Paper

Specific presynaptic functions require distinct Drosophila Cav2 splice isoforms

Ryglewski, Stefanie, Kobler, Oliver, Rickert, Christof, Gottschalk, Daniel, Deneke, Lea, Duch, Carsten, Strauss, Julia, Bell, Christopher, Kern, Hanna, Heine, Martin, Kilo, Lukas, Jashar Arian

2024

Overview

The multiplicity of neural circuits that accommodate the sheer infinite number of computations conducted by brains requires diverse synapse and neuron types. At many vertebrate synapses release probability and other aspects of presynaptic function are tuned by different combinations of Cav2.1, Cav2.2, and Cav2.3 channels. By contrast, most invertebrate genomes contain only one Cav2 gene. The one Drosophila Cav2 homolog, cacophony (cac), localizes to presynaptic active zones (AZs) to induce synaptic vesicle release. We hypothesize that Drosophila cac functional diversity is enhanced by two specific exon pairs that are mutually exclusively spliced and not conserved in vertebrates, one in the voltage sensor and one in the intracellular loop containing the binding site(s) for Caβ and G-protein βγ subunits. We test our hypothesis by combining opto- and electrophysiological with neuroanatomical approaches at a fast glutamatergic model synapse, the Drosophila larval neuromuscular junction. We find that alternative splicing in the voltage sensor affects channel activation voltage and is imperative for normal synapse function. Only the isoform with the higher activation voltage localizes to the presynaptic AZ and mediates evoked release. Removal of these cac splice isoforms renders fast glutamatergic synapses non-functional. By contrast, alternative splicing at the other alternative exon that encodes the intracellular loop between the first and the second homologous repeats does not affect cac presynaptic AZ localization, but it tunes multiple aspects of presynaptic function. While expression of one exon yields normal transmission, expression of the other exon reduces channel number in the AZ and thus release probability. This also abolishes presynaptic homeostatic plasticity. Moreover, reduced channel number upon selective exon excision increases paired pulse ratios and the variability of synaptic depression during low frequency stimulation trains (1 and 10 Hz), and thus affects short term plasticity. Effects on short term plasticity can be rescued by increasing the external calcium concentration to match release probability to control. In sum, in Drosophila alternative splicing provides a mechanism to regulate different aspects of presynaptic functions with only one Cav2 gene.Competing Interest StatementThe authors have declared no competing interest.Footnotes* The revised manuscript provides additional data strengthening the claims we made. Particularly, excision of one of two mutually exclusive exon pairs encoding part of the voltage sensor of the Drosophila Cav2 channel cacophony leads to a lack of channels at the neuromuscular synapse and thus to lethality while the other mutually exclusive exon at this site does not participate in cacophony channels at this fast chemical synapse. Our new data support our interpretation that this finding is not due to effects such as protein degradation upon unintended effects during CRISPR mediated exon excision. In fact, we find cacophony protein using the exon that is not found in cacoophony channels at the neuromuscular synapse elsewhere in the nervous system.

• Publisher: Cold Spring Harbor Laboratory Press,Cold Spring Harbor Laboratory
• Subject: Alternative splicing / Calcium channels (voltage-gated) / CRISPR / Drosophila / Glutamatergic transmission / Homeostatic plasticity / Insects / Intracellular / Isoforms / Lethality / Localization / Nervous system / Neural networks / Neuroscience / Presynaptic plasticity / Proteins / Sensors / Synaptic depression / Synaptic plasticity