br Although earlier studies had reported conflicting results

Although earlier studies had reported conflicting results regarding the role of Ca2+ in regulating SV endocytosis, more recent data suggest that Ca2+ influx facilitates SV endocytosis in hippocampal neurons, the calyx of Held, and in inner hair lomitapide synthesis (Dittman and Ryan, 2009, Hosoi et al., 2009, Moser and Beutner, 2000, Sankaranarayanan and Ryan, 2001, Wu et al., 2014a, Wu et al., 2009, Wu et al., 2014b, Zefirov et al., 2006). Moreover, the endocytic rate constant correlates with the presynaptic Ca2+ current at the calyx of Held (Wu et al., 2009), and depletion of Ca2+ results in the accumulation of endocytic intermediates at reticulospinal synapses of the lamprey (Gad et al., 1998). As Ca2+ influx is mediated predominantly by AZ-localized voltage-dependent Ca2+ channels, the presynaptic AZ may provide a means for the spatiotemporal coupling of SV membrane endocytosis to the exocytic fusion process (Gundelfinger and Fejtova, 2012, Kittel et al., 2006b) − a scenario discussed in more detail below. Recent studies suggest that the Ca2+-binding protein calmodulin is a major Ca2+ sensor for endocytosis (Wu et al., 2009). Knockdown of calmodulin or application of calmodulin inhibitors (e.g. cyclosporine A or FK506) has been reported to impair endocytosis of SV membranes in several systems including the calyx of Held, although other studies have yielded conflicting results (see (Igarashi and Watanabe, 2007, Wu et al., 2014a) for review). Moreover, knockdown or knockout of the protein phosphatase calcineurin, a major downstream target of calmodulin, has been shown to inhibit various forms of SV endocytosis at the calyx of Held and at hippocampal synapses (Wu et al., 2014b). Active calmodulin-calcineurin dephosphorylates numerous endocytic proteins such as the GTPase dynamin 1 (Anggono et al., 2006, Lai et al., 1999), the inositol phosphatase synaptojanin 1, the phosphatidylinositol 4-phosphate 5-kinase Iγ (PIPKIγ), the ubiquitin-binding adaptors Eps15 and epsin, and the synaptobrevin-specific adaptor AP180, collectively referred to as dephosphins (Cousin and Robinson, 2001), thereby facilitating SV endocytosis and reformation (Fig. 1A). Calmodulin also activates myosin light chain kinase, which regulates the pool of fast-releasing SVs at the calyx of Held (Srinivasan et al., 2008) and has been suggested to facilitate SV endocytosis in response to high-frequency stimulation at mouse neuromuscular junctions (Maeno-Hikichi et al., 2011). Recently, calmodulin was shown to directly regulate endocytic bin-amphiphysin-rvs (BAR) domain-containing proteins like endophilin A thereby enhancing their membrane shaping activity (Myers et al., 2016). In addition to calmodulin and calcineurin the kinetics of SV endocytosis and/or reformation may be regulated by Ca2+ binding to the C2 domains of the SV protein synaptotagmin, a major Ca2+ sensor for SV exocytosis (Martin, 2015, Sudhof and Rothman, 2009, Wu et al., 2014a). Loss of synaptotagmin 1 in hippocampal neurons or Drosophila and C. elegans neuromuscular junctions slows SV endocytosis (Jorgensen et al., 1995, Littleton et al., 2001, Nicholson-Tomishima and Ryan, 2004, Poskanzer et al., 2003, Yao et al., 2011). A similar, though less pronounced endocytic phenotype has been reported for Ca2+-binding-defective mutant synaptotagmin 1 (Poskanzer et al., 2006, Yao et al., 2011). In chromaffin cells synaptotagmin 1 was shown to mediate the Ca2+ dependence of vesicle fission dynamics (Yao et al., 2012). The exact molecular mechanism by which synaptotagmin isoforms regulate SV endocytosis in neurons is unclear, but may relate to exocytic-endocytic coupling as asynchronous release of SVs in the absence of synaptotagmin 1 has been shown to selectively target fused SVs toward a slow retrieval mode facilitated by the asynchronous release Ca2+ sensor synaptotagmin 7 (Li et al., 2017). Synchronous fusion of SVs, thus, appears to generate signals that target fused SV membranes for fast endocytic retrieval.