A Tripartite Interaction Among the Calcium Channel α(1)- and β-Subunits and F-Actin Increases the Readily Releasable Pool of Vesicles and Its Recovery After Depletion

Neurotransmitter release is initiated by the influx of Ca(2+) via voltage-gated calcium channels. The accessory β-subunit (Ca(V)β) of these channels shapes synaptic transmission by associating with the pore-forming subunit (Ca(V)α(1)) and up-regulating presynaptic calcium currents. Besides Ca(V)α(1,) Ca(V)β 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 Ca(V)β/F-actin association on synaptic transmission has not yet been explored. We here study how Ca(V)β(4), 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 Ca(V)β(4) 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 Ca(V)α(1). We found that exogenously expressed wild-type Ca(V)β(4) enhances spontaneous and depolarization-evoked excitatory postsynaptic currents (EPSCs) without altering synaptogenesis. Ca(V)β(4) 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 Ca(V)β(4) is abolished upon disruption of the actin cytoskeleton. The Ca(V)α(1) association-deficient Ca(V)β(4) 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 Ca(V)β(4) and F-actin also support the recruitment of SVs to the RRP in a Ca(V)α(1)-independent manner. Our studies show an emerging role of Ca(V)β 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.