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GABAB receptor phosphorylation regulates KCTD12-induced K⁺ current desensitization.

Adelfinger L, Turecek R, Ivankova K, Jensen AA, Moss SJ, Gassmann M, Bettler B - Biochem. Pharmacol. (2014)

Bottom Line: Receptor-activated K(+) currents desensitize in the sustained presence of agonist to avoid excessive effects on neuronal activity.GABAB receptor activity reduces protein kinase-A activity, which reduces phosphorylation of serine-892 in GABAB2 and promotes receptor degradation.This cross-regulation of serine-892 phosphorylation and KCTD12 activity sharpens the response during repeated receptor activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedicine, University of Basel, 4056 Basel, Switzerland.

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Basal phosphorylation of S892 is decreased by inhibiting PKA in hippocampal neurons of WT mice. Western blot of cultured neurons in the presence and absence of H89 (10 µM, 2 h pre-incubation; left). Inhibition of PKA does not affect the amount of GB2 protein but decreases the amount of S892 phosphorylation (GB2-pS892). β-III-Tubulin was used as a loading control. Bar graph summarizing the amount of phosphorylated S892 (GB2-pS892 in %; right). The total amount of GB2 and GB2-pS892 were normalized to β-III-tubulin on the same blot. Data are means ± SD, n = 6. *, p < 0.05; t test.
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Figure 5: Basal phosphorylation of S892 is decreased by inhibiting PKA in hippocampal neurons of WT mice. Western blot of cultured neurons in the presence and absence of H89 (10 µM, 2 h pre-incubation; left). Inhibition of PKA does not affect the amount of GB2 protein but decreases the amount of S892 phosphorylation (GB2-pS892). β-III-Tubulin was used as a loading control. Bar graph summarizing the amount of phosphorylated S892 (GB2-pS892 in %; right). The total amount of GB2 and GB2-pS892 were normalized to β-III-tubulin on the same blot. Data are means ± SD, n = 6. *, p < 0.05; t test.

Mentions: PKA inhibition with H89 or PKI accelerates fast desensitization of K+ currents in WT hippocampal neurons. Endogenous PKA activity must therefore provide a high level of basal S892 phosphorylation. Indeed, Western blot analysis of hippocampal neurons revealed that GB2 is highly phosphorylated at S892 (Fig. 5), which is reduced by inhibition of PKA with H89. We next addressed whether phosphorylation of S892 in GB2 is essential for PKA effects on baclofen-induced K+ current desensitization in neurons. We generated S892A knock-in mice carrying a S892 to alanine mutation in the GB2 gene using standard gene targeting techniques (Fig. 6A and B). S892A mice display no overt behavioral abnormalities. Western blot analysis revealed similar levels of GB1a, GB1b and GB2 protein in S892A and WT brain extracts (Fig. 6C). Recordings of baclofen-induced K+ currents from cultured hippocampal neurons revealed that the desensitization was significantly faster in S892A compared to WT neurons (Fig. 6D and E). Activation of PKA with 8-Br-cAMP did not significantly increase τ1 of the desensitization in S892A neurons, in contrast to control WT neurons (Fig. 6E). The τ2 of the desensitization was similar in both genotypes and did not change upon activation of PKA with 8-Br-cAMP (Fig. 6E). The lack of PKA effect in S892A neurons indicates that S892 phosphorylation is mandatory for PKA-mediated attenuation of fast desensitization. In summary, our results show that basal PKA-mediated phosphorylation of S892 slows KCTD12-induced desensitization in neurons.


GABAB receptor phosphorylation regulates KCTD12-induced K⁺ current desensitization.

Adelfinger L, Turecek R, Ivankova K, Jensen AA, Moss SJ, Gassmann M, Bettler B - Biochem. Pharmacol. (2014)

Basal phosphorylation of S892 is decreased by inhibiting PKA in hippocampal neurons of WT mice. Western blot of cultured neurons in the presence and absence of H89 (10 µM, 2 h pre-incubation; left). Inhibition of PKA does not affect the amount of GB2 protein but decreases the amount of S892 phosphorylation (GB2-pS892). β-III-Tubulin was used as a loading control. Bar graph summarizing the amount of phosphorylated S892 (GB2-pS892 in %; right). The total amount of GB2 and GB2-pS892 were normalized to β-III-tubulin on the same blot. Data are means ± SD, n = 6. *, p < 0.05; t test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 5: Basal phosphorylation of S892 is decreased by inhibiting PKA in hippocampal neurons of WT mice. Western blot of cultured neurons in the presence and absence of H89 (10 µM, 2 h pre-incubation; left). Inhibition of PKA does not affect the amount of GB2 protein but decreases the amount of S892 phosphorylation (GB2-pS892). β-III-Tubulin was used as a loading control. Bar graph summarizing the amount of phosphorylated S892 (GB2-pS892 in %; right). The total amount of GB2 and GB2-pS892 were normalized to β-III-tubulin on the same blot. Data are means ± SD, n = 6. *, p < 0.05; t test.
Mentions: PKA inhibition with H89 or PKI accelerates fast desensitization of K+ currents in WT hippocampal neurons. Endogenous PKA activity must therefore provide a high level of basal S892 phosphorylation. Indeed, Western blot analysis of hippocampal neurons revealed that GB2 is highly phosphorylated at S892 (Fig. 5), which is reduced by inhibition of PKA with H89. We next addressed whether phosphorylation of S892 in GB2 is essential for PKA effects on baclofen-induced K+ current desensitization in neurons. We generated S892A knock-in mice carrying a S892 to alanine mutation in the GB2 gene using standard gene targeting techniques (Fig. 6A and B). S892A mice display no overt behavioral abnormalities. Western blot analysis revealed similar levels of GB1a, GB1b and GB2 protein in S892A and WT brain extracts (Fig. 6C). Recordings of baclofen-induced K+ currents from cultured hippocampal neurons revealed that the desensitization was significantly faster in S892A compared to WT neurons (Fig. 6D and E). Activation of PKA with 8-Br-cAMP did not significantly increase τ1 of the desensitization in S892A neurons, in contrast to control WT neurons (Fig. 6E). The τ2 of the desensitization was similar in both genotypes and did not change upon activation of PKA with 8-Br-cAMP (Fig. 6E). The lack of PKA effect in S892A neurons indicates that S892 phosphorylation is mandatory for PKA-mediated attenuation of fast desensitization. In summary, our results show that basal PKA-mediated phosphorylation of S892 slows KCTD12-induced desensitization in neurons.

Bottom Line: Receptor-activated K(+) currents desensitize in the sustained presence of agonist to avoid excessive effects on neuronal activity.GABAB receptor activity reduces protein kinase-A activity, which reduces phosphorylation of serine-892 in GABAB2 and promotes receptor degradation.This cross-regulation of serine-892 phosphorylation and KCTD12 activity sharpens the response during repeated receptor activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedicine, University of Basel, 4056 Basel, Switzerland.

Show MeSH
Related in: MedlinePlus