Abstract
α-Latrotoxin (αLTX) causes exhaustive release of neurotransmitters from nerve terminals in the absence of extracellular Ca(2+) (Ca(2+)(e)). To investigate the mechanisms underlying this effect, we loaded mouse neuromuscular junctions with BAPTA-AM. This membrane-permeable Ca(2+)-chelator demonstrates that Ca(2+)(e)-independent effects of αLTX require an increase in cytosolic Ca(2+) (Ca(2+)(cyt)). We also show that thapsigargin, which depletes Ca(2+) stores, induces neurotransmitter release, but inhibits the effect of αLTX. We then studied αLTX's effects on Ca(2+)(cyt) using neuroblastoma cells expressing signaling-capable or signaling-incapable variants of latrophilin-1, a G protein-coupled receptor of αLTX. Our results demonstrate that αLTX acts as a cation ionophore and a latrophilin agonist. In model cells at 0 Ca(2+)(e), αLTX forms membrane pores and allows the influx of Na(+); this reverses the Na(+)-Ca(2+) exchanger, leading to the release of stored Ca(2+) and inhibition of its extrusion. Concurrently, αLTX stimulates latrophilin signaling, which depletes a Ca(2+) store and induces transient opening of Ca(2+) channels in the plasmalemma that are sensitive to inhibitors of store-operated Ca(2+) entry. These results indicate that Ca(2+) release from intracellular stores and that Ca(2+) influx through latrophilin-activated store-operated Ca(2+) channels contributes to αLTX actions and may be involved in physiological control of neurotransmitter release at nerve terminals.