2. We also found presynaptic alterations in plasticity associated with Cdk5-mediated phosphorylation of CaV2.2 that included enhanced basal synaptic transmission, enhanced presynaptic release probability, and an overall reduction in short-term facilitation. Importantly, these effects were not observed with the Cdk5 phosphorylation mutant 8X CaV2.2, either alone or in the presence of Cdk5. Taken together, these studies demonstrate a pivotal role Apoptosis inhibitor for Cdk5-mediated posttranslational modifications of the N-type calcium channel in regulating

presynaptic function, and they highlight the close interaction between kinases and calcium channels in neurons. While this study shows that CaV2.2 is a Cdk5 substrate, previous work has implicated several kinases in the modulation of voltage-gated calcium channels (Bannister et al., 2005). The calcium-calmodulin kinase II (CaMKII) interacts with the P/Q-type calcium Selleckchem GDC 941 channel to facilitate transmitter release (Jiang et al., 2008), and the glycogen synthase kinase (GSK3β) phosphorylates the P/Q-type calcium channel in the intracellular II-III loop (Zhu et al., 2010) to inhibit vesicle exocytosis by disrupting SNARE complex formation. Other kinases that target CaV2.2

include protein kinases A and C (PKA and PKC), and both PKA- and PKC-mediated phosphorylation of CaV2.2 inhibit CaV2.2 interaction with SNARE complexes (Yokoyama et al., 1997). PKC-mediated phosphorylation of CaV2.2 also enhances N-type calcium current by reducing the G-protein inhibition of CaV2.2

(Swartz et al., 1993). Furthermore, PKC phosphorylation of CaV2.2 in the I-II linker region reduces the inhibitory effect of the Gβγ subunits on CaV2.2 (Zamponi et al., 1997). Notably, the CaV2.2 N-terminus, together with the I-II region, plays a fundamental role in modulating Gβγ inhibition (Agler et al., 2005). Altogether, these results provide a complex representation of signaling pathways involving kinases, second messengers such as Gβγ subunits, and synaptic release machinery such as SNARE proteins leading up to neurotransmitter release. Pharmacological inhibition of Cdk5 using roscovitine was previously used to examine much calcium channel function (Tomizawa et al., 2002). However, in addition to inhibiting Cdk5, roscovitine is an inhibitor of cyclin-dependent kinases 1, 2, 5, and 7 (Bach et al., 2005) and also acts directly on calcium channels by binding to the extracellular domain of L-type calcium channels (Yarotskyy and Elmslie, 2007). Furthermore, extracellular roscovitine application potentiates (P/Q-type) CaV2.1-mediated neurotransmitter release to slow deactivation kinetics (Yan et al., 2002) and increase the inactivation of CaV2.2 (Buraei et al., 2005).