Figure 7.

Calmodulin and its calcium binding domain are required for endocytosis at hippocampal synapses. A, Western blot of CaM, AP2, clathrin heavy chain (CHC), dynamin (Dyn), and β-actin in WT and CaM₂^−/− brain. B, CaM Western blot intensity (CaM Int, a.u.) from WT or CaM₂^−/− culture. C, F_SypH traces (normalized to baseline) and Rate_decay induced by Train_40Hz (bar) in WT (n = 14 experiments) or CaM₂^−/− (n = 8) hippocampal culture at 22–24°C (mean + SEM). D, Similar to C, but at 34–37°C (WT, n = 6; CaM₂^−/−, n = 4). E, Similar to C, but after a 10 s train at 20 Hz (WT, n = 16; CaM₂^−/−, n = 7). F, F_SypH traces and Rate_decay induced by Train_40Hz in WT hippocampal boutons (n = 14 experiments, with SypH transfection), CaM₂^−/− boutons (n = 8, with SypH transfection), CaM₂^−/− boutons transfected with a plasmid containing CaM and mCherry (mCherry for recognition, SypH was cotransfected, n = 4, CaM₂^−/−+CaM), and CaM₂^−/− boutons transfected with a plasmid containing CaM₁₂₃₄ and mCherry (n = 4, CaM₂^−/−+M). Temperature was 22–24°C. G, EM images of WT and CaM₂^−/− hippocampal boutons at rest (Rest) and at 0 min (K⁺), 3 min and 10 min after 1.5 min application of KCl and HRP (same arrangements as in Fig. 5A). H, I, The number of HRP(+) vesicles (H) and the bulk endosome area (I) per square micrometer of synaptic cross-section are plotted versus the time before (Rest) and at 0 min (K⁺), 3 min, and 10 min after KCl/HRP application in WT and CaM₂^−/− hippocampal cultures (22–24°C). Data are expressed as mean + SEM; each group was from 100–132 synaptic profiles from 4–12 mice. J, K, Similar to H and I, respectively, except that the temperature was 37°C.