Differential control of presynaptic CaMKII activation and translocation to active zones

Abstract

The release of neurotransmitters, neurotrophins, and neuropeptides is modulated by Ca(2+) mobilization from the endoplasmic reticulum (ER) and activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Furthermore, when neuronal cultures are subjected to prolonged depolarization, presynaptic CaMKII redistributes from the cytoplasm to accumulate near active zones (AZs), a process that is reminiscent of CaMKII translocation to the postsynaptic side of the synapse. However, it is not known how presynaptic CaMKII activation and translocation depend on neuronal activity and ER Ca(2+) release. Here these issues are addressed in Drosophila motoneuron terminals by imaging a fluorescent reporter of CaMKII activity and subcellular distribution. We report that neuronal excitation acts with ER Ca(2+) stores to induce CaMKII activation and translocation to a subset of AZs. Surprisingly, activation is slow, reflecting T286 autophosphorylation and the function of presynaptic ER ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs). Furthermore, translocation is not simply proportional to CaMKII activity, as T286 autophosphorylation promotes activation, but does not affect translocation. In contrast, RNA interference-induced knockdown of the AZ scaffold protein Bruchpilot disrupts CaMKII translocation without affecting activation. Finally, RyRs comparably stimulate both activation and translocation, but IP3Rs preferentially promote translocation. Thus, Ca(2+) provided by different presynaptic ER Ca(2+) release channels is not equivalent. These results suggest that presynaptic CaMKII activation depends on autophosphorylation and global Ca(2+) in the terminal, while translocation to AZs requires Ca(2+) microdomains generated by IP3Rs.

Figure 1.

Activation of CaMKII in synaptic boutons. A, Pseudo-color image showing the change in the CFP/YFP FRET ratio in a type Ib bouton induced by nerve stimulation for 15 s at 70 Hz. An increase in CaMKII activity is shown as a shift to warmer colors. Scale bar, 2 μm. B, Quantification of FRET responses to stimulation for 15 s at 70 Hz in control boutons (Con) (n = 8), control boutons treated with 10 μm KN92 (n = 5), 10 μm KN93 (n = 9), or 20 nm PLTX (n = 6), and in T305/306D mutants (n = 6). *p < 0.05, ***p < 0.001. C, FRET responses to 70 Hz nerve stimulation with 100 μm ryanodine (Ryan) (n = 6) or expression of RyR-targeted RNAi constructs RyR109631 (n = 7) and RyR10844R-3 (n = 11). Note that an increase in the plotted ratio corresponds to activation.

Figure 2.

Localization of CaMKII clusters and active zones. A, Wide-field fluorescence images of fixed preparations showing Camui localization before (Con) and after stimulation for 60 s at 70 Hz (Stim), BRP immunofluorescence detected with Nc82 antibody, and a superposition of CaMKII (green) and BRP (magenta) signals (Merged). B, Wide-field images of a live bouton showing CaMKII indicator (green) before (Con) and after stimulation (Stim), strawberry-labeled Brp-short (magenta, BRP), and merged superposition of CaMKII and BRP signals acquired after stimulation (Merged). Scale bars, 2 μm.

Figure 3.

Presynaptic CaMKII activity kinetics. A, CaMKII activation in response to stimulation at 30 Hz (n = 5), 50 Hz (n = 5), 70 Hz (n = 8), and 100 Hz (n = 7). B, Deactivation time course of CaMKII stimulated at 70 Hz for 15 s (n = 4) and 60 s (n = 8). For 15 s, τ = 5.38 s. For 60 s, τ = 8.93 s. Note persistent activity after 60 s stimulation. C, Time course of intracellular Ca²⁺ (gray trace shows mean from 10 experiments) and CaMKII activity in controls (closed circles, n = 8) and T286A mutants (open circles, n = 6) during 1 min of 70 Hz activity. Error bars indicate SEM.

Figure 4.

Activity-induced translocation of presynaptic CaMKII. A, Sequence of images of a type Ib bouton showing the localization of Camui before, during, and after stimulation at 70 Hz for 120 s (indicated by bar on left). Fluorescein optics were used. Scale bar, 2 μm. B, Appearance and aggregation of Camui puncta (green) adjacent to an AZ marked with the strawberry-tagged BRP-short (magenta). Scale bar, 0.5 μm. Only part of a bouton is shown. C, Appearance and growth of a Camui punctum near an AZ. Scale bar, 0.5 μm. Only part of a bouton is shown. D, Movement of Camui puncta. Three sequential time-lapse images taken 3 s apart were presented in different colors (red, green, and blue) and then superimposed so that immobile signal is white and motion is indicated by other colors. Scale bar, 2 μm. E, Time courses for three CaMKII clusters from a single bouton induced by 60 s stimulation at 70 Hz. F (a.u.), Punctum fluorescence in arbitrary units. F, G, Sequences of fluorescence images acquired through the cuticle of undissected larvae show CaMKII clustering (F) and dispersal (G) in different boutons. Scale bars, 2 μm. For all images, numbers show time in seconds.

Figure 5.

Presynaptic CaMKII regulation by the RyR and T286 autophosphorylation. A, FRET responses (open bars) after 33 s of 70 Hz stimulation, which produces peak activation in control boutons (Con) (n = 8), for boutons treated with 100 μm ryanodine (Ryan) (n = 6) and boutons expressing RyR-targeted RNAi constructs RyR 109631 (n = 7), and RyR 10844 (n = 11) are compared with C_max (closed bars) for Con (n = 11), Ryan (n = 8), RyR 109631 (n = 7), and RyR 10844 (n = 12). *p < 0.05, **p < 0.01, ***p < 0.001. B, C_max is plotted versus FRET at 30 s. The line and 95% confidence limits (dashed lines) were generated by linear regression between Con, DMSO, and KN92 data points (upper right black circles) and unstimulated (Unstim) and PLTX-treated data points (lower left black circles). Red circles show data from T286A and KN93, which inhibit autophosphorylation. Note that inhibiting RyRs (blue circles) produces results that fall near the line, but that inhibiting autophosphorylation produces results in the upper left quadrant beyond the confidence limits.

Figure 6.

Inhibition of activity-induced CaMKII translocation by RNAi-induced knockdown of BRP. A, BRP immunofluorescence in control boutons and boutons expressing RNAi BRP 25891. Similar results were obtained with BRP 107748. Scale bar, 2 μm. Gray line shows terminal profile. B, FRET responses at 30 s (white bars) and C_max (black bars) in control boutons (Con, n = 8) and boutons expressing BRP RNAi constructs BRP25891 (n = 9) and BRP107748 (n = 8). ***p < 0.001. FRET responses did not change significantly with BRP knockdown, but C_max was reduced significantly.

Figure 7.

Regulation of CaMKII activation and translocation by IP3Rs. A, FRET responses in the presence of 0.1% DMSO (n = 8), 20 μm thapsigargin in DMSO (Tg, n = 5), 0.1 μm xestospongin C in DMSO (Xesto, n = 9), or with neuronal expression of IP3R RNAi (n = 10). B, FRET induced by 30 s of stimulation at 70 Hz (white bars) is compared with C_max. *p < 0.05, ***p < 0.001. C, C_max versus FRET plot showing that IP3R inhibition (shaded triangles) or BRP knockdown (open circles) produces results that fall in the lower right quadrant. Thus, IP3Rs and BRP preferentially affect CaMKII translocation.

Figure 8.

Model of CaMKII activation and translocation in the nerve terminal. Global Ca²⁺ from Ca_v channels, IP3Rs, and RyRs combine to activate presynaptic CaMKII (red arrows). Then, CaMKII dodecamers cluster near BRP-containing AZs (black arrows), where Ca²⁺ microdomains from Ca_v channels and ER IP3Rs are integrated.