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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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Lack of S892 phosphorylation in S892A knock-in mice accelerates fast desensitization of GABAB-activated K+ currents. (A) WT and mutated GB2 alleles. The S892 to alanine mutation (tcc → gcc) and a silent diagnostic NheI restriction site (gctagc) were introduced into exon 19 using homologous recombination in Balb/c embryonic stem (ES) cells. Mutated nucleotides are shown in italic. A neomycin marker (neo) flanked by loxP sites (arrowheads) was used for selection of ES cells. Correctly targeted ES cells (S892A + neo allele) were injected into C57BL/6 blastocysts. A founder mouse was crossed with a Balb/c mouse expressing Cre-recombinase to excise the neomycin cassette, leaving one loxP site behind (S892A allele). The hybridization probe used in the Southern blot in (B) is indicated. A, alanine; N, NheI restriction sites; S, serine. (B) Southern blot of NheI cut genomic DNA from correctly targeted ES cells. The probe labels a 15.3 kb fragment for the WT allele and a 4.3 kb fragment for the S892A + neo allele. (C) Western blot analysis of brain extracts showing that S892A mice express normal levels of GB2, GB1a and GB1b proteins. S892 was phosphorylated in brain extracts of WT but not S892A mice as shown with an antibody specific for phosphorylated S892 (GB2-pS892). Brain extracts of GB2-deficient mice (GB2−/−) [38] confirm the specificity of the GB2 and GB2-pS892 antibodies. β-III-Tubulin was used as a loading control. (D) Representative GABAB-activated K+ currents recorded at −50 mV in response to baclofen application (100 µM) from cultured hippocampal neurons of S892A mice. PKA was activated by pre-incubation for 30 min with 8-Br-cAMP (1 mM; grey trace). Controls represent recordings from untreated neurons (black trace). The desensitization time constants τ1 and τ2 were derived from double-exponential fits to the decay phase of K+ currents during baclofen application (enlarged on the right). (E) Bar graph summarizing the time constants τ1 and τ2 of baclofen-induced K+ current desensitization in WT and S892A neurons. Data are means ± SD, n = 5–7. *,p < 0.05; ***, p < 0.001; Sidak’s multiple comparison test.
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Figure 6: Lack of S892 phosphorylation in S892A knock-in mice accelerates fast desensitization of GABAB-activated K+ currents. (A) WT and mutated GB2 alleles. The S892 to alanine mutation (tcc → gcc) and a silent diagnostic NheI restriction site (gctagc) were introduced into exon 19 using homologous recombination in Balb/c embryonic stem (ES) cells. Mutated nucleotides are shown in italic. A neomycin marker (neo) flanked by loxP sites (arrowheads) was used for selection of ES cells. Correctly targeted ES cells (S892A + neo allele) were injected into C57BL/6 blastocysts. A founder mouse was crossed with a Balb/c mouse expressing Cre-recombinase to excise the neomycin cassette, leaving one loxP site behind (S892A allele). The hybridization probe used in the Southern blot in (B) is indicated. A, alanine; N, NheI restriction sites; S, serine. (B) Southern blot of NheI cut genomic DNA from correctly targeted ES cells. The probe labels a 15.3 kb fragment for the WT allele and a 4.3 kb fragment for the S892A + neo allele. (C) Western blot analysis of brain extracts showing that S892A mice express normal levels of GB2, GB1a and GB1b proteins. S892 was phosphorylated in brain extracts of WT but not S892A mice as shown with an antibody specific for phosphorylated S892 (GB2-pS892). Brain extracts of GB2-deficient mice (GB2−/−) [38] confirm the specificity of the GB2 and GB2-pS892 antibodies. β-III-Tubulin was used as a loading control. (D) Representative GABAB-activated K+ currents recorded at −50 mV in response to baclofen application (100 µM) from cultured hippocampal neurons of S892A mice. PKA was activated by pre-incubation for 30 min with 8-Br-cAMP (1 mM; grey trace). Controls represent recordings from untreated neurons (black trace). The desensitization time constants τ1 and τ2 were derived from double-exponential fits to the decay phase of K+ currents during baclofen application (enlarged on the right). (E) Bar graph summarizing the time constants τ1 and τ2 of baclofen-induced K+ current desensitization in WT and S892A neurons. Data are means ± SD, n = 5–7. *,p < 0.05; ***, p < 0.001; Sidak’s multiple comparison 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)

Lack of S892 phosphorylation in S892A knock-in mice accelerates fast desensitization of GABAB-activated K+ currents. (A) WT and mutated GB2 alleles. The S892 to alanine mutation (tcc → gcc) and a silent diagnostic NheI restriction site (gctagc) were introduced into exon 19 using homologous recombination in Balb/c embryonic stem (ES) cells. Mutated nucleotides are shown in italic. A neomycin marker (neo) flanked by loxP sites (arrowheads) was used for selection of ES cells. Correctly targeted ES cells (S892A + neo allele) were injected into C57BL/6 blastocysts. A founder mouse was crossed with a Balb/c mouse expressing Cre-recombinase to excise the neomycin cassette, leaving one loxP site behind (S892A allele). The hybridization probe used in the Southern blot in (B) is indicated. A, alanine; N, NheI restriction sites; S, serine. (B) Southern blot of NheI cut genomic DNA from correctly targeted ES cells. The probe labels a 15.3 kb fragment for the WT allele and a 4.3 kb fragment for the S892A + neo allele. (C) Western blot analysis of brain extracts showing that S892A mice express normal levels of GB2, GB1a and GB1b proteins. S892 was phosphorylated in brain extracts of WT but not S892A mice as shown with an antibody specific for phosphorylated S892 (GB2-pS892). Brain extracts of GB2-deficient mice (GB2−/−) [38] confirm the specificity of the GB2 and GB2-pS892 antibodies. β-III-Tubulin was used as a loading control. (D) Representative GABAB-activated K+ currents recorded at −50 mV in response to baclofen application (100 µM) from cultured hippocampal neurons of S892A mice. PKA was activated by pre-incubation for 30 min with 8-Br-cAMP (1 mM; grey trace). Controls represent recordings from untreated neurons (black trace). The desensitization time constants τ1 and τ2 were derived from double-exponential fits to the decay phase of K+ currents during baclofen application (enlarged on the right). (E) Bar graph summarizing the time constants τ1 and τ2 of baclofen-induced K+ current desensitization in WT and S892A neurons. Data are means ± SD, n = 5–7. *,p < 0.05; ***, p < 0.001; Sidak’s multiple comparison test.
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Figure 6: Lack of S892 phosphorylation in S892A knock-in mice accelerates fast desensitization of GABAB-activated K+ currents. (A) WT and mutated GB2 alleles. The S892 to alanine mutation (tcc → gcc) and a silent diagnostic NheI restriction site (gctagc) were introduced into exon 19 using homologous recombination in Balb/c embryonic stem (ES) cells. Mutated nucleotides are shown in italic. A neomycin marker (neo) flanked by loxP sites (arrowheads) was used for selection of ES cells. Correctly targeted ES cells (S892A + neo allele) were injected into C57BL/6 blastocysts. A founder mouse was crossed with a Balb/c mouse expressing Cre-recombinase to excise the neomycin cassette, leaving one loxP site behind (S892A allele). The hybridization probe used in the Southern blot in (B) is indicated. A, alanine; N, NheI restriction sites; S, serine. (B) Southern blot of NheI cut genomic DNA from correctly targeted ES cells. The probe labels a 15.3 kb fragment for the WT allele and a 4.3 kb fragment for the S892A + neo allele. (C) Western blot analysis of brain extracts showing that S892A mice express normal levels of GB2, GB1a and GB1b proteins. S892 was phosphorylated in brain extracts of WT but not S892A mice as shown with an antibody specific for phosphorylated S892 (GB2-pS892). Brain extracts of GB2-deficient mice (GB2−/−) [38] confirm the specificity of the GB2 and GB2-pS892 antibodies. β-III-Tubulin was used as a loading control. (D) Representative GABAB-activated K+ currents recorded at −50 mV in response to baclofen application (100 µM) from cultured hippocampal neurons of S892A mice. PKA was activated by pre-incubation for 30 min with 8-Br-cAMP (1 mM; grey trace). Controls represent recordings from untreated neurons (black trace). The desensitization time constants τ1 and τ2 were derived from double-exponential fits to the decay phase of K+ currents during baclofen application (enlarged on the right). (E) Bar graph summarizing the time constants τ1 and τ2 of baclofen-induced K+ current desensitization in WT and S892A neurons. Data are means ± SD, n = 5–7. *,p < 0.05; ***, p < 0.001; Sidak’s multiple comparison 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