Familial hemiplegic migraine Ca _V2.1 channel mutation R192Q enhances ATP-gated P2X ₃ receptor activity of mouse sensory ganglion neurons mediating trigeminal pain

Abstract

Background

The R192Q mutation of the CACNA1A gene, encoding for the α1 subunit of voltage-gated P/Q Ca ²⁺ channels (Ca _v2.1), is associated with familial hemiplegic migraine-1. We investigated whether this gain-of-function mutation changed the structure and function of trigeminal neuron P2X ₃ receptors that are thought to be important contributors to migraine pain.

Results

Using in vitro trigeminal sensory neurons of a mouse genetic model knockin for the CACNA1A R192Q mutation, we performed patch clamp recording and intracellular Ca ²⁺ imaging that showed how these knockin ganglion neurons generated P2X ₃ receptor-mediated responses significantly larger than wt neurons. These enhanced effects were reversed by the Ca _v2.1 blocker ω-agatoxin. We, thus, explored intracellular signalling dependent on kinases and phosphatases to understand the molecular regulation of P2X ₃ 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 P2X ₃ phenotype. Although no significant difference in membrane expression of knockin receptors was found, serine phosphorylation of knockin P2X ₃ 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 P2X ₃ receptor responses of knockin neurons and increased their serine phosphorylation.

Conclusions

The present results suggest that the CACNA1A mutation conferred a novel molecular phenotype to P2X ₃ 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.