Familial Hemiplegic Migraine Ca_V2.1 Channel Mutation R192Q Enhances ATP-gated P2X₃ Receptor Activity of Mouse Sensory Ganglion Neurons Mediating Trigeminal Pain

Abstract

BackgroundThe R192Q mutation of the CACNA1A gene, encoding for the α1 subunit of voltage-gated P/Q Ca2+ channels (Cav2.1), is associated with familial hemiplegic migraine-1. We investigated whether this gain-of-function mutation changed the structure and function of trigeminal neuron P2X3 receptors that are thought to be important contributors to migraine pain.ResultsUsing in vitro trigeminal sensory neurons of a mouse genetic model knockin for the CACNA1A R192Q mutation, we performed patch clamp recording and intracellular Ca2+ imaging that showed how these knockin ganglion neurons generated P2X3 receptor-mediated responses significantly larger than wt neurons. These enhanced effects were reversed by the Cav2.1 blocker ω-agatoxin. We, thus, explored intracellular signalling dependent on kinases and phosphatases to understand the molecular regulation of P2X3 receptors of knockin neurons. In such cells we observed strong activation of CaMKII reversed by ω-agatoxin treatment. The CaMKII inhibitor KN-93 blocked CaMKII phosphorylation and the hyperesponsive P2X3 phenotype. Although no significant difference in membrane expression of knockin receptors was found, serine phosphorylation of knockin P2X3 receptors was constitutively decreased and restored by KN-93. No change in threonine or tyrosine phosphorylation was detected. Finally, pharmacological inhibitors of the phosphatase calcineurin normalized the enhanced P2X3 receptor responses of knockin neurons and increased their serine phosphorylation.ConclusionsThe present results suggest that the CACNA1A mutation conferred a novel molecular phenotype to P2X3 receptors of trigeminal ganglion neurons via CaMKII-dependent activation of calcineurin that selectively impaired the serine phosphorylation state of such receptors, thus potentiating their effects in transducing trigeminal nociception.

Nucleotide homeostasis and purinergic nociceptive signaling in rat meninges in migraine-like conditions

“…We showed previously CGRP induced upregulation of ATP-gated P2X3 receptors [21][22][23] and the inhibitory action of CGRP on nicotinic receptors [46] suggesting various effects of this neuropeptide related to migraine pathology. We also found a significantly enhanced P2X3-mediated signaling in trigeminal neurons of transgenic mouse expressing human mutated gene responsible for familial type migraine [16] and the inhibitory action of the popular anti-migraine agent naproxen on P2X3 receptors [47]. Consistent with the concept of ATP-driven migraine pain, we found now that CGRP increased the level of the pronociceptive nucleotides ATP and ADP in various parts of the trigeminal nociceptive system and increased responses to low concentrations of ATP.…”

“…This commonly observed opposite action of two purines suggests that both the concentration of released ATP and the kinetics of its breakdown play a pivotal role in setting a fine balance between pain and analgesia. In line with this view, the Bmigraine mouse^which expresses a mutated human gene associated with familial hemiplegic migraine type 1 has (i) enhanced responses to the stable ATP analogues acting via P2X3 receptors [16] and (ii) decreased susceptibility to inhibition by adenosine [17]. Activation of P2X3 receptors often associated with slowly desensitizing P2X2 receptors is underlying pronociceptive signaling in various tissues [18].…”

Calcitonin Gene-Related Peptide-Mediated Enhancement of Purinergic Neuron/Glia Communication by the Algogenic Factor Bradykinin in Mouse Trigeminal Ganglia from Wild-Type and R192Q Cav2.1 Knock-In Mice: Implications for Basic Mechanisms of Migraine Pain

“…Interestingly, neurons from Ca V 2.1 ␣1 R192Q mutant KI mice show P2X 3 receptor-mediated responses significantly larger than WT neurons (Nair et al, 2010), strengthening the link of this receptor channel with migraine pathophysiology. Thus far, eight P2Y receptor subtypes are known (named P2Y 1,2,4,6,11,12,13,14 ), with both neuronal and glial localization in all the central and peripheral stations of pain transducing systems (Burnstock, 2009).…”

“…A significant release of pro/anti-inflammatory cytokines is also observed (7), which could further contribute to cell-to-cell communication within the ganglion. A significantly higher basal and stimulated CGRP release, likely responsible for the upregulation of P2X 3 receptor signaling (Nair et al, 2010), together with an increased percentage of nucleotideresponding cells after exposure to BK were detected in trigeminal ganglia from Ca V 2.1 a1 R192Q mutant KI mice, which indicates the importance of generating and maintaining an enhanced proalgogenic tissue state in migraine (see Discussion for details).…”

P2Y purinergic receptors: New targets for analgesic and antimigraine drugs

“…A role for P2X3 receptors in migraine is further supported by the recent observations that P2X3 receptor segregation to lipid rafts is significantly increased in TG neurons in a genetic mouse model of migraine, the CACNA1A knock-in (KI) mice [21]. As a consequence, the increased amplitude of P2X3-mediated calcium responses and slower desensitization of the receptor are observed in KI mice than in wild-type (WT) mice [22]. The CACNA1A KI mice bear a gain-of-function mutation of the α subunit of the Ca v 2.1 neuronal calcium channel, which is typically detected in families suffering from familial hemiplegic migraine type 1 (FHM1), a severe and genetic form of migraine accompanied by hemiparesis [23].…”