Ca ²⁺ /calmodulin-dependent protein kinase II potentiates ATP responses by promoting trafficking of P2X receptors

Abstract

To elucidate the functional link between Ca 2؉ ͞calmodulin protein kinase II (CaMKII) and P2X receptor activation, we studied the effects of electrical stimulation, such as occurs in injurious conditions, on P2X receptor-mediated ATP responses in primary sensory dorsal root ganglion neurons. We found that endogenously active CaMKII upregulates basal P2X3 receptor activity in dorsal root ganglion neurons. Electrical stimulation causes prolonged increases in ATP currents that lasts up to Ϸ45 min. In addition, the total and phosphorylated CaMKII are also up-regulated. The enhancement of ATP currents depends on Ca 2؉ A TP-gated P2X3 receptors are prominently expressed in primary sensory dorsal root ganglion (DRG) neurons (1-5). The activation of P2X3 receptors facilitates transmission of nociceptive signals from the periphery to the spinal cord (6). P2X receptors play a particularly important role in signaling cell damage. Inflammation and tissue injuries cause a large increase in the expression of P2X3 receptors (7) and the release of cytosolic ATP from damaged cells (8). These changes greatly enhance P2X receptor activity, thus contributing to sensitization of DRG neurons (7,8) and exaggerated responses to nonnoxious and noxious stimuli (allodynia and hyperalgesia) (9-12). It has been shown that protein kinases are activated after tissue injuries (13,14). However the functional link between kinases and P2X receptor activation is poorly understood. Among serine͞ threonine kinases, Ca 2ϩ -and calmodulin (CaM)-dependent protein kinase II (CaMKII) is of particular interest because CaMKII is located in Ϸ45% of rat DRG neurons and most of them are involved in the processing of nociceptive information (15-17). Here, we determine the role of CaMKII in modulation of P2X3 receptor activity after relative high-frequency (10 Hz) electrical stimulation, a condition mimicking the injurious state in DRG neurons. We found that electrical stimulation produces a sustained potentiation of ATP responses. The potentiation can be completely blocked by the CaMKII inhibitor, 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) (KN-93). Furthermore, electrical stimulation promotes the expression of P2X3 receptors in the membrane, which is mediated by CaMKII. Materials and MethodsElectrophyiology. All experiments were approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch and are in accordance with the guidelines of the National Institutes of Health and the International Association for the Study of Pain. The procedures used for cell dissociation and current recordings were the same as described in refs. 7 and 18. Briefly, L4-6 DRGs were taken from adult (27-34 days old) Sprague-Dawley rats and put into an ice-cold, oxygenated dissecting solution [130 mM NaCl͞5 mM KCl͞2 mM KH 2 PO 4 ͞1.5 mM CaCl 2 ͞6 mM MgSO 4 ͞10 mM glucose͞10 mM Hepes, pH 7.2 (osmolarity, 305 mosM)]. After a 30-min recovery period, the ganglia were transferred to the dissectin...

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