Figure 4.

PKC and its calcium-binding domain are required for endocytosis at hippocampal synapses. A, B, Western blot of PKC_α, PKC_β, adaptor protein 2 (AP2), clathrin heavy chain (CHC), dynamin (Dyn), and β-actin in WT, PKC_α^−/− (A), and PKC_β^−/− (B) hippocampal culture. Results in A and B were repeated by 2–4 times. C, F_SypH traces (normalized to baseline, left) and Rate_decay (right) induced by Train_40Hz (bar) in WT (n = 14 experiments) or PKC_α^−/− (n = 28 experiments) hippocampal culture at 22–24°C. Data plotted as mean + SEM; *p < 0.05; **p < 0.01, t test (applies to all similar graphs). Throughout the study, each experiment contained 20–30 boutons; 1–3 experiments were taken from 1 culture; each culture was from 3–5 mice; each group was from 4–12 cultures. D, Applying MES solution (pH:5.5, bars) quenched F_SypH (mean + SEM) to a similar level (lowest dash line) before and after a 10 s train of stimuli in PKC_α^−/− boutons (n = 6 experiments, 22–24°C). ΔS, SypH at resting plasma membrane quenched by MES. E–G, Similar to C, but at 34–37°C (E), in PKC_β^−/− culture (F), or after a 10 s train at 20 Hz (G). E, WT, n = 6 experiments; PKC_α^−/−, n = 6. F, WT, n = 14; PKC_β^−/−, n = 5. G, WT, n = 16; PKC_α^−/−, n = 22. H, F_SypH traces and Rate_decay induced by Train_40Hz (bar) in WT boutons (n = 14), PKC_α^−/− boutons (PKC_α^−/−, n = 28), PKC_α^−/− boutons rescued with WT PKC_α (containing mCherry for recognition, PKC_α^−/−+PKC_α, n = 7), and in PKC_α^−/− boutons rescued with PKC_α^D/A and mCherry (PKC_α^−/−+PKC_α^D/A, n = 8). I, Protein sequence of PKC_α and PKC_α^D/A C2 domain. The Ca²⁺-coordinating aspartates of PKC_α (bold) were mutated to alanines (red) in PKC_α^D/A. J, We expressed PKC_α-GFP (left two panels) or PKC_α^D/A-GFP (right two panels) in HEK293T cells and monitored the subcellular distribution of the kinase. The Ca²⁺ ionophore, ionomycin (10 μm, 15 min), induced translocation of PKC_α-GFP toward the plasma membrane, but did not alter the intracellular distribution of PKC_α^D/A-GFP. Such results were observed in 3 experiments (each experiment had 2–3 cells). K, Left, PKC_α^−/− neurons rescued with WT PKC_α (containing mCherry for recognition, PKC_α^−/−+PKC_α), or with PKC_α^D/A and mCherry (PKC_α^−/−+PKC_α^D/A). Right, Fluorescence intensity of mCherry (F_mCherry) in PKC_α^−/−+PKC_α neurons (n = 10) and PKC_α^−/−+PKC_α^D/A neurons (n = 13). F_mCherry was measured from both soma and branches.