Data_Sheet_1_A Tripartite Interaction Among the Calcium Channel α1- and β-Subunits and F-Actin Increases the Readily Releasable Pool of Vesicles and Its Recovery After Depletion.pdf

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.

神经递质释放由电压门控钙通道（voltage-gated calcium channels）介导的钙离子内流所启动。这类通道的辅助β亚基（CaVβ）可通过与成孔亚基（CaVα1）结合并上调突触前钙电流，从而调控突触传递过程。除CaVα1外，CaVβ还可与包括肌动蛋白丝（F-actin）在内的多种蛋白伴侣相互作用。已知这类肌动蛋白丝可在突触前终末与突触囊泡（synaptic vesicles, SVs）结合，并协助囊泡在不同囊泡池间转运，但目前尚未明确CaVβ与F-actin的结合对突触传递的调控作用。本研究针对哺乳动物脑内主要的钙通道β亚型CaVβ4，探究其与F-actin协同作用对体外培养海马神经元突触传递的调控机制。我们通过在肌动蛋白细胞骨架被药物破坏前后，分别检测外源性CaVβ4的作用效果，并对比野生型亚基与丧失CaVα1结合能力的双突变亚基的功能差异，解析钙通道依赖与非依赖的调控通路。实验结果显示，外源性表达的野生型CaVβ4可增强自发性与去极化诱发的兴奋性突触后电流（EPSCs），且不影响突触发生过程。CaVβ4可在静息状态下扩大突触囊泡易释放池（readily releasable pool, RRP）的容量，并加速囊泡耗尽后的恢复进程。CaVβ4诱导的神经递质释放增强效应可被肌动蛋白细胞骨架的破坏所阻断。无法结合CaVα1的CaVβ4突变体仍可与肌动蛋白丝结合，但既不会改变突触后反应，也不会影响易释放池的恢复时程。此外，该突变体仍保留增大易释放池容量的能力。上述结果表明，CaVβ4与F-actin的相互作用还可通过不依赖CaVα1的方式，促进突触囊泡向易释放池的招募过程。本研究揭示了CaVβ在调控突触囊泡成熟至预激活状态以及释放后囊泡补充过程中的全新功能。我们推测，该亚基在囊泡循环过程中可耦合胞吐与胞吞过程。