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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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S892 phosphorylation slows KCTD12-induced desensitization of baclofen-activated K+ currents. (A) Scheme highlighting the close proximity of the PKA phosphorylation-site S892 and the KCTD12 binding-site Y902 in the C-terminal domain of GB2. (B) Representative traces of GIRK currents activated by baclofen (100 µM) and recorded at −50 mV from CHO-K1 cells expressing GB2-WT, GB1b, GIRK1/2 and KCTD12. Cells were pre-incubated without (Control, black trace) or with 8-Br-cAMP(1 mM, 30 min; grey trace). The desensitization time constants τ1 and τ2 were derived from double-exponential fits (blue trace) to the decay phase of K+ currents during baclofen application. (C) Representative traces as in (B) from CHO-K1 cells lacking KCTD12. (D–F) Representative traces as in (B) from CHO-K1 cells expressing the GB2 mutants S892A (D), S892E (E) or Y902A (F). (G) Bar graph summarizing the time constant τ (amplitude-weighted mean time constant calculated from τ1 and τ2) of baclofen-induced K+ current desensitization. Data are means ± SD, n = 5–16. *,p < 0.05; **,p < 0.01; ***,p < 0.001; Sidak’s multiple comparison test. (H) Repeated activation of GIRK currents in CHO-K1 cells expressing WT GABAB receptors (GB1,2-WT) together with KCTD12 results in a sharpening of the current response (left; 1st response black, 3rd response red). No sharpening of the current response upon repeated activation is observed with mutated GABAB receptors (GB1,2-S892A) that cannot be phosphorylated at S892 (right). Baclofen applications were for 60-s in intervals of 5 min. (I) Bar graph summarizing experiments as shown in (H). In experiments with GB2-WT, the τ of the 3rd response (τP3) was significantly reduced compared to the τ of the 1st response (τP1). No change in the desensitization of the current response upon repeated activation is seen (i) when S892 cannot be phosphorylated (S892A), (ii) with the phospho-mimetic S892E and (iii) when KCTD12 cannot bind to GB2 (Y902A). Data are means ± SD, n = 7–12. **, p < 0.01; t test.
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Figure 1: S892 phosphorylation slows KCTD12-induced desensitization of baclofen-activated K+ currents. (A) Scheme highlighting the close proximity of the PKA phosphorylation-site S892 and the KCTD12 binding-site Y902 in the C-terminal domain of GB2. (B) Representative traces of GIRK currents activated by baclofen (100 µM) and recorded at −50 mV from CHO-K1 cells expressing GB2-WT, GB1b, GIRK1/2 and KCTD12. Cells were pre-incubated without (Control, black trace) or with 8-Br-cAMP(1 mM, 30 min; grey trace). The desensitization time constants τ1 and τ2 were derived from double-exponential fits (blue trace) to the decay phase of K+ currents during baclofen application. (C) Representative traces as in (B) from CHO-K1 cells lacking KCTD12. (D–F) Representative traces as in (B) from CHO-K1 cells expressing the GB2 mutants S892A (D), S892E (E) or Y902A (F). (G) Bar graph summarizing the time constant τ (amplitude-weighted mean time constant calculated from τ1 and τ2) of baclofen-induced K+ current desensitization. Data are means ± SD, n = 5–16. *,p < 0.05; **,p < 0.01; ***,p < 0.001; Sidak’s multiple comparison test. (H) Repeated activation of GIRK currents in CHO-K1 cells expressing WT GABAB receptors (GB1,2-WT) together with KCTD12 results in a sharpening of the current response (left; 1st response black, 3rd response red). No sharpening of the current response upon repeated activation is observed with mutated GABAB receptors (GB1,2-S892A) that cannot be phosphorylated at S892 (right). Baclofen applications were for 60-s in intervals of 5 min. (I) Bar graph summarizing experiments as shown in (H). In experiments with GB2-WT, the τ of the 3rd response (τP3) was significantly reduced compared to the τ of the 1st response (τP1). No change in the desensitization of the current response upon repeated activation is seen (i) when S892 cannot be phosphorylated (S892A), (ii) with the phospho-mimetic S892E and (iii) when KCTD12 cannot bind to GB2 (Y902A). Data are means ± SD, n = 7–12. **, p < 0.01; t test.

Mentions: Since KCTD12 binds to GB2 in proximity of the PKA phosphorylation-site S892 (Fig. 1A), phosphorylation of S892 may influence the ternary receptor/KCTD12/G-protein complex and vice versa, KCTD12 may influence S892 phosphorylation. We therefore addressed whether PKA regulates KCTD12-induced K+ current desensitization. We found that PKA phosphorylation of S892 reduces KCTD12-induced K+ current desensitization in heterologous cells and cultured hippocampal neurons. Mechanistically, S892 phosphorylation induces a conformational change in the GABAB receptor/KCTD12 complex that slows KCTD12-induced desensitization. Reciprocally, the binding of KCTD12 promotes phosphorylation of S892, consistent with previous data showing that KCTD12 stabilizes receptors at the cell surface [21].


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)

S892 phosphorylation slows KCTD12-induced desensitization of baclofen-activated K+ currents. (A) Scheme highlighting the close proximity of the PKA phosphorylation-site S892 and the KCTD12 binding-site Y902 in the C-terminal domain of GB2. (B) Representative traces of GIRK currents activated by baclofen (100 µM) and recorded at −50 mV from CHO-K1 cells expressing GB2-WT, GB1b, GIRK1/2 and KCTD12. Cells were pre-incubated without (Control, black trace) or with 8-Br-cAMP(1 mM, 30 min; grey trace). The desensitization time constants τ1 and τ2 were derived from double-exponential fits (blue trace) to the decay phase of K+ currents during baclofen application. (C) Representative traces as in (B) from CHO-K1 cells lacking KCTD12. (D–F) Representative traces as in (B) from CHO-K1 cells expressing the GB2 mutants S892A (D), S892E (E) or Y902A (F). (G) Bar graph summarizing the time constant τ (amplitude-weighted mean time constant calculated from τ1 and τ2) of baclofen-induced K+ current desensitization. Data are means ± SD, n = 5–16. *,p < 0.05; **,p < 0.01; ***,p < 0.001; Sidak’s multiple comparison test. (H) Repeated activation of GIRK currents in CHO-K1 cells expressing WT GABAB receptors (GB1,2-WT) together with KCTD12 results in a sharpening of the current response (left; 1st response black, 3rd response red). No sharpening of the current response upon repeated activation is observed with mutated GABAB receptors (GB1,2-S892A) that cannot be phosphorylated at S892 (right). Baclofen applications were for 60-s in intervals of 5 min. (I) Bar graph summarizing experiments as shown in (H). In experiments with GB2-WT, the τ of the 3rd response (τP3) was significantly reduced compared to the τ of the 1st response (τP1). No change in the desensitization of the current response upon repeated activation is seen (i) when S892 cannot be phosphorylated (S892A), (ii) with the phospho-mimetic S892E and (iii) when KCTD12 cannot bind to GB2 (Y902A). Data are means ± SD, n = 7–12. **, p < 0.01; t test.
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Figure 1: S892 phosphorylation slows KCTD12-induced desensitization of baclofen-activated K+ currents. (A) Scheme highlighting the close proximity of the PKA phosphorylation-site S892 and the KCTD12 binding-site Y902 in the C-terminal domain of GB2. (B) Representative traces of GIRK currents activated by baclofen (100 µM) and recorded at −50 mV from CHO-K1 cells expressing GB2-WT, GB1b, GIRK1/2 and KCTD12. Cells were pre-incubated without (Control, black trace) or with 8-Br-cAMP(1 mM, 30 min; grey trace). The desensitization time constants τ1 and τ2 were derived from double-exponential fits (blue trace) to the decay phase of K+ currents during baclofen application. (C) Representative traces as in (B) from CHO-K1 cells lacking KCTD12. (D–F) Representative traces as in (B) from CHO-K1 cells expressing the GB2 mutants S892A (D), S892E (E) or Y902A (F). (G) Bar graph summarizing the time constant τ (amplitude-weighted mean time constant calculated from τ1 and τ2) of baclofen-induced K+ current desensitization. Data are means ± SD, n = 5–16. *,p < 0.05; **,p < 0.01; ***,p < 0.001; Sidak’s multiple comparison test. (H) Repeated activation of GIRK currents in CHO-K1 cells expressing WT GABAB receptors (GB1,2-WT) together with KCTD12 results in a sharpening of the current response (left; 1st response black, 3rd response red). No sharpening of the current response upon repeated activation is observed with mutated GABAB receptors (GB1,2-S892A) that cannot be phosphorylated at S892 (right). Baclofen applications were for 60-s in intervals of 5 min. (I) Bar graph summarizing experiments as shown in (H). In experiments with GB2-WT, the τ of the 3rd response (τP3) was significantly reduced compared to the τ of the 1st response (τP1). No change in the desensitization of the current response upon repeated activation is seen (i) when S892 cannot be phosphorylated (S892A), (ii) with the phospho-mimetic S892E and (iii) when KCTD12 cannot bind to GB2 (Y902A). Data are means ± SD, n = 7–12. **, p < 0.01; t test.
Mentions: Since KCTD12 binds to GB2 in proximity of the PKA phosphorylation-site S892 (Fig. 1A), phosphorylation of S892 may influence the ternary receptor/KCTD12/G-protein complex and vice versa, KCTD12 may influence S892 phosphorylation. We therefore addressed whether PKA regulates KCTD12-induced K+ current desensitization. We found that PKA phosphorylation of S892 reduces KCTD12-induced K+ current desensitization in heterologous cells and cultured hippocampal neurons. Mechanistically, S892 phosphorylation induces a conformational change in the GABAB receptor/KCTD12 complex that slows KCTD12-induced desensitization. Reciprocally, the binding of KCTD12 promotes phosphorylation of S892, consistent with previous data showing that KCTD12 stabilizes receptors at the cell surface [21].

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