic P2X2 receptors to trigger a functional cross-inhibition leading to reciprocal channel occlusion. Although this functional receptor–receptor coupling seems to serve a modulatory role on both channels, this might not be its main physiological purpose. Using primary cultures of rat hippocampal neurons as a quantitative model of polarized targeting, we show here a novel function for this interaction. In this model, 5-HT3A receptors did not exhibit by themselves the capability of distal targeting in dendrites and axons but required the presence of P2X2R for their proper subcellular localization. 5-HT3AR distal targeting occurred with a delayed time course and exhibited a neuron phenotype dependency. In the subpopulation of neurons expressing endogenous P2X2R, 5-HT3AR distal neuritic localization correlated with P2X2R expression and could be selectively inhibited by P2X2R RNA interference. Cotransfection of both receptors revealed a specific colocalization, cotrafficking in common surface clusters, and the axonal rerouting of 5-HT3AR. The physical association between the two receptors was dependent on the second intracellular loop of the 5-HT3A subunit, but not on the P2X2R C-terminal tail that triggers the functional cross-inhibition with the 5-HT3AR. Together, these data establish that 5-HT3AR distal targeting in axons and dendrites primarily depends on P2X2R expression. Because several P2XR have now been shown to functionally interact with several other members of the 4-TMD family of receptor channels, we propose to reconsider the real functional role for this receptor family, as trafficking partner proteins dynamically involved in other receptors targeting.>SIGNIFICANCE STATEMENT So far, receptor targeting mechanisms were found to involve intracellular partner proteins or supramolecular complexes that couple receptors to cytoskeletal elements and recruit them into cargo vesicles. In this paper, we describe a new trafficking mechanism for the neuronal serotonin 5-HT3A ionotropic channel receptor, in which the role of routing partner is endowed by a functionally interacting purinergic receptor: the P2X2 receptor. This work not only unveils the mechanism by which 5-HT3 receptors can reach their axonal localization required for the control of neurotransmitter release, but also suggests that, in addition to their modulatory role, the family of P2X receptors could have a previously undescribed functional role of trafficking partner proteins dynamically involved in the targeting of other receptors.
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