GABA blocks pathological but not acute TRPV1 pain signals.

Sensitization of the capsaicin receptor TRPV1 is central to the initiation of pathological forms of pain, and multiple signaling cascades are known to enhance TRPV1 activity under inflammatory conditions. How might detrimental escalation of TRPV1 activity be counteracted? Using a genetic-proteomic approach, we identify the GABAB1 receptor subunit as bona fide inhibitor of TRPV1 sensitization in the context of diverse inflammatory settings. We find that the endogenous GABAB agonist, GABA, is released from nociceptive nerve terminals, suggesting an autocrine feedback mechanism limiting TRPV1 sensitization. The effect of GABAB on TRPV1 is independent of canonical G protein signaling and rather relies on close juxtaposition of the GABAB1 receptor subunit and TRPV1. Activating the GABAB1 receptor subunit does not attenuate normal functioning of the capsaicin receptor but exclusively reverts its sensitized state. Thus, harnessing this mechanism for anti-pain therapy may prevent adverse effects associated with currently available TRPV1 blockers.

Pharmacological characterization of GABAB receptor subtypes assembled with auxiliary KCTD subunits.

GABAB receptors (GABABRs) are considered promising drug targets for the treatment of mental health disorders. GABABRs are obligate heteromers of principal GABAB1 and GABAB2 subunits. GABABRs can additionally associate with auxiliary KCTD8, 12, 12b and 16 subunits, which also bind the G-protein and differentially regulate G-protein signaling. It is unknown whether the KCTDs allosterically influence pharmacological properties of GABABRs. Here we show that KCTD8 and KCTD16 slightly but significantly increase GABA affinity at recombinant receptors. However, KCTDs clearly do not account for the 10-fold higher GABA affinity of native compared to recombinant GABABRs. The positive allosteric modulator (PAM) GS39783, which binds to GABAB2, increases both potency and efficacy of GABA-mediated G-protein activation ([(35)S]GTPγS binding, BRET between G-protein subunits), irrespective of whether KCTDs are present or not. Of note, the increase in efficacy was significantly larger in the presence of KCTD8, which likely is the consequence of a reduced tonic G-protein activation in the combined presence of KCTD8 and GABABRs. We recorded Kir3 currents to study the effects of GS39783 on receptor-activated G-protein ßγ-signaling. In transfected CHO cells and cultured hippocampal neurons GS39783 increased Kir3 current amplitudes activated by 1 µM of baclofen in the absence and presence of KCTDs. Our data show that auxiliary KCTD subunits exert marginal allosteric influences on principal GABABR subunits. PAMs at principal subunits will therefore not be selective for receptor subtypes owing to KCTD subunits. However, PAMs can differentially modulate the responses of receptor subtypes because the KCTDs differentially regulate G-protein signaling.

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.