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

ABSTRACTGain-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), IP3 receptor, 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.AUTHOR SUMMARYPrior research has demonstrated that gain-of-function mutations in a gene important for neurotransmission (CACNA1A) are known to cause migraine in humans. We attempted to mimic some of those gain-of-function mutations in a simple genetic model organism and to examine neurotransmission by electrophysiology. Our findings yield potential clues as to how particular migraine-causing mutations may impact neurophysiology on a cellular level. We used the fruit fly Drosophila melanogaster and its model synapse, the neuromuscular junction (NMJ) to perform our studies. We document three main advances: 1) characterization of fruit fly models harboring gain-of-function calcium channel alterations known to cause human familial hemiplegic migraine type 1 (FHM1); 2) characterization of hyperactive neurotransmission caused by one of these alterations; and 3) an ability to quell hyperactive neurotransmission by impairing intracellular Ca2+ store release, through both genetic and pharmacological means. Our work contributes to a broader understanding of how pathological mutations could impact cellular physiology. More generally, the utilization of genetic model organisms promises to uncover potential ways to reverse those impacts.