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Up-regulation of GABA(B) receptor signaling by constitutive assembly with the K+ channel tetramerization domain-containing protein 12 (KCTD12).

Ivankova K, Turecek R, Fritzius T, Seddik R, Prezeau L, Comps-Agrar L, Pin JP, Fakler B, Besseyrias V, Gassmann M, Bettler B - J. Biol. Chem. (2013)

Bottom Line: Glycosylation experiments support that association with KCTD12 does not influence maturation of the receptor complex.Immunoprecipitation and bioluminescence resonance energy transfer experiments demonstrate that KCTD12 remains associated with the receptor during receptor activity and receptor internalization from the cell surface.We further show that KCTD12 reduces constitutive receptor internalization and thereby increases the magnitude of receptor signaling at the cell surface.

View Article: PubMed Central - PubMed

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

ABSTRACT
GABA(B) receptors are the G-protein coupled receptors (GPCRs) for GABA, the main inhibitory neurotransmitter in the central nervous system. Native GABA(B) receptors comprise principle and auxiliary subunits that regulate receptor properties in distinct ways. The principle subunits GABA(B1a), GABA(B1b), and GABA(B2) form fully functional heteromeric GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Principal subunits regulate forward trafficking of the receptors from the endoplasmic reticulum to the plasma membrane and control receptor distribution to axons and dendrites. The auxiliary subunits KCTD8, -12, -12b, and -16 are cytosolic proteins that influence agonist potency and G-protein signaling of GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Here, we used transfected cells to study assembly, surface trafficking, and internalization of GABA(B) receptors in the presence of the KCTD12 subunit. Using bimolecular fluorescence complementation and metabolic labeling, we show that GABA(B) receptors associate with KCTD12 while they reside in the endoplasmic reticulum. Glycosylation experiments support that association with KCTD12 does not influence maturation of the receptor complex. Immunoprecipitation and bioluminescence resonance energy transfer experiments demonstrate that KCTD12 remains associated with the receptor during receptor activity and receptor internalization from the cell surface. We further show that KCTD12 reduces constitutive receptor internalization and thereby increases the magnitude of receptor signaling at the cell surface. Accordingly, knock-out or knockdown of KCTD12 in cultured hippocampal neurons reduces the magnitude of the GABA(B) receptor-mediated K(+) current response. In summary, our experiments support that the up-regulation of functional GABA(B) receptors at the neuronal plasma membrane is an additional physiological role of the auxiliary subunit KCTD12.

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KCTD12 increases the GABAB receptor-mediated peak K+ current amplitude.A, representative outward K+ currents evoked by fast application of baclofen to CHO-K1 cells expressing Kir3.1/3.2 channels and GABAB receptors in the presence (black traces) or absence (gray traces) of KCTD12. GABAB receptors were composed of either GABAB1 and GABAB2 (GB1+GB2) or GABAB1 and GABAB2Y902 (GB1+GB2Y902A) subunits. KCTD12 increases the peak K+ current at GB1+GB2 but not at GB1+GB2Y902A receptors. Whole-cell recordings were made at a holding potential of −50 mV. B, bar graph illustrating that KCTD12 enhances the K+ current density at the peak of the baclofen response in cells expressing GB1+GB2(GB1+GB2 + KCTD12, 11.8 ± 1.9 pA/pF; GB1+GB2, 7.12 ± 0.94 pA/pF; *, p < 0.05) but not in cells expressing GB1+GB2Y902A (GB1+GB2Y902A+KCTD12, 5.10 ± 0.78 pA/pF; GB1+GB2Y902A, 7.30 ± 1.31 pA/pF; p > 0.05). Each bar is the mean ± S.E. of 19–52 cells. ns, not significant.
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Figure 2: KCTD12 increases the GABAB receptor-mediated peak K+ current amplitude.A, representative outward K+ currents evoked by fast application of baclofen to CHO-K1 cells expressing Kir3.1/3.2 channels and GABAB receptors in the presence (black traces) or absence (gray traces) of KCTD12. GABAB receptors were composed of either GABAB1 and GABAB2 (GB1+GB2) or GABAB1 and GABAB2Y902 (GB1+GB2Y902A) subunits. KCTD12 increases the peak K+ current at GB1+GB2 but not at GB1+GB2Y902A receptors. Whole-cell recordings were made at a holding potential of −50 mV. B, bar graph illustrating that KCTD12 enhances the K+ current density at the peak of the baclofen response in cells expressing GB1+GB2(GB1+GB2 + KCTD12, 11.8 ± 1.9 pA/pF; GB1+GB2, 7.12 ± 0.94 pA/pF; *, p < 0.05) but not in cells expressing GB1+GB2Y902A (GB1+GB2Y902A+KCTD12, 5.10 ± 0.78 pA/pF; GB1+GB2Y902A, 7.30 ± 1.31 pA/pF; p > 0.05). Each bar is the mean ± S.E. of 19–52 cells. ns, not significant.

Mentions: We addressed whether increased GABAB receptor surface expression in the presence of KCTD12 results in increased receptor-mediated Kir3 current amplitudes. We recorded outward K+ currents induced by fast application of the GABAB receptor agonist baclofen (100 μm) to CHO-K1 cells expressing GABAB receptors and Kir3.1/3.2 effector channels. In agreement with earlier data (5), we observed a shorter rise time and a pronounced rapid desensitization of baclofen-induced K+ currents in the presence of KCTD12 (Fig. 2A). Consistent with increased receptor surface expression in the presence of KCTD12, we observed a significantly increased K+ current density at the peak of the response (Fig. 2B). An increase in the peak K+ current density was not seen with receptors lacking the KCTD-binding site (GB1+GB2Y902A, Fig. 2B). Thus, electrophysiological experiments in heterologous cells support that binding of KCTD12 to GABAB receptors increases the peak receptor response, consistent with the observed increase in receptor cell surface expression.


Up-regulation of GABA(B) receptor signaling by constitutive assembly with the K+ channel tetramerization domain-containing protein 12 (KCTD12).

Ivankova K, Turecek R, Fritzius T, Seddik R, Prezeau L, Comps-Agrar L, Pin JP, Fakler B, Besseyrias V, Gassmann M, Bettler B - J. Biol. Chem. (2013)

KCTD12 increases the GABAB receptor-mediated peak K+ current amplitude.A, representative outward K+ currents evoked by fast application of baclofen to CHO-K1 cells expressing Kir3.1/3.2 channels and GABAB receptors in the presence (black traces) or absence (gray traces) of KCTD12. GABAB receptors were composed of either GABAB1 and GABAB2 (GB1+GB2) or GABAB1 and GABAB2Y902 (GB1+GB2Y902A) subunits. KCTD12 increases the peak K+ current at GB1+GB2 but not at GB1+GB2Y902A receptors. Whole-cell recordings were made at a holding potential of −50 mV. B, bar graph illustrating that KCTD12 enhances the K+ current density at the peak of the baclofen response in cells expressing GB1+GB2(GB1+GB2 + KCTD12, 11.8 ± 1.9 pA/pF; GB1+GB2, 7.12 ± 0.94 pA/pF; *, p < 0.05) but not in cells expressing GB1+GB2Y902A (GB1+GB2Y902A+KCTD12, 5.10 ± 0.78 pA/pF; GB1+GB2Y902A, 7.30 ± 1.31 pA/pF; p > 0.05). Each bar is the mean ± S.E. of 19–52 cells. ns, not significant.
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Figure 2: KCTD12 increases the GABAB receptor-mediated peak K+ current amplitude.A, representative outward K+ currents evoked by fast application of baclofen to CHO-K1 cells expressing Kir3.1/3.2 channels and GABAB receptors in the presence (black traces) or absence (gray traces) of KCTD12. GABAB receptors were composed of either GABAB1 and GABAB2 (GB1+GB2) or GABAB1 and GABAB2Y902 (GB1+GB2Y902A) subunits. KCTD12 increases the peak K+ current at GB1+GB2 but not at GB1+GB2Y902A receptors. Whole-cell recordings were made at a holding potential of −50 mV. B, bar graph illustrating that KCTD12 enhances the K+ current density at the peak of the baclofen response in cells expressing GB1+GB2(GB1+GB2 + KCTD12, 11.8 ± 1.9 pA/pF; GB1+GB2, 7.12 ± 0.94 pA/pF; *, p < 0.05) but not in cells expressing GB1+GB2Y902A (GB1+GB2Y902A+KCTD12, 5.10 ± 0.78 pA/pF; GB1+GB2Y902A, 7.30 ± 1.31 pA/pF; p > 0.05). Each bar is the mean ± S.E. of 19–52 cells. ns, not significant.
Mentions: We addressed whether increased GABAB receptor surface expression in the presence of KCTD12 results in increased receptor-mediated Kir3 current amplitudes. We recorded outward K+ currents induced by fast application of the GABAB receptor agonist baclofen (100 μm) to CHO-K1 cells expressing GABAB receptors and Kir3.1/3.2 effector channels. In agreement with earlier data (5), we observed a shorter rise time and a pronounced rapid desensitization of baclofen-induced K+ currents in the presence of KCTD12 (Fig. 2A). Consistent with increased receptor surface expression in the presence of KCTD12, we observed a significantly increased K+ current density at the peak of the response (Fig. 2B). An increase in the peak K+ current density was not seen with receptors lacking the KCTD-binding site (GB1+GB2Y902A, Fig. 2B). Thus, electrophysiological experiments in heterologous cells support that binding of KCTD12 to GABAB receptors increases the peak receptor response, consistent with the observed increase in receptor cell surface expression.

Bottom Line: Glycosylation experiments support that association with KCTD12 does not influence maturation of the receptor complex.Immunoprecipitation and bioluminescence resonance energy transfer experiments demonstrate that KCTD12 remains associated with the receptor during receptor activity and receptor internalization from the cell surface.We further show that KCTD12 reduces constitutive receptor internalization and thereby increases the magnitude of receptor signaling at the cell surface.

View Article: PubMed Central - PubMed

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

ABSTRACT
GABA(B) receptors are the G-protein coupled receptors (GPCRs) for GABA, the main inhibitory neurotransmitter in the central nervous system. Native GABA(B) receptors comprise principle and auxiliary subunits that regulate receptor properties in distinct ways. The principle subunits GABA(B1a), GABA(B1b), and GABA(B2) form fully functional heteromeric GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Principal subunits regulate forward trafficking of the receptors from the endoplasmic reticulum to the plasma membrane and control receptor distribution to axons and dendrites. The auxiliary subunits KCTD8, -12, -12b, and -16 are cytosolic proteins that influence agonist potency and G-protein signaling of GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Here, we used transfected cells to study assembly, surface trafficking, and internalization of GABA(B) receptors in the presence of the KCTD12 subunit. Using bimolecular fluorescence complementation and metabolic labeling, we show that GABA(B) receptors associate with KCTD12 while they reside in the endoplasmic reticulum. Glycosylation experiments support that association with KCTD12 does not influence maturation of the receptor complex. Immunoprecipitation and bioluminescence resonance energy transfer experiments demonstrate that KCTD12 remains associated with the receptor during receptor activity and receptor internalization from the cell surface. We further show that KCTD12 reduces constitutive receptor internalization and thereby increases the magnitude of receptor signaling at the cell surface. Accordingly, knock-out or knockdown of KCTD12 in cultured hippocampal neurons reduces the magnitude of the GABA(B) receptor-mediated K(+) current response. In summary, our experiments support that the up-regulation of functional GABA(B) receptors at the neuronal plasma membrane is an additional physiological role of the auxiliary subunit KCTD12.

Show MeSH
Related in: MedlinePlus