(2011) showed that the RIM1 and RIM2 PDZ domains directly bind to the C-terminal sequences of the P/Q- and N-type Ca2+ channel α subunits and that the PDZ domain was required to rescue the decreased Ca2+ transients and the abnormal Ca2+ channel localization. In addition, the proline-rich domain of RIM1/2 was also necessary for the rescue, suggesting that a tripartite interaction between RIM1/2, RIM-BPs (which bind to the proline-rich

domain of RIMs; Hibino et al., 2002) and the Ca2+ channel α subunits is critical for holding Ca2+ channel at the active zone. This, together with the present work, identifies RIM1/2 as a presynaptic scaffolding proteins with a clear role in maintaining a high Ca2+ channel density at the presynaptic active zone. Previous work

has suggested a role for Bruchpilot, a Drosophila gene related to ELKS/CAST proteins, in maintaining presynaptic Ca2+ selleck chemicals channel density and structural entirety at Drosophila presynaptic active zones ( Kittel et al., 2006). Conditional removal of RIM1/2 did not significantly change the relative contribution of P/Q-type and N-type channels to the total presynaptic Ca2+ current, despite a strongly reduced total Ca2+ current. Anti-infection Compound Library This finding is different to the situation in P/Q-type (α1A subunit) KO mice (Jun et al., 1999), in which a strong compensatory upregulation of presynaptic N-type channels was observed at the calyx of Held (Inchauspe et al., 2004 and Ishikawa et al., 2005). Thus, in the absence of RIM1/2, N-type Ca2+ channels are not capable of compensating for the missing P/Q-type channels; therefore, the absence of RIM1/2 probably affects both Ca2+ channel α subunits equally. The calyx synapse has been an ideal preparation to functionally define a fast and a slow subpool of the readily releasable pool Org 27569 (Sakaba and Neher, 2001, Wölfel et al., 2007 and Wadel et al., 2007; see Neher, 2006 for a review). Here, we find that conditional removal of all major RIM isoforms leads to a strong, ∼70% decrease of the readily releasable pool size as defined by various types of pool-depleting Ca2+ stimuli (Figure 3, Figure 4 and Figure 5).

Importantly, we found a very similar decrease of the number of docked vesicles at the active zone (by ∼70%; Figure 6), demonstrating a genetic manipulation that leads to a parallel decrease in the number of docked vesicles and of the readily releasable pool size determined functionally. Thus, at the calyx of Held, the amount of vesicle docking seems to determine the size of the readily releasable pool. This conclusion is further supported by the reasonable quantitative agreement between the number of docked vesicles on the one hand (∼seven docked vesicle per active zone ∗ 500 ≈3500; assuming that a calyx has ∼500 active zones; Sätzler et al., 2002 and Taschenberger et al., 2002), and the sum of FRP and SRP vesicles on the other hand (∼3000–3500; see Figure 5).