SPECIFIC ALTERNATIVE <EM >CACOPHONY</EM> SPLICE ISOFORMS ARE NECESSARY FOR FAST VERSUS GRADED SYNAPTIC TRANSMISSION IN <EM>DROSOPHILA</EM>

Transmitter release in chemical synapses is induced by calcium influx into the active zone through voltage gated calcium channels, whereby synapses of fast action potential (AP)-triggered and graded membrane potential-dependent vesicle release can be distinguished. While fast transmitter release in vertebrate central synapses is predominantly mediated by Cav2.1 and Cav2.2 channels, graded synaptic transmission in photoreceptors and cochlear hair cells is facilitated by specialized active zones and L-type Cav1 channels. In Drosophila, the sole Cav2 homologue Cacophony (Cac) mediates both fast transmission at the neuromuscular junction and graded transmission in the visual system. Thus, Cac channels must support both AP-triggered and graded membrane potential-dependent Ca²⁺ influx to meet the requirements of these specific synapses. This may be possible through alternatively spliced Cac isoforms and their auxiliary subunits.We reduced the isoform variability of 18 potential Cac isoforms using CRISPR/Cas9 by excising a single exon of a mutually exclusive exon pair, namely exon IS4A or IS4B, and generate exon-out mutants (Bell et al., 2025). By using immunocytochemical and electrophysiological methods, we demonstrate that Cac^IS4B isoforms are necessary for fast synaptic transmission at the NMJ, while graded transmission in photoreceptors is mediated by Cac^IS4A isoforms. We augment our findings by measuring whole cell Ca²⁺ currents of recordings of heterologously expressed exon-specific Cac isoforms in SF9 cell culture. Based on our data, we propose that differential expression of Cac isoforms containing either one or the other mutually exclusive exon provides functional diversity of Cacophony suited for fast versus graded synaptic transmission in Drosophila.