Regulation of presynaptic Ca2+ channel abundance at active zones through a balance of delivery and turnover

Abstract

Voltage-gated Ca2+ channels (VGCCs) mediate Ca2+ influx to trigger neurotransmitter release at specialized presynaptic sites termed active zones (AZs). The abundance of VGCCs at AZs regulates neurotransmitter release probability (Pr), a key presynaptic determinant of synaptic strength. Although biosynthesis, delivery and recycling cooperate to establish AZ VGCC abundance, experimentally isolating these distinct regulatory processes has been difficult. Here we describe how the AZ levels of Cacophony (Cac), the sole VGCC mediating synaptic transmission in Drosophila, are determined. We also analyzed the relationship between Cac, the conserved VGCC regulatory subunit α2δ, and the core AZ scaffold protein Bruchpilot (BRP) in establishing a functional AZ. We find Cac and BRP are independently regulated at growing AZs, as Cac is dispensable for AZ formation and structural maturation, and BRP abundance is not limiting for Cac accumulation. Additionally, AZs stop accumulating Cac after an initial growth phase, whereas BRP levels continue to increase given extended developmental time. AZ Cac is also buffered against moderate increases or decreases in biosynthesis, whereas BRP lacks this buffering. To probe mechanisms that determine AZ Cac abundance, intravital FRAP and Cac photoconversion were used to separately measure delivery and turnover at individual AZs over a multi-day period. Cac delivery occurs broadly across the AZ population, correlates with AZ size, and is rate-limited by α2δ. Although Cac does not undergo significant lateral transfer between neighboring AZs over the course of development, Cac removal from AZs does occur and is promoted by new Cac delivery, generating a cap on Cac accumulation at mature AZs. Together these findings reveal how Cac biosynthesis, synaptic delivery, and recycling set the abundance of VGCCs at individual AZs throughout synapse development and maintenance.

Physiologic and nanoscale distinctions define glutamatergic synapses in tonic vs phasic neurons

“…It would appear counterintuitive, that the "strong" phasic release sites would be represented by less scaffold protein in more compact AZ structures relative to "weak" tonic AZs. AZs represented by a greater presence of scaffold, such as BRP and RBP are typically interpreted as having an enhanced release probability at the fly NMJ (Cunningham et al, 2022;Gratz et al, 2019;Newman et al, 2017) However, our analysis highlights two key aspects of AZ nanostructure that likely contribute to enhanced release at phasic AZs despite their reduced size. First, while BRP and RBP scaffold proteins are indeed reduced at Is vs Ib AZs, Cac, the channel that directly gates Ca 2+ entry at AZs, is present in similar amounts.…”

Mechanisms controlling the trafficking, localization, and abundance of presynaptic Ca2+ channels

“…Indeed, Ca v 2.2 channels partially compensate in ca v 2.1 knockout mice at the mature calyx of Held, but Ca v 2.1 does not increase in ca v 2.2 mutant hippocampal neurons (Kim et al, 2001;Inchauspe et al, 2004;Ishikawa et al, 2005;Jeon et al, 2007). Additionally, Ca v 2 α1 subunit overexpression in dissociated rat neurons and Drosophila NMJs does not increase Ca v 2 levels at synapses, providing further support for a competition model (Hoppa et al, 2012;Cunningham et al, 2022).…”

“…Like Ca v ß, α2δ is required for proper surface expression of the channel. While the exact mechanism is unknown, this role of α2δ is likely performed by promoting forward trafficking rather than preventing channel endocytosis (Cantí et al, 2001;Dickman et al, 2008;Ly et al, 2008;Saheki and Bargmann, 2009;Hoppa et al, 2012;Cassidy et al, 2014;Cunningham et al, 2022). Understanding the structure and function of α2δ is of special importance due to its pharmacological targeting by the widely prescribed drugs gabapentin and pregabalin (Gee et al, 1996;Taylor et al, 2007;Eroglu et al, 2009;Dolphin, 2016).…”

“…Unlike mammalian CAST/ ELKS which is not always required for AZ morphology and structure (Dong et al, 2018), brp null mutants lack the AZ "T-bar" (Kittel et al, 2006;Fouquet et al, 2009). Despite this requirement for BRP in clustering VGCCs at the AZ, BRP is not a rate-limiting regulator of VGCC abundance, as ~35% reductions in AZ BRP have no impact on VGCC abundance and do not change single AP-evoked SV release (Müller et al, 2015;Cunningham et al, 2022).…”

“…Drosophila BRP is well situated to be a slot protein, as it binds directly to the Ca v 2 channel and is required for channel accumulation and stabilization at AZs (Fouquet et al, 2009;Ghelani et al, 2022). However, reductions in AZ BRP abundance have no effect on AZ VGCC levels, indicating this protein is likely not a ratelimiting regulator of VGCCs at AZs (Cunningham et al, 2022). RIMs and RBPs initially were compelling candidates for a slot protein because they both bind to VGCCs and are required (albeit redundantly at different synapses) for VGCC localization (Han et al, 2011;Kaeser et al, 2011;Liu et al, 2011;Graf et al, 2012;Jung et al, 2015;Oh et al, 2021).…”