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 well-known FHM1 amino-acid substitutions S218L (SL) and R192Q (RQ) into transgenes of Drosophila melanogaster Cav2/cacophony. We expressed the transgenes panneuronally. Phenotypes were mild for RQ-expressing animals. By contrast, single mutant SL-and double mutant (DM) SL, RQ-expressing animals showed severe phenotypes, including sharply decreased viability. By electrophysiology, SL- and DM-expressing neuromuscular junctions (NMJs) exhibited enhanced evoked discharges, epileptiform supernumerary discharges when single electrical pulses were applied to the motor nerves, and a dramatic increase in the amplitudes and frequencies of spontaneous miniature 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 giant events were elicited by spontaneous presynaptic firing. Interestingly, gain-of-function electrophysiological phenotypes were markedly lessened after genetic 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 inhibition of intracellular Ca2+ store release produced similar effects. Our data suggest inhibition of intracellular Ca2+ signaling could counteract hyperexcitability induced by gain-of-function Cav2.1 amino-acid substitutions.
AUTHOR SUMMARY Prior research has demonstrated that gain-of-function mutations in a gene important for neurotransmission 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 examine neurotransmission by electrophysiology. Our findings yield potential clues as to how particular 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. Here 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, though 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.
