Genetic dissection of the mechanisms and functions of post-transcriptional calcium channel modifications in Drosophila

• Applicant: Dr. Stefanie Ryglewski
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells
• Term: since 2015
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 275581320

Voltage gated calcium channels (VGCCs) play essential roles in all neuron-types and all sub-neuronal compartments, from synaptic vesicle (SV) release via shaping the action potential (AP) and dendritic computations to regulating activity dependent transcription and translation. In vertebrates, 10 genes categorized into three families (Cav1, Cav2, Cav3) encode the pore-forming subunit of VGCCs. VGCC functional diversity is augmented by association with accessory alpha2delta and beta subunits resulting in more than 100 possible VGCC complexes that are further diversified by alternative splicing. In Drosophila, only three genes, each of which being homologous to one entire family of vertebrate VGCCs, and a smaller number of accessory subunits orchestrate VGCC function. The Drosophila Cav2 homolog cacophony (cac) is expressed in all sub-neuronal compartments and mediates SV release, participates in dendritic input computation, shapes APs in axons, and mediates transcriptional control. Cac contains two mutually exclusive exon pairs, one in the voltage sensor and one in the intracellular linker with binding sites for VGCC beta and G-protein subunits. By employing the genetic tool kit and experimental advantages of the Drosophila model system, this proposal will test how cac differential splicing tunes Cav2 localization and properties to accommodate vastly different functions. In the previous funding period, we have shown that cac pairing with different alpha2delta subunits mediates neuronal compartment specific functions. To address the role of differential splicing for cac localization and function, we generated exon out mutants of tagged and untagged cac by CRISPR. We found differential splicing in the intracellular linker to affect short term and presynaptic homeostatic plasticity. Only one of the two exons in the voltage sensor is essential for fast glutamatergic synaptic transmission, while our preliminary data suggest that only the other exon is essential for graded synaptic transmission. Finally, in situ voltage clamp recordings indicate that differential splicing tunes channel properties. Based on our data, we now propose to (1) characterize cac splice isoform biophysical properties together with different accessory subunits expressed in a heterologous expression system. This will reveal how Cav2 differential splicing affects channel properties and interactions with accessory subunits. (2) We will test the hypothesis that cac alternative splicing tunes channel localization and function for fast versus grades SV release. This would imply a different solution than in vertebrate graded synapses, which often use Cav1 for SV release. To address the function of dendritic cac channels we will (3) unravel which cac splice isoforms participate in synaptic input integration in dendrites and how they shape input/output operation.

• DFG Programme: Research Grants