Abstract
Neurotransmitter release is initiated by the influx of Ca2+ via voltage-gated calcium channels. The accessory β-subunit (CaVβ) of these channels shapes synaptic transmission by associating with the pore-forming subunit (CaVα1) and up-regulating presynaptic calcium currents. Besides CaVα1, CaVβ interacts with several partners including actin filaments (F-actin). These filaments are known to associate with synaptic vesicles (SVs) at the presynaptic terminals and support their translocation within different pools, but the role of CaVβ/F-actin association on synaptic transmission has not yet been explored. We here study how CaVβ4, the major calcium channel β isoform in mamalian brain, modifies synaptic transmission in concert with F-actin in cultured hippocampal neurons. We analyzed the effect of exogenous CaVβ4 before and after pharmacological disruption of the actin cytoskeleton and dissected calcium channel-dependent and -independent functions by comparing the effects of the wild-type subunit with the one bearing a double mutation that impairs binding to CaVα1. We found that exogenously expressed wild-type CaVβ4 enhances spontaneous and depolarization-evoked excitatory postsynaptic currents (EPSCs) without altering synaptogenesis. CaVβ4 increases the size of the readily releasable pool (RRP) of SVs at resting conditions and accelerates their recovery after depletion. The enhanced neurotransmitter release induced by CaVβ4 is abolished upon disruption of the actin cytoskeleton. The CaVα1 association-deficient CaVβ4 mutant associates with actin filaments, but neither alters postsynaptic responses nor the time course of the RRP recovery. Furthermore, this mutant protein preserves the ability to increase the RRP size. These results indicate that the interplay between CaVβ4 and F-actin also support the recruitment of SVs to the RRP in a CaVα1-independent manner. Our studies show an emerging role of CaVβ in determining SV maturation toward the priming state and its replenishment after release. We envision that this subunit plays a role in coupling exocytosis to endocytosis during the vesicle cycle.
Highlights
Calcium entry through CaV2.x high-voltage activated calcium channels is a pivotal step during action potential-evoked neurotransmitter release and synaptic plasticity (Wheeler et al, 1994; Cao and Tsien, 2010; Simms and Zamponi, 2014; Nanou and Catterall, 2018)
The CaVβ family belongs to the membrane-associated guanylate kinases (MAGUKs) class of scaffolding proteins encompassing two highly conserved domains, a Src 3 homology (SH3) domain and a guanylate kinase (GK) domain that are flanked by variable regions (Chen et al, 2004; Opatowsky et al, 2004; Van Petegem et al, 2004)
We examined if the interaction of CaVβ with F-actin and CaVα1 are mutually exclusive or not
Summary
Calcium entry through CaV2.x high-voltage activated calcium channels is a pivotal step during action potential-evoked neurotransmitter release and synaptic plasticity (Wheeler et al, 1994; Cao and Tsien, 2010; Simms and Zamponi, 2014; Nanou and Catterall, 2018). The CaV2.x calcium channel core complex in the mammalian brain is composed of one CaVα1 and one CaVβ subunit (Müller et al, 2010). The four CaVβ isoforms described until now, CaVβ1 to CaVβ4, associate with a highly conserved sequence among the high-voltage activated CaVα1 referred to as α1 interaction domain (AID) that is located within the intracellular loop joining the transmembrane domains I and II (Pragnell et al, 1994). In mouse hippocampal neurons exogenously expressed CaVβ4, the predominant CaVβ isoform associated with the CaV2.x core complex (Müller et al, 2010), increases neurotransmitter release via slowing down voltage-dependent inactivation and promoting CaV2.x channel cell surface expression (Wittemann et al, 2000; Xie et al, 2007; Etemad et al, 2014)
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