Migraine is a common, disabling, multifactorial, episodic neurovascular disorder of largely unknown etiology. It affects more than 20% of the general population. Despite recent progress in drug therapy for preventing and treating it, migraine remains unsatisfactorily controlled in many patients. One problem that slows the development of new therapeutic approaches is our limited understanding of migraine basic pathophysiology. Recent studies suggest that ligand gated ion channels may be considered as novel targets for the development of future analgesic drugs. In particular, ATP-gated P2X3 receptors of sensory neurons are strongly implicated in mediating nociception and chronic pain in various animal models of neuropathic syndromes. Several reports suggest an important role of P2X3 receptors and their modulation in generating an attack of migraine and sensitizing trigeminal sensory neurons. There is, however, a dearth of information about the mechanisms regulating P2X3 receptors. The present work was, therefore, focused on certain aspects relevant to basic processes of migraine pain. Thus, I investigated P2X3 receptor compartmentalization in membrane lipid rafts of trigeminal neurons, the functional consequence of this subdomain segregation, and the changes detectable in a genetic mouse model of familial hemiplegic migraine (R192Q mutant KI mouse). In general, P2X3 receptors in lipid rafts produced stronger functional responses. In KI ganglion neurons even larger lipid raft expression of P2X3 receptors was observed, a result probably caused by constitutively higher calcium influx (reversed by ω-agatoxin pretreatment). To study interactors that facilitated location of P2X3 receptors in lipid rafts, I examined the role of the endogenous kinase CASK (Calcium/Calmodulin dependent serine protein kinase). CASK is known to be scaffolding protein operating in concert with CAMKII kinase. In wildtype neurons, CASK was coprecipitated with purified P2X3 receptors and preferentially expressed in lipid raft fractions. In R192Q KI trigeminal sensory neurons, higher level of membrane CASK expression and even stronger interaction with P2X3 receptors in lipid rafts was observed, a phenomenon dependent on intracellular calcium influx and CAMKII/calmodulin activity. CASK silencing experiments carried out on trigeminal sensory neurons demonstrated that CASK silenced neurons expressed less P2X3 receptor protein because of unopposed proteasomal degradation (reversed by proteasomal inhibitor MG-132). Rapid upregulation of P2X3 receptors by NGF or their downregulation by receptor desensitization was accompanied by parallel changes in CASK/P2X3 interaction. Silencing CASK not only decreased P2X3 membrane expression, but it also significantly depressed P2X3-mediated currents. These data provide new insights into the basic mechanisms of P2X3 receptor regulation and show novel changes in this protein expression and function in relation to a genetic migraine model.
|Titolo:||Molecular mechanisms regulating P2X3 receptors in trigeminal ganglia of wild type and migraine-model mice|
|Data di pubblicazione:||30-ott-2012|
|Appare nelle tipologie:||8.1 PhD thesis|