Targeting neurotransmitter release machinery to the active zones of presynaptic nerve terminals

Abstract

Neurotransmitter release requires the coordinated activity of multiple macromolecular complexes within the presynaptic terminal. For example, voltage-gated calcium channels (CaVs) generate calcium transients that drive synaptic vesicle exocytosis, and active zone proteins tether these vesicles nearby to the presynaptic membrane and ‘prime’ them into a fusion-competent state. For many such complexes, their overall importance to neurotransmitter release is well-defined, but the underlying molecular mechanisms and the functions of their individual components are not. Necessity experiments that perturb neurotransmitter release have yielded compelling insight into the structure-function of its protein machinery, but the mechanistic models they inform are complicated by the functional redundancy and interdependency among presynaptic proteins. Here, I instead reconstruct presynaptic function in release-deficient neurons using two complementary approaches to determine proteins and protein domains essential for neurotransmitter release. First, I re-targeted endogenous but non-synaptic proteins to the presynaptic terminal to define protein targeting mechanisms; and second, I generated an artificial secretory complex to define the minimal protein machinery necessary for fusion. In Chapter 2, I establish the intracellular CaV C-terminus as the primary determinant for CaV compartment targeting. I demonstrated that translocation of the C-terminus from presynaptic CaV2.1s onto somatodendritic CaV1.3s confers active zone targeting and function to CaV1.3s. In CaV2 triple-knockout neurons, chimeric CaV1.3s with CaV2.1 C-termini rescued neurotransmitter release and rendered it fully sensitive to CaV1 pharmacological blockade. Next, in Chapter 3, we constructed a minimal release-promoting protein comprising the auxiliary CaVβ4 subunit and the MUN and lipid-binding C2C domains of Munc-13 after genetically ablating the active zone in hippocampal synapses. This artificial protein primes synaptic vesicles for exocytosis and enables effective calcium triggering of release, thus bypassing the need for an intact active zone complex. Altogether, this work reveals a surprising simplicity to the presynaptic release machinery and the targeting interactions that localize it to the active zone – little is needed of its molecular and mechanistic complexity to achieve functional neurotransmitter release.