Familial hemiplegic migraine Ca(v)2.1 channel mutation R192Q enhances ATP-gated P2X3 receptor activity of mouse sensory ganglion neurons mediating trigeminal pain.
BACKGROUND
The R192Q mutation of the CACNA1A gene, encoding for the α1 subunit of voltage-gated P/Q Ca2+ channels (Ca(v)2.1), 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.


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 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 Ca(v)2.1 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.


CONCLUSIONS
The 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.
Nair
Asha
A
Neurobiology Sector, International School for Advanced Studies (SISSA), Via Bonomea 265, 34136 Trieste, Italy.
Simonetti
Manuela
M
Birsa
Nicol
N
Ferrari
Michel D
MD
van den Maagdenberg
Arn M J M
AM
Giniatullin
Rashid
R
Nistri
Andrea
A
Fabbretti
Elsa
E
D000255:Adenosine Triphosphate / Q000378:metabolism; D060848:Agatoxins; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D019703:Calcineurin / Q000378:metabolism; D020864:Calcium Channels, N-Type; D020865:Calcium Channels, P-Type / Q000235:genetics* / Q000378:metabolism; D020907:Calcium Channels, Q-Type / Q000235:genetics* / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D017950:Ganglia, Sensory / Q000187:drug effects / Q000378:metabolism*; D055879:Gene Knock-In Techniques; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D020325:Migraine with Aura / Q000150:complications / Q000235:genetics* / Q000503:physiopathology; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D010146:Pain / Q000150:complications / Q000503:physiopathology*; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D058477:Receptors, Purinergic P2X3 / Q000378:metabolism*; D011984:Sensory Receptor Cells / Q000187:drug effects / Q000378:metabolism; D013111:Spider Venoms / Q000494:pharmacology; D014276:Trigeminal Nerve / Q000187:drug effects / Q000201:enzymology / Q000503:physiopathology
D000255:Adenosine Triphosphate; D060848:Agatoxins; D019703:Calcineurin; D020864:Calcium Channels, N-Type; D020865:Calcium Channels, P-Type; D020907:Calcium Channels, Q-Type; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D050505:Mutant Proteins; D010768:Phosphoserine; D011188:Potassium; D058477:Receptors, Purinergic P2X3; D013111:Spider Venoms; C060465:omega-agatoxin I