Scaffolding Mechanisms Supporting Fast Neurotransmitter Release at the Presynaptic Active Zone

Abstract

Synaptic vesicle exocytosis from presynaptic nerve terminals occurs with extreme temporal and spatial precision, on the order of microseconds and nanometers. Within the presynaptic terminal, a protein complex known as the active zone enables this and serves as the site of vesicle fusion. Extensive research has identified individual protein components of the active zone and the important roles they play in controlling fusion. Active zone proteins are responsible for ‘docking’ vesicles to the plasma membrane, for generating fusion competent ‘readily releasable’ vesicles, and for positioning those vesicles in close proximity to Ca2+ channels to increase their release probability. Despite this functional knowledge, major questions remain about the structure of the active zone protein complex itself. First, the molecular mechanisms that establish and maintain the structural integrity of the active zone have remained unclear. Second, the scaffolding interactions that are responsible for localizing essential proteins to the active zone are incompletely understood. This work was designed to address these two questions using gene knockouts of important active zone protein families. I first analyzed the effects of knocking out ELKS, a protein family that may mediate active zone scaffolding. While these studies revealed no independent structural roles for ELKS, I found surprising diversity in the synaptic mechanisms that underlie release deficits in ELKS knockouts. In subsequent work, both ELKS and another active zone protein, RIM, were genetically removed. This resulted in near complete disassembly of the active zone protein complex, revealing surprising, strong scaffolding redundancy between ELKS and RIM. Finally, I determined how interactions between active zone proteins and presynaptic Ca2+ channels contribute to active zone scaffolding. Removing all CaV2 channels revealed that CaV2 channels themselves do not scaffold active zone proteins. Instead, multiple redundant interactions of the CaV2 C-terminus tether the channels to release sites. This work highlights a fundamental organizational principle of the active zone: highly redundant protein interactions ensure the structural integrity of the active zone. Such an organizational scheme can increase the functional reliability of this molecular machine and allow specific interactions and functions to be favored depending on the local context, enabling synapse-specific release properties.