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.

At synaptic terminals, high voltage-activated Ca(v)2.1 and Ca(v)2.2 calcium channels have an essential and joint role in coupling the presynaptic action potential to neurotransmitter release. Here we show that membrane-tethered toxins allowed cell-autonomous blockade of each channel individually or simultaneously in mouse neurons in vivo. We report optimized constitutive, inducible and Cre recombinase-dependent lentiviral vectors encoding fluorescent recombinant toxins, and we also validated the toxin-based strategy in a transgenic mouse model. Toxins delivered by lentiviral vectors selectively inhibited the dopaminergic nigrostriatal pathway, and transgenic mice with targeted expression in nociceptive peripheral neurons displayed long-lasting suppression of chronic pain. Optimized tethered toxins are tools for cell-specific and temporal manipulation of ion channel-mediated activities in vivo, including blockade of neurotransmitter release.

Venoms are evolutionarily fine-tuned mixtures of small molecules, peptides, and proteins-referred to as toxins-that have evolved to specifically modulate and interfere with the function of diverse molecular targets within the envenomated animal. Many of the identified toxin targets are membrane receptors and ion channels. Due to their high specificity, toxins have emerged as an invaluable tool set for the molecular characterization of ion channels, and a selected group of toxins even have been developed into therapeutics. More recently, TRP ion channels have been included as targets for venomous toxins. In particular, a number of apparently unrelated peptide toxins target the capsaicin receptor TRPV1 to produce inflammatory pain. These toxins have turned out to be invaluable for structural and functional characterizations of the capsaicin receptor. If toxins will serve similar roles for other TRP ion channels, only future will tell.

Signalling requires precise spatial and temporal regulation of molecular interactions, which is frequently orchestrated by disordered scaffolding proteins, such as A-kinase anchoring protein 5 (AKAP5). AKAP5 contains multiple Short Linear Motifs (SLiMs) that assemble the necessary components, including the phosphatase Calcineurin, which is anchored via a well-characterised PxIxIT SLiM. Here we show, using a combination of biochemical and biophysical approaches, that Calcineurin also recognises additional lower-affinity SLiMs C-terminal to the PxIxIT motif. Moreover, we demonstrate that the assembly is in reality a complex system in which AKAP SLiMs spanning a wide affinity range act cooperatively to maintain distinct pools of anchored and more loosely held enzyme, analogous to the well-understood transcription factor search complexes on DNA, and compatible with the requirement for both stable anchoring and responsive downstream signalling. We conclude that the AKAP5 C-terminus is enriched in lower-affinity/mini-SLiMs that cooperate to maintain a structurally disordered but tightly regulated signalosome.