Flower/FLWR-1 regulates neuronal activity via the plasma membrane Ca2+ ATPase to promote recycling of synaptic vesicles

The Flower protein was suggested to couple the fusion of synaptic vesicles (SVs) to their recycling in different model organisms. It is supposed to trigger activity-dependent bulk endocytosis by conducting Ca²⁺ at endocytic sites. However, this mode of action is debated. Here, we investigated the role of the <i>Caenorhabditis elegans</i> homologue FLWR-1 in neurotransmission. Our results confirm that FLWR-1 facilitates the recycling of SVs at the neuromuscular junction (NMJ). Ultrastructural analysis of synaptic boutons after hyperstimulation revealed an accumulation of large endocytic structures in <i>flwr-1</i> mutants. These findings do not support a role of FLWR-1 in the formation of bulk endosomes but rather a function in their breakdown. Unexpectedly, the loss of FLWR-1 led to increased neuronal Ca²⁺ levels in axon terminals during stimulation, particularly in GABAergic motor neurons, causing excitation-inhibition imbalance. We found that this increased NMJ transmission might be caused by deregulation of MCA-3, the nematode orthologue of the plasma membrane Ca²⁺ ATPase (PMCA). <i>In vivo</i> molecular interactions indicated that FLWR-1 may be a positive regulator of the PMCA and might influence its recycling through modification of plasma membrane levels of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂).