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Kainate receptor activation induces glycine receptor endocytosis through PKC deSUMOylation.

Sun H, Lu L, Zuo Y, Wang Y, Jiao Y, Zeng WZ, Huang C, Zhu MX, Zamponi GW, Zhou T, Xu TL, Cheng J, Li Y - Nat Commun (2014)

Bottom Line: Here we show that activation of kainate receptors (KARs) causes GlyR endocytosis in a calcium- and protein kinase C (PKC)-dependent manner, leading to reduced GlyR-mediated synaptic activity in cultured spinal cord neurons and the superficial dorsal horn of rat spinal cord slices.This effect requires SUMO1/sentrin-specific peptidase 1 (SENP1)-mediated deSUMOylation of PKC, indicating that the crosstalk between KARs and GlyRs relies on the SUMOylation status of PKC.SENP1-mediated deSUMOylation of PKC is involved in the kainate-induced GlyR endocytosis and thus plays an important role in the anti-homeostatic regulation between excitatory and inhibitory ligand-gated ion channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.

ABSTRACT
Surface expression and regulated endocytosis of glycine receptors (GlyRs) play a critical function in balancing neuronal excitability. SUMOylation (SUMO modification) is of critical importance for maintaining neuronal function in the central nervous system. Here we show that activation of kainate receptors (KARs) causes GlyR endocytosis in a calcium- and protein kinase C (PKC)-dependent manner, leading to reduced GlyR-mediated synaptic activity in cultured spinal cord neurons and the superficial dorsal horn of rat spinal cord slices. This effect requires SUMO1/sentrin-specific peptidase 1 (SENP1)-mediated deSUMOylation of PKC, indicating that the crosstalk between KARs and GlyRs relies on the SUMOylation status of PKC. SENP1-mediated deSUMOylation of PKC is involved in the kainate-induced GlyR endocytosis and thus plays an important role in the anti-homeostatic regulation between excitatory and inhibitory ligand-gated ion channels. Altogether, we have identified a SUMOylation-dependent regulatory pathway for GlyR endocytosis, which may have important physiological implications for proper neuronal excitability.

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Kainate induces GlyR internalization through activation of PKC.(a) Effect of PKC activator (PMA, 1 μM, 1 h) or PKC inhibitor (calphostin C, 1 μM, 1 h) alone or in combination with kainate (KA) on the endocytosis of GlyRs. Surface GlyRs assessed by the antibody-feeding assay under non-permeabilized conditions in neurons treated with drugs as indicated and quantification obtained as in Fig. 1a. Data are means±s.e.m. from four experiments; the total numbers of neurons analysed (n) ranged from 29 to 55 cells per condition. Scale bar, 10 μm. (b) Histograms depict the effects of PKA activator (8-Br-cAMP 100 μM plus IBMX 100 μM, 1 h) and inhibitor (H89, 10 μM, 1 h) on the puncta number (left) and intensity (right) of surface GlyRs following kainate (KA) stimulation. Data are normalized to untreated controls and shown as means±s.e.m. from three experiments; the total numbers of neurons analysed (n) ranged from 14 to 40 cells per condition. (c) Bar graph showing the effect of Gö6976 (0.5 μM, 1 h) on puncta number (left) and intensity (right) of surface GlyRs following kainate (KA) treatment. Data are normalized to untreated controls and shown as means±s.e.m. from at least two experiments; the total numbers of neurons analysed (n) ranged from 10 to 25 cells per condition. *P<0.05, ***P<0.001 compared with control, by one-way analysis of variance with pairwise comparison by Tukey’s post hoc test.
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f4: Kainate induces GlyR internalization through activation of PKC.(a) Effect of PKC activator (PMA, 1 μM, 1 h) or PKC inhibitor (calphostin C, 1 μM, 1 h) alone or in combination with kainate (KA) on the endocytosis of GlyRs. Surface GlyRs assessed by the antibody-feeding assay under non-permeabilized conditions in neurons treated with drugs as indicated and quantification obtained as in Fig. 1a. Data are means±s.e.m. from four experiments; the total numbers of neurons analysed (n) ranged from 29 to 55 cells per condition. Scale bar, 10 μm. (b) Histograms depict the effects of PKA activator (8-Br-cAMP 100 μM plus IBMX 100 μM, 1 h) and inhibitor (H89, 10 μM, 1 h) on the puncta number (left) and intensity (right) of surface GlyRs following kainate (KA) stimulation. Data are normalized to untreated controls and shown as means±s.e.m. from three experiments; the total numbers of neurons analysed (n) ranged from 14 to 40 cells per condition. (c) Bar graph showing the effect of Gö6976 (0.5 μM, 1 h) on puncta number (left) and intensity (right) of surface GlyRs following kainate (KA) treatment. Data are normalized to untreated controls and shown as means±s.e.m. from at least two experiments; the total numbers of neurons analysed (n) ranged from 10 to 25 cells per condition. *P<0.05, ***P<0.001 compared with control, by one-way analysis of variance with pairwise comparison by Tukey’s post hoc test.

Mentions: Direct phosphorylation of receptor subunits regulates endocytosis of glutamate receptors, including that of AMPARs, NMDARs and KARs62122404142. It has recently been shown that diffusion properties of GlyRs and the interaction between GlyRs and gephyrin were regulated through PKC phosphorylation of the GlyR β-subunit20. To test whether protein phosphorylation was involved in kainate-evoked GlyR endocytosis, we examined the effects of selective activators and inhibitors of PKA (protein kinase A) and PKC on kainate-induced GlyR endocytosis. Although the expressed GluK2 subunits in mammalian cells was directly phosphorylated and modulated by PKA4344, neither the PKA activator 8-Br-cAMP (8-bromo cyclic AMP) plus IBMX (3-isobutyl-1-methylxanthine) nor the PKA inhibitor H89 affected kainate-evoked GlyR endocytosis in cultured spinal cord neurons (Fig. 4b). These results indicate that PKA plays no role in kainate-evoked GlyR internalization. In contrast, inhibitors of PKC calphostin C or Gö6976 (selectively inhibits Ca2+-dependent isoforms of PKC) had remarkable effects on kainate-evoked GlyR endocytosis. Treatment with calphostin C (Fig. 4a) or Gö6976 (Fig. 4c) nearly abolished all endocytosis, whereas activation of PKC by phorbol ester (PMA) had no further effect on the kainate-induced GlyR internalization (Fig. 4a). Interestingly, PMA alone also significantly increased GlyR internalization (Fig. 4a), indicating that PKC may directly regulate GlyR endocytosis under the present experimental conditions. Altogether, our data suggest that PKC activation is required for kainate-evoked GlyR internalization.


Kainate receptor activation induces glycine receptor endocytosis through PKC deSUMOylation.

Sun H, Lu L, Zuo Y, Wang Y, Jiao Y, Zeng WZ, Huang C, Zhu MX, Zamponi GW, Zhou T, Xu TL, Cheng J, Li Y - Nat Commun (2014)

Kainate induces GlyR internalization through activation of PKC.(a) Effect of PKC activator (PMA, 1 μM, 1 h) or PKC inhibitor (calphostin C, 1 μM, 1 h) alone or in combination with kainate (KA) on the endocytosis of GlyRs. Surface GlyRs assessed by the antibody-feeding assay under non-permeabilized conditions in neurons treated with drugs as indicated and quantification obtained as in Fig. 1a. Data are means±s.e.m. from four experiments; the total numbers of neurons analysed (n) ranged from 29 to 55 cells per condition. Scale bar, 10 μm. (b) Histograms depict the effects of PKA activator (8-Br-cAMP 100 μM plus IBMX 100 μM, 1 h) and inhibitor (H89, 10 μM, 1 h) on the puncta number (left) and intensity (right) of surface GlyRs following kainate (KA) stimulation. Data are normalized to untreated controls and shown as means±s.e.m. from three experiments; the total numbers of neurons analysed (n) ranged from 14 to 40 cells per condition. (c) Bar graph showing the effect of Gö6976 (0.5 μM, 1 h) on puncta number (left) and intensity (right) of surface GlyRs following kainate (KA) treatment. Data are normalized to untreated controls and shown as means±s.e.m. from at least two experiments; the total numbers of neurons analysed (n) ranged from 10 to 25 cells per condition. *P<0.05, ***P<0.001 compared with control, by one-way analysis of variance with pairwise comparison by Tukey’s post hoc test.
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Related In: Results  -  Collection

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f4: Kainate induces GlyR internalization through activation of PKC.(a) Effect of PKC activator (PMA, 1 μM, 1 h) or PKC inhibitor (calphostin C, 1 μM, 1 h) alone or in combination with kainate (KA) on the endocytosis of GlyRs. Surface GlyRs assessed by the antibody-feeding assay under non-permeabilized conditions in neurons treated with drugs as indicated and quantification obtained as in Fig. 1a. Data are means±s.e.m. from four experiments; the total numbers of neurons analysed (n) ranged from 29 to 55 cells per condition. Scale bar, 10 μm. (b) Histograms depict the effects of PKA activator (8-Br-cAMP 100 μM plus IBMX 100 μM, 1 h) and inhibitor (H89, 10 μM, 1 h) on the puncta number (left) and intensity (right) of surface GlyRs following kainate (KA) stimulation. Data are normalized to untreated controls and shown as means±s.e.m. from three experiments; the total numbers of neurons analysed (n) ranged from 14 to 40 cells per condition. (c) Bar graph showing the effect of Gö6976 (0.5 μM, 1 h) on puncta number (left) and intensity (right) of surface GlyRs following kainate (KA) treatment. Data are normalized to untreated controls and shown as means±s.e.m. from at least two experiments; the total numbers of neurons analysed (n) ranged from 10 to 25 cells per condition. *P<0.05, ***P<0.001 compared with control, by one-way analysis of variance with pairwise comparison by Tukey’s post hoc test.
Mentions: Direct phosphorylation of receptor subunits regulates endocytosis of glutamate receptors, including that of AMPARs, NMDARs and KARs62122404142. It has recently been shown that diffusion properties of GlyRs and the interaction between GlyRs and gephyrin were regulated through PKC phosphorylation of the GlyR β-subunit20. To test whether protein phosphorylation was involved in kainate-evoked GlyR endocytosis, we examined the effects of selective activators and inhibitors of PKA (protein kinase A) and PKC on kainate-induced GlyR endocytosis. Although the expressed GluK2 subunits in mammalian cells was directly phosphorylated and modulated by PKA4344, neither the PKA activator 8-Br-cAMP (8-bromo cyclic AMP) plus IBMX (3-isobutyl-1-methylxanthine) nor the PKA inhibitor H89 affected kainate-evoked GlyR endocytosis in cultured spinal cord neurons (Fig. 4b). These results indicate that PKA plays no role in kainate-evoked GlyR internalization. In contrast, inhibitors of PKC calphostin C or Gö6976 (selectively inhibits Ca2+-dependent isoforms of PKC) had remarkable effects on kainate-evoked GlyR endocytosis. Treatment with calphostin C (Fig. 4a) or Gö6976 (Fig. 4c) nearly abolished all endocytosis, whereas activation of PKC by phorbol ester (PMA) had no further effect on the kainate-induced GlyR internalization (Fig. 4a). Interestingly, PMA alone also significantly increased GlyR internalization (Fig. 4a), indicating that PKC may directly regulate GlyR endocytosis under the present experimental conditions. Altogether, our data suggest that PKC activation is required for kainate-evoked GlyR internalization.

Bottom Line: Here we show that activation of kainate receptors (KARs) causes GlyR endocytosis in a calcium- and protein kinase C (PKC)-dependent manner, leading to reduced GlyR-mediated synaptic activity in cultured spinal cord neurons and the superficial dorsal horn of rat spinal cord slices.This effect requires SUMO1/sentrin-specific peptidase 1 (SENP1)-mediated deSUMOylation of PKC, indicating that the crosstalk between KARs and GlyRs relies on the SUMOylation status of PKC.SENP1-mediated deSUMOylation of PKC is involved in the kainate-induced GlyR endocytosis and thus plays an important role in the anti-homeostatic regulation between excitatory and inhibitory ligand-gated ion channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.

ABSTRACT
Surface expression and regulated endocytosis of glycine receptors (GlyRs) play a critical function in balancing neuronal excitability. SUMOylation (SUMO modification) is of critical importance for maintaining neuronal function in the central nervous system. Here we show that activation of kainate receptors (KARs) causes GlyR endocytosis in a calcium- and protein kinase C (PKC)-dependent manner, leading to reduced GlyR-mediated synaptic activity in cultured spinal cord neurons and the superficial dorsal horn of rat spinal cord slices. This effect requires SUMO1/sentrin-specific peptidase 1 (SENP1)-mediated deSUMOylation of PKC, indicating that the crosstalk between KARs and GlyRs relies on the SUMOylation status of PKC. SENP1-mediated deSUMOylation of PKC is involved in the kainate-induced GlyR endocytosis and thus plays an important role in the anti-homeostatic regulation between excitatory and inhibitory ligand-gated ion channels. Altogether, we have identified a SUMOylation-dependent regulatory pathway for GlyR endocytosis, which may have important physiological implications for proper neuronal excitability.

Show MeSH
Related in: MedlinePlus