CAST/ELKS Proteins Control Voltage-Gated Ca2+ Channel Density and Synaptic Release Probability at a Mammalian Central Synapse

Abstract

In the presynaptic terminal, the magnitude and location of Ca entry through voltage-gated Ca channels (VGCCs) regulate the efficacy of neurotransmitter release. However, how presynaptic active zone proteins control mammalian VGCC levels and organization is unclear. To address this, we deleted the CAST/ELKS protein family at the calyx of Held, a Ca2.1 channel-exclusive presynaptic terminal. We found that loss of CAST/ELKS reduces the Ca2.1 current density with concomitant reductions in Ca2.1 channel numbers and clusters. Surprisingly, deletion of CAST/ELKS increases release probability while decreasing the readily releasable pool, with no change in active zone ultrastructure. In addition, Ca channel coupling is unchanged, but spontaneous release rates are elevated. Thus, our data identify distinct roles for CAST/ELKS as positive regulators of Ca2.1 channel density and suggest that they regulate release probability through a post-priming step that controls synaptic vesicle fusogenicity.

Impaired experience-dependent maternal care in presynaptic active zone protein CAST-deficient dams

“…Here, we first expected that neglect in CAST KO dams might be caused by impaired OXT release. Indeed, previous studies indicated an essential role for CAST in recruiting Ca 2+ channels that can trigger neurotransmitter release at the active zone 21,22,25,[41][42][43] , as well as the OXT release depends on Ca 2+ influx into the axon terminal 44,45 . Our findings showed that magnocellular neuron terminals in the posterior pituitary were intensely labelled for CAST and OXT (Fig.…”

“…Previous work has shown that mutant forms of CAST/ELKS homologs in C. elegans (ELKS) and Drosophila (bruchpilot) exhibit distinct phenotypes, indicating they are required for synapse formation in C. elegans and for the promotion of active zone assembly in Drosophila 19,20 . Furthermore, analysis of CAST and/or ELKS deficient mice indicated that these proteins affect calcium channel-associated release machinery in retinal photoreceptor neurons, hippocampal neurons, calyx of the Held/MNTB synapses, and pancreatic β cells [21][22][23][24][25][26] . However, despite the importance of presynaptic events, only a few studies have reported the contribution of presynaptic neurotransmitter release to maternal behaviour 2,27 .…”

Presynaptic development is controlled by the core active zone proteins CAST/ELKS

“…Genetic studies of mammalian retinal ribbon synapses in vivo which deleted both CAST/ELKS identified roles for CAST/ELKS regulation of synapse formation and AZ morphology (Hagiwara et al 2018). However, deletion of both CAST/ELKS at the mature calyx of Held in vivo, a large Ca V 2.1 exclusive glutamatergic presynapse with many conventional AZs in parallel (Baydyuk et al 2016), led to no changes in presynaptic morphology or AZ ultrastructure (Dong et al 2018). In addition, studies on in vitro cultured hippocampal neurons deleted for both CAST/ELKS found no changes in AZ ultrastructure in GABAergic neurons (Liu et al 2014) or in glutamatergic neurons (Held et al 2016).…”

“…Therefore, it is unclear if the roles of CAST/ELKS in regulating synapse development and AZ ultrastructure are unique to ribbon synapses, or whether these roles are confined to a specific developmental stage of synapse maturation. In addition, although a role for CAST/ELKS was found for regulating Ca V 2.1 current levels and channel numbers in a Ca V 2.1 exclusive presynaptic terminal (Dong et al 2018), it is not known if CAST/ELKS regulate all presynaptic Ca V 2 subtype levels or if they are solely Ca V 2.1-specific.…”

“…During early development synaptic transmission at the calyx of Held relies on three subtypes of Ca V 2 channels which makes it ideal for studying the effects of CAST/ELKS on specific Ca 2+ subtypes. We used Cast/Erc2 knockout (KO) (Cast −/− ) and Elk1s/Erc1 conditional knockout (CKO) transgenic mice (Dong et al 2018;Hagiwara et al 2018) in conjunction with our ability to conditionally ablate ELKS expression in the calyx of Held (Dong et al 2018) and demonstrated that ELKS was significantly reduced by P7 and completely ablated by P9. Using confocal and electron microscopy we found that combined loss of CAST and ELKS resulted in significant reductions in calyx size.…”