The C-terminal Src Inhibitory Kinase (Csk)-mediated Tyrosine Phosphorylation Is a Novel Molecular Mechanism to Limit P2X3 Receptor Function in Mouse Sensory Neurons

On sensory neurons, sensitization of P2X₃ receptors gated by extracellular ATP contributes to chronic pain. We explored the possibility that receptor sensitization may arise from down-regulation of an intracellular signal negatively controlling receptor function. In view of the structural modeling between the Src region phosphorylated by the C-terminal Src inhibitory kinase (Csk) and the intracellular C terminus domain of the P2X₃ receptor, we investigated how Csk might regulate receptor activity. Using HEK cells and the in vitro kinase assay, we observed that Csk directly phosphorylated the tyrosine 393 residue of the P2X₃ receptor and strongly inhibited receptor currents. On mouse trigeminal sensory neurons, the role of Csk was tightly controlled by the extracellular level of nerve growth factor, a known algogen. Furthermore, silencing endogenous Csk in HEK or trigeminal cells potentiated P2X₃ receptor responses, confirming constitutive Csk-mediated inhibition. The present study provides the first demonstration of an original molecular mechanism responsible for negative control over P2X₃ receptor function and outlines a potential new target for trigeminal pain suppression.ATP-activated P2X₃ receptors are expressed almost exclusively by mammalian sensory neurons to play an important role in the transduction of painful stimuli to the central nervous system (1). Activation of P2X₃ receptors by ATP released during acute and chronic pain is thought to send nociceptive signals to central pain-related networks (2). In view of the multitude of environmental stimuli normally reaching sensory terminals, the question then arises how inappropriate activation of P2X₃ receptors is normally prevented. This process may contribute to suppression of continuous pain sensation in conjunction with central synaptic inhibition.The molecular pathways triggered by algogenic substances and responsible for modulating P2X₃ receptor structure and function remain incompletely understood. This topic is of particular interest because it can provide original clues for novel approaches related to treat pain. The nerve growth factor, NGF,² is one of the most powerful endogenous substances which elicit pain and inflammation via the tyrosine kinase receptor TrkA (3). This neurotrophin stimulates an intracellular cascade that elicits PKC-dependent P2X₃ receptor phosphorylation with ensuing facilitation of receptor currents. Conversely, suppression of NGF signaling powerfully down-regulates P2X₃ receptor function (4). These observations are consistent with the raised NGF levels in acute or inflammatory pain conditions (3). The molecular mechanisms underlying these effects remain, however, unclear.A dynamic balance between tyrosine phosphorylation and dephosphorylation is a major factor controlling the activity of many neurotransmitter receptors (5). TrkA stimulation activates intracellular signaling including Src tyrosine kinases (6) that, in neurons, are important modulators of ligand-gated receptors like nicotinic (7), NMDA receptors (8), and TRPV1 receptors (9). All these receptors are involved in mediating various types of pain in the spinal cord and sensory ganglia. There is, however, no available data on the role of tyrosine phosphorylation on P2X₃ receptor function.The fundamental regulator of Src signaling is the C-terminal Src kinase (Csk) that blocks it via tyrosine phosphorylation (Tyr-527, Refs. 10, 11). We explored whether tyrosine phosphorylation might regulate P2X₃ receptors of sensory neurons by focusing on the P2X₃ C-terminal domain Tyr-393 residue, which is included in a region with significant similarity with the Csk-phosphorylating region of Src. Our data demonstrate that Csk activation induced an increased tyrosine (Tyr-393 residue) P2X₃ receptor phosphorylation with decreased receptor function, observed both in mouse trigeminal sensory neurons as well as a cell expression system. We, thus, propose that Csk-mediated P2X₃ receptor inhibition is a novel mechanism to limit overactivation of P2X₃ receptors.