Drosophila CaV2 channels harboring human migraine mutations cause synapse hyperexcitability that can be suppressed by inhibition of a Ca2+store release pathway

Abstract

Gain-of-function mutations in the human CaV2.1 gene CACNA1A cause familial hemiplegic migraine type 1 (FHM1). To characterize cellular problems potentially triggered by CaV2.1 gains of function, we engineered mutations encoding FHM1 amino-acid substitutions S218L (SL) and R192Q (RQ) into transgenes of Drosophila melanogaster CaV2/cacophony. We expressed the transgenes pan-neuronally. Phenotypes were mild for RQ-expressing animals. By contrast, single mutant SL- and complex allele RQ,SL-expressing animals showed overt phenotypes, including sharply decreased viability. By electrophysiology, SL- and RQ,SL-expressing neuromuscular junctions (NMJs) exhibited enhanced evoked discharges, supernumerary discharges, and an increase in the amplitudes and frequencies of spontaneous events. Some spontaneous events were gigantic (10–40 mV), multi-quantal events. Gigantic spontaneous events were eliminated by application of TTX–or by lowered or chelated Ca2+–suggesting that gigantic events were elicited by spontaneous nerve firing. A follow-up genetic approach revealed that some neuronal hyperexcitability phenotypes were reversed after knockdown or mutation of Drosophila homologs of phospholipase Cβ (PLCβ), IP3receptor, or ryanodine receptor (RyR)–all factors known to mediate Ca2+release from intracellular stores. Pharmacological inhibitors of intracellular Ca2+store release produced similar effects. Interestingly, however, the decreased viability phenotype was not reversed by genetic impairment of intracellular Ca2+release factors. On a cellular level, our data suggest inhibition of signaling that triggers intracellular Ca2+release could counteract hyperexcitability induced by gains of CaV2.1 function.