At many vertebrate synapses, presynaptic functions are tuned by expression of different Ca_v2 channels. Most invertebrate genomes contain only one Ca_v2 gene. The Drosophila Ca_v2 homolog, cacophony (cac), induces synaptic vesicle release at presynaptic active zones (AZs). We hypothesize that Drosophila cac functional diversity is enhanced by two mutually exclusive exon pairs that are not conserved in vertebrates, one in the voltage sensor and one in the loop binding Ca_β and G_βγ subunits. We find that alternative splicing in the voltage sensor affects channel activation voltage. Only the isoform with the higher activation voltage localizes to AZs at the glutamatergic Drosophila larval neuromuscular junction and is imperative for normal synapse function. By contrast, alternative splicing at the other alternative exon pair tunes multiple aspects of presynaptic function. While expression of one exon yields normal transmission, expression of the other reduces channel number in the AZ and thus release probability. This also abolishes presynaptic homeostatic plasticity. Moreover, reduced channel number affects short-term plasticity, which is 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 Ca_v2 gene.

We present near-atomic-resolution cryoEM structures of the mammalian voltage-gated potassium channel Kv1.2 in open, C-type inactivated, toxin-blocked and sodium-bound states at 3.2 Å, 2.5 Å, 3.2 Å, and 2.9 Å. These structures, all obtained at nominally zero membrane potential in detergent micelles, reveal distinct ion-occupancy patterns in the selectivity filter. The first two structures are very similar to those reported in the related Shaker channel and the much-studied Kv1.2–2.1 chimeric channel. On the other hand, two new structures show unexpected patterns of ion occupancy. First, the toxin α-Dendrotoxin, like Charybdotoxin, is seen to attach to the negatively-charged channel outer mouth, and a lysine residue penetrates into the selectivity filter, with the terminal amine coordinated by carbonyls, partially disrupting the outermost ion-binding site. In the remainder of the filter two densities of bound ions are observed, rather than three as observed with other toxin-blocked Kv channels. Second, a structure of Kv1.2 in Na⁺ solution does not show collapse or destabilization of the selectivity filter, but instead shows an intact selectivity filter with ion density in each binding site. We also attempted to image the C-type inactivated Kv1.2 W366F channel in Na⁺ solution, but the protein conformation was seen to be highly variable and only a low-resolution structure could be obtained. These findings present new insights into the stability of the selectivity filter and the mechanism of toxin block of this intensively studied, voltage-gated potassium channel.