Protein Kinase C and Calmodulin Serve As Calcium Sensors for Calcium-Stimulated Endocytosis at Synapses

Abstract

Calcium influx triggers and facilitates endocytosis, which recycles vesicles and thus sustains synaptic transmission. Despite decades of studies, the underlying calcium sensor remained not well understood. Here, we examined two calcium binding proteins, protein kinase C (PKC) and calmodulin. Whether PKC is involved in endocytosis was unclear; whether calmodulin acts as a calcium sensor for endocytosis was neither clear, although calmodulin involvement in endocytosis had been suggested. We generated PKC (α or β-isoform) and calmodulin (calmodulin 2 gene) knock-out mice of either sex and measured endocytosis with capacitance measurements, pHluorin imaging and electron microscopy. We found that these knock-outs inhibited slow (∼10-30 s) and rapid (<∼3 s) endocytosis at large calyx-type calyces, and inhibited slow endocytosis and bulk endocytosis (forming large endosome-like structures) at small conventional hippocampal synapses, suggesting the involvement of PKC and calmodulin in three most common forms of endocytosis-the slow, rapid and bulk endocytosis. Inhibition of slow endocytosis in PKC or calmodulin 2 knock-out hippocampal synapses was rescued by overexpressing wild-type PKC or calmodulin, but not calcium-binding-deficient PKC or calmodulin mutant, respectively, suggesting that calcium stimulates endocytosis by binding with its calcium sensor PKC and calmodulin. PKC and calmodulin 2 knock-out inhibited calcium-dependent vesicle mobilization to the readily releasable pool, suggesting that PKC and calmodulin may mediate calcium-dependent facilitation of vesicle mobilization. These findings shed light on the molecular signaling link among calcium, endocytosis and vesicle mobilization that are crucial in maintaining synaptic transmission and neuronal network activity.SIGNIFICANCE STATEMENT Vesicle fusion releases neurotransmitters to mediate synaptic transmission. To sustain synaptic transmission, fused vesicles must be retrieved via endocytosis. Accumulating evidence suggests that calcium influx triggers synaptic vesicle endocytosis. However, how calcium triggers endocytosis is not well understood. Using genetic tools together with capacitance measurements, optical imaging and electron microscopy, we identified two calcium sensors, including protein kinase C (α and β isoforms) and calmodulin, for the most commonly observed forms of endocytosis: slow, rapid, and bulk. We also found that these two proteins are involved in calcium-dependent vesicle mobilization to the readily releasable pool. These results provide the molecular signaling link among calcium, endocytosis, and vesicle mobilization that are essential in sustaining synaptic transmission and neuronal network activity.

Keywords: calmodulin; capacitance measurement; electron microscopy; endocytosis; pHluorin imaging; protein kinase C.

Figure 1.

PKC knock-out mouse generation and immunostaining. A, Schematic illustration of the generation of PKC_β^−/− mice. PKC_β gene has five exons, three of which (3, 4, and 5) are shown. Targeted embryonic stem (ES) cells (Prkcb^{tm1a(EUCOMM)Wtsi} line EPD0233_5_F09) were obtained from The International Mouse Phenotyping Consortium and were injected into C57BL/6J blastocysts to generate chimeras. Chimeric mice were then bred with C57BL/6J to generate PKC_β targeted germline mice (PKC_β^+/loxP). PKC_β^+/loxP mice were bred with CMV-Cre mice (The Jackson Laboratory, 006054) to delete exon 4, generating PKC_β^−/+ mice, which were used to establish the PKC_β^−/− mouse line. Mouse genotypes were determined by PCR. B, Antibody staining of PKC_α and vGluT₁ in P9 WT and PKC_α^−/− calyces (“overlay”). C, Antibody staining of PKC_β and vGluT₁ in P9 WT and PKC_β^−/− calyces (“overlay”). D, Left, PKC_α immunostaining staining intensity (F_PKCα; a.u., arbitrary units; mean + SEM) in P7–P10 WT (14 calyces, 4 mice) and PKC_α^−/− calyces (15 calyces, 4 mice). **p < 0.01 (t test). Right, PKC_β immunostaining staining intensity (F_PKCβ) in P7–P10 WT (24 calyces, 3 mice) and PKC_β^−/− calyces (16 calyces, 3 mice). **p < 0.01 (t test).

Figure 2.

PKC_α or PKC_β knock-out inhibits slow endocytosis at calyces. A, Sampled I_Ca (left) and C_m (right) induced by depol_20ms (arrow) in a WT calyx. The red curve is a mono-exponential fit of the C_m decay with a τ of 19.1 s. (B) I_Ca (mean + SEM) and C_m (mean + SEM) induced by depol_20ms (arrow) in WT (black, 9 calyces, 9 mice, abbreviated as 9c/9m), PKC_α^−/− (14c/14m, red) and PKC_β^−/− (6c/6m, green) calyces. Data from P7–P10 mice at 22–24°C; SEM plotted every 2 ms for I_Ca and 1 s for C_m (applies to all similar graphs). C, Rate_decay, the capacitance jump (ΔC_m) and the I_Ca charge (QI_Ca) induced by depol_20ms in WT (9c/9m), PKC_α^−/− (14c/14m) and PKC_β^−/− (6c/6m) calyces (P7–P10, 22–24°C). Bar graphs are plotted as mean + SEM (applies to all bar graphs) *p < 0.05; **p < 0.01; t test compared with WT (applies to all bar graphs). D, E, Similar to B and C, respectively, except that the stimulus was 20 APe at 100 Hz (WT, 8c/8m; PKC_α^−/−, 10c/10m; PKC_β^−/−, 8c/8m; P7–P10, 22–24°C). F, G, C_m and Rate_decay (mean + SEM) induced by depol_20ms (arrow) in WT (black, 7c/7m) and PKC_α^−/− calyces at 34–37°C (F; WT, 7c/7m; PKC_α^−/−, 4c/4m; P7–P10 mice) or at P13–P14 mice (G: WT, 7c/7m; PKC_α^−/−, 9c/9m; 22–24°C). (H) C_m and Rate_decay (mean + SEM) induced by depol_20ms in control (Ctrl, 11c/11m) or in the presence of BIS (11c/11m) or PMA (8c/8m) at 22–24°C in P7–P10 mice.

Figure 3.

PKC_α or PKC_β knock-out inhibits rapid endocytosis and vesicle mobilization to the readily releasable pool at calyces. A–H, Similar arrangements as Figure 2A–H, respectively, except that the stimulus was depol_20msX10 (A–C, F–H) or 200 APe at 100 Hz (D,E) for inducing rapid endocytosis. A, Red curve is a biexponential fit of the C_m decay with a τ of 1.4 s and 14.3 s, respectively (I_Ca not shown). B, C, WT, 9c/9m; PKC_α^−/−, 14c/14m; PKC_β^−/−, 6c/6m. D, E, WT, 8c/8m; PKC_α^−/−, 11c/11m; PKC_β^−/−, 8c/8m. F, WT, 6c/6m; PKC_α^−/−, 4c/4m. G, WT, 7c/7m; PKC_α^−/−, 8c/8m. H, Ctrl, 11c/11m; BIS, 11c/11m; PMA, 8c/8m. I, Left, Sampled C_m induced by depol_20msX10 (each arrow: 1 depol_20ms) from WT, PKC_α^−/− and PKC_β^−/− calyces. ΔC_m induced by the first depol_20ms was normalized. Middle and right, ΔC_m (middle) and accumulated ΔC_m (ΣΔC_m, right) induced by each of the 10 depol_20ms during depol_20msX10 in WT (9c/9m), PKC_α^−/− (14c/14m) and PKC_β^−/− (6c/6m) calyces. Data (mean ± SEM) are normalized to ΔC_m induced by the first depol_20ms. ΣΔC_m was significantly higher for the WT group (*p < 0.05; **p < 0.01).

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.

Figure 5.

PKC_α knock-out affects endocytosis examined with EM at hippocampal synapses. A, EM images of WT and PKC_α^−/− hippocampal boutons at rest (Rest) and at 0 min (K⁺), 3 min and 10 min after 1.5 min 90 mm KCl application. For Rest, HRP was included for 1.5 min; for KCl application, HRP was included only during KCl application (see labels). B, C, Number of HRP(+) vesicles (B) and the bulk endosome area (C) 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 the end of KCl application in WT and PKC_α^−/− hippocampal cultures (mean + SEM, each group was from 100–132 synaptic profiles from 4–12 mice). The temperature before fixation was 22–24°C. ***p < 0.001; **p < 0.01; *p < 0.05 (t test). D, E, Similar to B and C, respectively, except that the temperature was 37°C before fixation.

Figure 6.

CaM 2 knock-out inhibits slow endocytosis, rapid endocytosis, and vesicle mobilization to the readily releasable pool at calyces. A, Generation of Calm2^loxP mice (Calm2: Calmodulin 2 gene). sgRNAs were designed by using CRISPR Design (https://zlab.bio/guide-design-resources) to identify unique target sites throughout the mouse genome. sgRNAs were transcribed in vitro using the MEGAshortscript T7 Transcription Kit (Life Technologies) from synthetic double-strand DNAs purchased from IDT (Integrated DNA Technologies) and purified using MEGAclear kit (Life Technologies). A mixture of Cas9 mRNA (TriLink Biotechnologies, 100 ng/μl), sgRNAs (50 ng/μl), and ssDNA templates (100 ng/μl, synthesized by IDT) was injected into the cytoplasm of one cell-stage fertilized embryos harvested from C57BL/6J mice (The Jackson Laboratory, 000664). Viable two-cell stage embryos were transferred into the oviducts of female surrogates to generate founder mice. Founders with loxP inserts were identified by PCR and sequencing, and were subsequently bred with C57BL/6J mice to generate heterozygous mice. The primers used to identify the 5′ and 3′ loxP insertions were Calm2 mtF: 5′-CCATGAACCTTGAACCTGTAGGATCCA-3′ and Calm2 mtR: 5′-ATGCTACATTCAACTTGTCACCATTCGAATTCA-3′. B, Top, Antibody staining of CaM and vGluT₁ (labeling calyx) in a P9 WT (upper) and a CaM₂^−/− (lower) calyx (images superimposed in the right). Bottom, CaM staining intensity (mean + SEM, a.u., arbitrary unit) in P7–P10 WT (47 calyces, 4 mice) and CaM₂^−/− calyces (53 calyces, 4 mice). **p < 0.01 (t test). C, I_Ca, C_m and Rate_decay (mean + SEM) induced by depol_20ms (arrow) in WT (8c/8m, black) and CaM₂^−/− (9c/9m) calyces (P7–P10, 22–24°C). D, Similar to C, but with depol_20msX10 (WT, 8c/8m; CaM₂^−/−, 9c/9m). E, F, Similar to C and D, respectively, but at 34–37°C. E, WT, 6c/6m; CaM₂^−/−, 6c/6m. F, WT, 6c/6m; CaM₂^−/−, 6c/6m. G, H, Similar to C and D, respectively, but from P13–P14 calyces. G, WT, 7c/7m; CaM₂^−/−, 8c/8m. H, WT, 7c/7m; CaM₂^−/−, 8c/8m.

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.