GSK-3 beta inhibits presynaptic vesicle exocytosis by phosphorylating P/Q-type calcium channel and interrupting SNARE complex formation

Abstract

Glycogen synthase kinase-3 (GSK-3), a Ser/Thr protein kinase abundantly expressed in neurons, plays diverse functions in physiological and neurodegenerative conditions. Our recent study shows that upregulation of GSK-3 suppresses long-term potentiation and presynaptic release of glutamate; however, the underlying mechanism is elusive. Here, we show that activation of GSK-3beta retards the synaptic vesicle exocytosis in response to membrane depolarization. Using calcium imaging, whole-cell patch-clamp, as well as specific Ca(2+) channel inhibitors, we demonstrate that GSK-3beta phosphorylates the intracellular loop-connecting domains II and III (L(II-III)) of P/Q-type Ca(2+) channels, which leads to a decrease of intracellular Ca(2+) rise through the P/Q-type voltage-dependent calcium channel. To further illustrate the mechanisms of GSK-3beta's action, we show that activation of GSK-3beta interferes with the formation of the soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) complex through: (1) weakening the association of synaptobrevin with SNAP25 and syntaxin; (2) reducing the interactions among the phosphorylated L(II-III) and synaptotagmin, SNAP25, and syntaxin; and (3) inhibiting dissociation of synaptobrevin from synaptophysin I. These results indicate that GSK-3beta negatively regulates synaptic vesicle fusion events via interfering with Ca(2+)-dependent SNARE complex formation.

Figure 1.

A–H, GSK-3β inhibits presynaptic exocytosis. K⁺-evoked release of FM4-64 was recorded by confocal microscope. Dissociated hippocampal neurons transfected with EGFP-vector or EGFP-dnGSK-3β or EGFP-wtGSK-3β were measured by decline of FM4-64 for the vesicle release at the axon terminals (boxes). A, C, E, The images were captured before (Pre) and 5 min after (Post) K⁺ treatment. Scale bar, 50 μm. B, D, F, The kinetic curves were recorded from ∼100 s before to ∼5 min after K⁺ stimulation. G, H, Single-exponential decay functions were fitted to the diagrams with fluorescence intensity changes of FM4-64 analyzed by ImageJ software (G) and the time constant τ (H). I–L, Mean ± SD, ANOVA, Student's t test; **p < 0.01 versus EGFP; ##p < 0.01 versus wtGSK (n = 10∼12). I, J, K⁺-evoked release of pHluorin-Syb recorded by TIRFM: N2a cells were cotransfected with pHluorin-Syb and pcDNA3.0 (I) or wtGSK-3β (J) for 24 h. I, J, Consecutive images with enhanced fluorescence intensity after K⁺ stimulation were collected and amplified (from the boxes). Scale bar: 10 μm; 1 μm for amplification. K, L, The intensity of pHluorin was plotted and fitted to an exponential growth curve. The results show that overexpression of wtGSK-3β retards the K⁺-stimulated exocytosis.

Figure 2.

A, B, GSK-3β inhibits high voltage-dependent Ca²⁺ influx. Calcium imaging: the hippocampal neurons with (arrows) or without (arrowheads) expression of wtGSK-3β were loaded with 10 μm Fluo3Am, the images (A) and fluorescence intensity at the axons (B) were recorded from ∼2 min before (Pre) to 3 min after (Post) the K⁺ stimulation. C, D, The Ca²⁺ current recorded by patch clamp: neurons were cultured for 8 DIV and then treated with Wort, SB, or vehicle (DMSO) 30 min before recording. The representative Ca²⁺ current (C) and the I–V curves were analyzed by EPC 10 (D). The results show that upregulation of GSK-3β suppresses the calcium influx.

Figure 3.

GSK-3β inhibits Ca²⁺ influx through P/Q-type calcium channels. A, Calcium images were recorded as described in Figure 2, except that the P/Q-type (Aga) or N-type (CgTx) Ca²⁺ channel inhibitors were added 30 min before recording. The Fluo3Am increment (ΔF) were calculated by subtracting the preimage from the postimage of Fluo3Am and transferred into the fluorescence gradient images using Image Pro Plus 4.5 and LSM510. Scale bar, 20 μm. B, C, Quantitative analysis of the relative increment. ΔF/Δt (F, fluorescence intensity; t, time). The results show that only inhibition of P/Q-type calcium channel retards the dnGSK-3β-induced calcium influx. Mean ± SD, ANOVA, Student's t test; **p < 0.01 untransfected versus dnGSK-3β-transfected; ^## p < 0.01 DMSO versus inhibitors in untransfected neurons; ^$$ p < 0.01 DMSO versus inhibitors in dnGSK-3β-transfected neurons (n = 6).

Figure 4.

GSK-3β phosphorylates P/Q-type calcium channels. A, In vitro phosphorylation of L_II-III by GSK-3β: the purified L_II-III (20 μg) was incubated with GSK-3β in the presence of 100 μm [γ-³²P]ATP for autoradiography and Western blotting with the antibodies against GSK-3β, L_II-III, pThr, and phosphoserine (pSer), respectively (N= rat brain cortex extract). B, Phosphorylation of L_II-III by GSK-3 in synaptosome: the purified synaptosome (P2) from rat brain hippocampi was incubated with DMSO, Wort, or SB, or Wort plus SB for 30 min, then the suspension was immunoprecipitated with anti-L_II-III antibody and the supernatant (S), and the pellets were probed by anti-pThr or anti-L_II-III (M = marker). X = 20. Note that GSK-3β can phosphorylate P/Q-type calcium channel at Thr residues.

Figure 5.

A, B, GSK-3 disrupts the interactions of synaptic proteins. GSK-3 activation inhibits association of Syb with SNAP25 and syntaxin: the purified hippocampal synaptosome (P2) was incubated with DMSO, or Wort, or SB, or Wort plus SB for 30 min, then immunoprecipitated with anti-Syb and probed by anti-SNAP25, syntaxin Ia, and Syb (A) and quantitative analysis (B). Mean ± SD, ANOVA, Student's t test; **p < 0.01 versus DMSO; ##p < 0.01 versus Wort (n = 3). C, D, Phosphorylated L_II-III by GSK-3β is incompetent in association with synaptotagmin, SNAP25, and syntaxin: purified L_II-III peptide was phosphorylated by GSK-3β and then coimmunoprecipitated with P2 fraction for Western blotting (C) and quantitative analysis (D). E–G, GSK-3 activation hinders dissociation of Syb from SypI by FRET: the hippocampal neurons (6 DIV) were cotransfected with EGFP-labeled GSK-3β and ECFP-Syb or EYFP-SypI as indicated, then the dissociation of Syb from SypI was detected at 8 DIV. The images were recorded before (Pre) and after (Post) K⁺ stimulation, and the axon terminals as marked were amplified. H–K, GSK-3 activation inhibits dissociation of Syb from SypI by immunoprecipitation: the purified P2 was incubated with DMSO, Wort, or Wort plus SB for 30 min, then immunoprecipitated with anti-Syp I (H) and probed by anti-Syb, or vice versa (J), and quantitative analysis was performed (I, K). Note that GSK-3 activation disturbed the interaction of synaptic proteins during exocytosis.

Figure 6.

A schematic diagram showing the mechanisms underlying the GSK-3-inhibited presynaptic exocytosis of vesicles. According to the results presented in the current study, we propose that activation of GSK-3β may inhibit the exocytosis by phosphorylating/inactivating P/Q-type VDCC and arresting Ca²⁺ influx, which disrupts the interaction of synaptic proteins, including decreasing the association of Syb with t-SNARE, decreasing the dissociation of Syb from SypI, and decreasing the association of L_II-III with synaptotagmin and the t-SNARE (t-SNARE, syntaxin, and SNAP25).