Dissecting the Forward Trafficking of Presynaptic Voltage Gated Calcium Channels

ABSTRACT Voltage-gated calcium channels (CaVs) are indispensable components of the presynaptic active zone because they directly drive synaptic transmission. However, the expression of CaVs within the presynaptic terminal presents a considerable trafficking dilemma. Presynaptic CaVs are synthesized at the cell body where they are often separated from their final destinations by hundreds of millimeters. CaVs en route to the presynaptic terminal must therefore navigate multiple trafficking checkpoints that restrict passage of misdirected cargoes including export from the Golgi apparatus, entry into the axon compartment, cargo capture at presynaptic terminals, and finally anchoring within the active zone. Further, all neuronal CaVs exhibit a significant degree of sequence and structural homology, including those that sort exclusively into somatic and dendritic compartments. In consequence, the mechanistic pathways that target CaVs specifically to the presynaptic terminals remain obscure, despite their central importance to neuronal activity. In this proposal I will focus on dissecting the trafficking of CaV2.1s, a major presynaptic CaV isoform. Recent studies have established the CaV2.1 C-terminus as a critical locus of targeting sequences. In addition to motifs that tether CaV2.1s to the active zone scaffold, my preliminary data reveals that the proximal region of the CaV2.1 C-terminus contains key sequences that are together necessary for presynaptic expression. Further, I show the compartment targeting of CaVs is determined primarily by its C-terminal sequences. I therefore hypothesize that the CaV2.1 proximal C-terminus is a key determinant for CaV2.1 active zone targeting and function. I will test this hypothesis through two specific aims. In aim 1, I will identify the trafficking checkpoints that require the proximal C-terminus. My preliminary data establish that mutations within the proximal C-terminus abolish presynaptic localization of CaV2.1s. My goal is to identify the specific trafficking checkpoint that is disrupted. I will evaluate how proximal C-terminus mutations affect the Golgi export, axon targeting, presynaptic capture, and active zone anchoring of CaV2.1 cargoes by visualizing CaV2.1s in the somas and axons of neurons and at the plasma membrane in cell lines. In aim 2, I will define the minimal CaV2.1 sequences for active zone localization and function. In preliminary experiments, I show that the transfer of key targeting sequences from CaV2.1 to CaV1 can redirect these channels from the soma and dendrites to the presynaptic active zone. I will generate further chimeric CaVs to isolate the trafficking functions of individual CaV2.1 sequences. I will comprehensively probe for rescue of synaptic transmission in neurons that re-express CaV chimeras by means STED microscopy, calcium imaging and electrophysiology. In summary, this proposal addresses a fundamental gap in our understanding of presynaptic CaV trafficking. The findings from these experiments will reveal novel regulatory mechanisms in CaV trafficking and they may in turn inform healthcare of neuropathies that stem from the aberrant expression and trafficking of presynaptic CaVs.