Frequenin/NCS-1 and the Ca²⁺-channel ₁-subunit co-regulate synaptic transmission and nerve-terminal growth

Drosophila Frequenin (Frq) and its mammalian and worm homologue, NCS-1, are Ca²⁺-binding proteins involved in neurotransmission. Using site-specific recombination in Drosophila, we created two deletions that removed the entire frq1 gene and part of the frq2 gene, resulting in no detectable Frq protein. Frq-null mutants were viable, but had defects in larval locomotion, deficient synaptic transmission, impaired Ca²⁺ entry and enhanced nerve-terminal growth. The impaired Ca²⁺ entry was sufficient to account for reduced neurotransmitter release. We hypothesized that Frq either modulates Ca²⁺ channels, or that it regulates the PI4K pathway as described in other organisms. To determine whether Frq interacts with PI4K with consequent effects on Ca²⁺ channels, we first characterized a PI4K-null mutant and found that PI4K was dispensable for synaptic transmission and nerve-terminal growth. Frq gain-of-function phenotypes remained present in a PI4K-null background. We conclude that the effects of Frq are not due to an interaction with PI4K. Using flies that were trans-heterozygous for a null frq allele and a null cacophony (encoding the ₁-subunit of voltage-gated Ca²⁺ channels) allele, we show a synergistic effect between these proteins in neurotransmitter release. Gain-of-function Frq phenotypes were rescued by a hypomorphic cacophony mutation. Overall, Frq modulates Ca²⁺ entry through a functional interaction with the ₁ voltage-gated Ca²⁺-channel subunit; this interaction regulates neurotransmission and nerve-terminal growth.