<i>C. elegans</i> Clarinet/CLA-1 recruits RIMB-1/RIM-binding protein and UNC-13 to orchestrate presynaptic neurotransmitter release.

Abstract

Synaptic transmission requires the coordinated activity of multiple synaptic proteins that are localized at the active zone (AZ). We previously identified a <i>Caenorhabditis elegans</i> protein named Clarinet (CLA-1) based on homology to the AZ proteins Piccolo, Rab3-interactingmolecule (RIM)/UNC-10 and Fife. At the neuromuscular junction (NMJ), <i>cla-1 null</i> mutants exhibit release defects that are greatly exacerbated in <i>cla-1;unc-10 double</i> mutants. To gain insights into the coordinated roles of CLA-1 and UNC-10, we examined the relative contributions of each to the function and organization of the AZ. Using a combination of electrophysiology, electron microscopy, and quantitative fluorescence imaging we explored the functional relationship of CLA-1 to other key AZ proteins including: RIM1, Cav2.1 channels, RIM1-binding protein, and Munc13 (<i>C. elegans</i> UNC-10, UNC-2, RIMB-1 and UNC-13, respectively). Our analyses show that CLA-1 acts in concert with UNC-10 to regulate UNC-2 calcium channel levels at the synapse via recruitment of RIMB-1. In addition, CLA-1 exerts a RIMB-1-independent role in the localization of the priming factor UNC-13. Thus <i>C. elegans</i> CLA-1/UNC-10 exhibit combinatorial effects that have overlapping design principles with other model organisms: RIM/RBP and RIM/ELKS in mouse and Fife/RIM and BRP/RBP in <i>Drosophila</i>. These data support a semiconserved arrangement of AZ scaffolding proteins that are necessary for the localization and activation of the fusion machinery within nanodomains for precise coupling to Ca²⁺ channels.