Native GABA(B) receptors are heteromultimers with a family of auxiliary subunits.

GABA(B) receptors are the G-protein-coupled receptors for gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. They are expressed in almost all neurons of the brain, where they regulate synaptic transmission and signal propagation by controlling the activity of voltage-gated calcium (Ca(v)) and inward-rectifier potassium (K(ir)) channels. Molecular cloning revealed that functional GABA(B) receptors are formed by the heteromeric assembly of GABA(B1) with GABA(B2) subunits. However, cloned GABA(B(1,2)) receptors failed to reproduce the functional diversity observed with native GABA(B) receptors. Here we show by functional proteomics that GABA(B) receptors in the brain are high-molecular-mass complexes of GABA(B1), GABA(B2) and members of a subfamily of the KCTD (potassium channel tetramerization domain-containing) proteins. KCTD proteins 8, 12, 12b and 16 show distinct expression profiles in the brain and associate tightly with the carboxy terminus of GABA(B2) as tetramers. This co-assembly changes the properties of the GABA(B(1,2)) core receptor: the KCTD proteins increase agonist potency and markedly alter the G-protein signalling of the receptors by accelerating onset and promoting desensitization in a KCTD-subtype-specific manner. Taken together, our results establish the KCTD proteins as auxiliary subunits of GABA(B) receptors that determine the pharmacology and kinetics of the receptor response.

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

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.

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

GABAB receptors assemble from GABAB1 and GABAB2 subunits. GABAB2 additionally associates with auxiliary KCTD subunits (named after their K(+) channel tetramerization-domain). GABAB receptors couple to heterotrimeric G-proteins and activate inwardly-rectifying K(+) channels through the ßγ subunits released from the G-protein. Receptor-activated K(+) currents desensitize in the sustained presence of agonist to avoid excessive effects on neuronal activity. Desensitization of K(+) currents integrates distinct mechanistic underpinnings. GABAB receptor activity reduces protein kinase-A activity, which reduces phosphorylation of serine-892 in GABAB2 and promotes receptor degradation. This form of desensitization operates on the time scale of several minutes to hours. A faster form of desensitization is induced by the auxiliary subunit KCTD12, which interferes with channel activation by binding to the G-protein ßγ subunits. Here we show that the two mechanisms of desensitization influence each other. Serine-892 phosphorylation in heterologous cells rearranges KCTD12 at the receptor and slows KCTD12-induced desensitization. Likewise, protein kinase-A activation in hippocampal neurons slows fast desensitization of GABAB receptor-activated K(+) currents while protein kinase-A inhibition accelerates fast desensitization. Protein kinase-A fails to regulate fast desensitization in KCTD12 knock-out mice or knock-in mice with a serine-892 to alanine mutation, thus demonstrating that serine-892 phosphorylation regulates KCTD12-induced desensitization in vivo. Fast current desensitization is accelerated in hippocampal neurons carrying the serine-892 to alanine mutation, showing that tonic serine-892 phosphorylation normally limits KCTD12-induced desensitization. Tonic serine-892 phosphorylation is in turn promoted by assembly of receptors with KCTD12. This cross-regulation of serine-892 phosphorylation and KCTD12 activity sharpens the response during repeated receptor activation.

Auxiliary GABAB receptor subunits uncouple G protein ßγ subunits from effector channels to induce desensitization.

Activation of K(+) channels by the G protein ßγ subunits is an important signaling mechanism of G-protein-coupled receptors. Typically, receptor-activated K(+) currents desensitize in the sustained presence of agonists to avoid excessive effects on cellular activity. The auxiliary GABAB receptor subunit KCTD12 induces fast and pronounced desensitization of the K(+) current response. Using proteomic and electrophysiological approaches, we now show that KCTD12-induced desensitization results from a dual interaction with the G protein: constitutive binding stabilizes the heterotrimeric G protein at the receptor, whereas dynamic binding to the receptor-activated Gßγ subunits induces desensitization by uncoupling Gßγ from the effector K(+) channel. While receptor-free KCTD12 desensitizes K(+) currents activated by other GPCRs in vitro, native KCTD12 is exclusively associated with GABAB receptors. Accordingly, genetic ablation of KCTD12 specifically alters GABAB responses in the brain. Our results show that GABAB receptors are endowed with fast and reversible desensitization by harnessing KCTD12 that intercepts Gßγ signaling.