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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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PKA activation slows fast desensitization of baclofen-activated K+ currents in cultured hippocampal neurons of WT but not of KCTD12−/− mice. (A) Representative baclofen-activated K+ currents recorded at −50 mV from WT hippocampal neurons. PKA activity was modulated by pre-incubation for 30 min with 8-Br-cAMP (1 mM; bright grey trace) or H89 (2 µM; dark 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). (B) Bar graph summarizing the time constants τ1 and τ2 of baclofen-induced K+ current desensitization for the indicated treatments. Data are means ± SD, n = 7–35. **, p < 0.01; ***, p < 0.001; Dunnett’s multiple comparison test, compared to Ctl. Ctl, control; FSK, forskolin; cAMP, 8-Br-cAMP. (C) Representative baclofen-activated K+ current responses from hippocampal KCTD12−/− neurons pre-incubated with 8-Br-cAMP (grey trace) or untreated KCTD12−/− neurons (control, black trace) as in (A). Traces were fitted as in (A). (D) Bar graph summarizing the time constants τ1 and τ2 of baclofen-induced K+ current desensitization for the indicated treatments. Data are means ± SD, n = 5–17, Dunnett’s multiple comparison test, compared to Ctl.
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Figure 4: PKA activation slows fast desensitization of baclofen-activated K+ currents in cultured hippocampal neurons of WT but not of KCTD12−/− mice. (A) Representative baclofen-activated K+ currents recorded at −50 mV from WT hippocampal neurons. PKA activity was modulated by pre-incubation for 30 min with 8-Br-cAMP (1 mM; bright grey trace) or H89 (2 µM; dark 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). (B) Bar graph summarizing the time constants τ1 and τ2 of baclofen-induced K+ current desensitization for the indicated treatments. Data are means ± SD, n = 7–35. **, p < 0.01; ***, p < 0.001; Dunnett’s multiple comparison test, compared to Ctl. Ctl, control; FSK, forskolin; cAMP, 8-Br-cAMP. (C) Representative baclofen-activated K+ current responses from hippocampal KCTD12−/− neurons pre-incubated with 8-Br-cAMP (grey trace) or untreated KCTD12−/− neurons (control, black trace) as in (A). Traces were fitted as in (A). (D) Bar graph summarizing the time constants τ1 and τ2 of baclofen-induced K+ current desensitization for the indicated treatments. Data are means ± SD, n = 5–17, Dunnett’s multiple comparison test, compared to Ctl.

Mentions: We next investigated whether PKA modulates the desensitization of GABAB-activated K+ currents in cultured hippocampal neurons, which are known to express KCTD12 [18,19,23]. With WT neurons, K+ currents elicited by 60-s long applications of baclofen showed a steady-state desensitization of 53.3 ±9.3% (n = 41, Fig. 4A). The time course of desensitization was approximated by a double exponential function with time constants of 1.5 ± 0.3 s (τ1) and 24.4 ± 6.4 s (τ2) (Fig. 4B), values that are similar as in earlier experiments [18,19]. Activation of PKA with 8-Br-cAMP or forskolin significantly increased the fast component τ1 of the desensitization, while inhibition of PKA with H89 or PKI had the opposite effect (Fig. 4B). Neither activation nor inhibition of PKA had a significant effect on the slow component τ2 of the desensitization (Fig. 4B). The relative contribution of the fast and slow components (τ1 and τ2, respectively) to the desensitization did not change with any of the treatments (τ1 of control: 46.5 ±10.0%; cAMP: 36.3 ±15.4%; FSK: 40.3 ±17.8%; H89: 55.7 ± 12.2%; PKI: 47.7 ± 7.7%; p>0.05; Dun-nett’s multiple comparison test). These results reveal that PKA activity specifically influences the fast component of baclofen-induced K+ current desensitization in WT 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)

PKA activation slows fast desensitization of baclofen-activated K+ currents in cultured hippocampal neurons of WT but not of KCTD12−/− mice. (A) Representative baclofen-activated K+ currents recorded at −50 mV from WT hippocampal neurons. PKA activity was modulated by pre-incubation for 30 min with 8-Br-cAMP (1 mM; bright grey trace) or H89 (2 µM; dark 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). (B) Bar graph summarizing the time constants τ1 and τ2 of baclofen-induced K+ current desensitization for the indicated treatments. Data are means ± SD, n = 7–35. **, p < 0.01; ***, p < 0.001; Dunnett’s multiple comparison test, compared to Ctl. Ctl, control; FSK, forskolin; cAMP, 8-Br-cAMP. (C) Representative baclofen-activated K+ current responses from hippocampal KCTD12−/− neurons pre-incubated with 8-Br-cAMP (grey trace) or untreated KCTD12−/− neurons (control, black trace) as in (A). Traces were fitted as in (A). (D) Bar graph summarizing the time constants τ1 and τ2 of baclofen-induced K+ current desensitization for the indicated treatments. Data are means ± SD, n = 5–17, Dunnett’s multiple comparison test, compared to Ctl.
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Figure 4: PKA activation slows fast desensitization of baclofen-activated K+ currents in cultured hippocampal neurons of WT but not of KCTD12−/− mice. (A) Representative baclofen-activated K+ currents recorded at −50 mV from WT hippocampal neurons. PKA activity was modulated by pre-incubation for 30 min with 8-Br-cAMP (1 mM; bright grey trace) or H89 (2 µM; dark 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). (B) Bar graph summarizing the time constants τ1 and τ2 of baclofen-induced K+ current desensitization for the indicated treatments. Data are means ± SD, n = 7–35. **, p < 0.01; ***, p < 0.001; Dunnett’s multiple comparison test, compared to Ctl. Ctl, control; FSK, forskolin; cAMP, 8-Br-cAMP. (C) Representative baclofen-activated K+ current responses from hippocampal KCTD12−/− neurons pre-incubated with 8-Br-cAMP (grey trace) or untreated KCTD12−/− neurons (control, black trace) as in (A). Traces were fitted as in (A). (D) Bar graph summarizing the time constants τ1 and τ2 of baclofen-induced K+ current desensitization for the indicated treatments. Data are means ± SD, n = 5–17, Dunnett’s multiple comparison test, compared to Ctl.
Mentions: We next investigated whether PKA modulates the desensitization of GABAB-activated K+ currents in cultured hippocampal neurons, which are known to express KCTD12 [18,19,23]. With WT neurons, K+ currents elicited by 60-s long applications of baclofen showed a steady-state desensitization of 53.3 ±9.3% (n = 41, Fig. 4A). The time course of desensitization was approximated by a double exponential function with time constants of 1.5 ± 0.3 s (τ1) and 24.4 ± 6.4 s (τ2) (Fig. 4B), values that are similar as in earlier experiments [18,19]. Activation of PKA with 8-Br-cAMP or forskolin significantly increased the fast component τ1 of the desensitization, while inhibition of PKA with H89 or PKI had the opposite effect (Fig. 4B). Neither activation nor inhibition of PKA had a significant effect on the slow component τ2 of the desensitization (Fig. 4B). The relative contribution of the fast and slow components (τ1 and τ2, respectively) to the desensitization did not change with any of the treatments (τ1 of control: 46.5 ±10.0%; cAMP: 36.3 ±15.4%; FSK: 40.3 ±17.8%; H89: 55.7 ± 12.2%; PKI: 47.7 ± 7.7%; p>0.05; Dun-nett’s multiple comparison test). These results reveal that PKA activity specifically influences the fast component of baclofen-induced K+ current desensitization in WT 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