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BACKGROUND AND PURPOSE Activation of GIRK channels via G protein-coupled GABAB receptors has been shown to attenuate nociceptive transmission. The analgesic α-conotoxin Vc1.1 activates GABAB receptors resulting in inhibition of Cav 2.2 and Cav 2.3 channels in mammalian primary afferent neurons. Here, we investigated the effects of analgesic α-conotoxins on recombinant and native GIRK-mediated K+ currents and on neuronal excitability. EXPERIMENTAL APPROACH The effects of analgesic α-conotoxins, Vc1.1, RgIA, and PeIA, were investigated on inwardly-rectifying K+ currents in HEK293T cells recombinantly co-expressing either heteromeric human GIRK1/2 or homomeric GIRK2 subunits, with GABAB receptors. The effects of α-conotoxin Vc1.1 and baclofen were studied on GIRK-mediated K+ currents and the passive and active electrical properties of adult mouse dorsal root ganglion neurons. KEY RESULTS Analgesic α-conotoxins Vc1.1, RgIA, and PeIA potentiate inwardly-rectifying K+ currents in HEK293T cells recombinantly expressing human GIRK1/2 channels and GABAB receptors. GABAB receptor-dependent GIRK channel potentiation by Vc1.1 and baclofen occurs via a pertussis toxin-sensitive G protein and is inhibited by the selective GABAB receptor antagonist CGP 55845. In adult mouse dorsal root ganglion neurons, GABAB receptor-dependent GIRK channel potentiation by Vc1.1 and baclofen hyperpolarizes the cell membrane potential and reduces excitability. CONCLUSIONS AND IMPLICATIONS This is the first report of GIRK channel potentiation via allosteric α-conotoxin Vc1.1-GABAB receptor agonism, leading to decreased neuronal excitability. Such action potentially contributes to the analgesic effects of Vc1.1 and baclofen observed in vivo.

αO-Conotoxin GeXIVA is a 28 amino acid peptide derived from the venom of the marine snail Conus generalis. The presence of four cysteine residues in the structure of GeXIVA allows it to have three different disulfide isomers, that is, the globular, ribbon or bead isomer. All three isomers are active at α9α10 nicotinic acetylcholine receptors, with the bead isomer, GeXIVA[1,2], being the most potent and exhibiting analgesic activity in animal models of neuropathic pain. The original report of GeXIVA activity failed to observe any effect of the isomers on high voltage-activated (HVA) calcium channel currents in rat dorsal root ganglion (DRG) neurons. In this study, we report, for the first time, the activity of globular GeXIVA[1,3] at G protein-coupled GABA receptors (GABAR) inhibiting HVA N-type calcium (Cav2.2) channels and reducing membrane excitability in mouse DRG neurons. The inhibition of HVA Ba currents and neuroexcitability by GeXIVA[1,3] was partially reversed by the selective GABAR antagonist CGP 55845. In transfected HEK293T cells co-expressing human GABAR1 and R2 subunits and Cav2.2 channels, both GeXIVA[1,3] and GeXIVA[1,4] inhibited depolarization-activated Ba currents mediated by Cav2.2 channels, whereas GeXIVA[1,2] had no effect. The effects of three cyclized GeXIVA[1,4] ribbon isomers were also tested, with cGeXIVA GAG being the most potent at human GABAR-coupled Cav2.2 channels. Interestingly, globular GeXIVA[1,3] also reversibly potentiated inwardly-rectifying K currents mediated by human GIRK1/2 channels co-expressed with GABAR in HEK293T cells. This study highlights GABAR as a potentially important receptor target for the activity of αO-conotoxin GeXIVA to mediate analgesia. (Figure presented.).

Macrocyclic, disulfide-rich peptides have found widespread applications in drug design and development. Current peptide production strategies rely heavily on solid phase peptide synthesis (SPPS) requiring large amounts of hazardous/toxic reagents and solvents which have negative environmental impacts. A possible solution is to develop a sustainable hybrid production platform incorporating recombinant production of cyclic peptide precursors in yeast followed by enzymatic maturation of these precursors into cyclic peptides using asparaginyl endopeptidases in vitro. Harnessing the efficient secretory pathway of Pichia pastoris, peptide precursors, cloned downstream of the α-mating factor secretion signal, were purified from culture supernatant mitigating the need for complex purification. To demonstrate the broad utility of the platform, three distinct classes of cyclic peptides were produced; two were structurally validated by NMR and shown to be functionally equivalent to their synthetically produced versions. Furthermore, using this platform we report the first recombinant production of any α-conotoxin in its native “globular” conformation. Using scale-up production in bioreactors, cyclic peptide yields of 85–97 mg L−1 of culture were achieved, far exceeding the highest yields so far achieved for cyclic disulfide-rich peptides in any recombinant process. This platform can potentially unlock production and facilitate applications of cyclic disulfide-rich peptides previously inaccessible through large-scale chemical synthesis and reduce their environmental burden.

αO-Conotoxin GeXIVA is a 28 amino acid peptide derived from the venom of the marine snail Conus generalis. The presence of four cysteine residues in the structure of GeXIVA allows it to have three different disulfide isomers, that is, the globular, ribbon or bead isomer. All three isomers are active at α9α10 nicotinic acetylcholine receptors, with the bead isomer, GeXIVA[1,2], being the most potent and exhibiting analgesic activity in animal models of neuropathic pain. The original report of GeXIVA activity failed to observe any effect of the isomers on high voltage-activated (HVA) calcium channel currents in rat dorsal root ganglion (DRG) neurons. In this study, we report, for the first time, the activity of globular GeXIVA[1,3] at G protein-coupled GABA

Activation of GIRK channels via G protein-coupled GABAThe effects of analgesic α-conotoxins, Vc1.1, RgIA, and PeIA, were investigated on inwardly-rectifying KAnalgesic α-conotoxins Vc1.1, RgIA, and PeIA potentiate inwardly-rectifying KThis is the first report of GIRK channel potentiation via allosteric α-conotoxin Vc1.1-GABA

Activation of G protein-coupled inwardly rectifying potassium (GIRK or Kir3) channels leads to membrane hyperpolarization and dampening of neuronal excitability. Here we show that the analgesic α-conotoxin Vc1.1 potentiates inwardly rectifying K+currents (IKir) mediated through native and recombinant GIRK1/2 channels by activation of the G protein-coupled GABABreceptor (GABABR) via aPertussistoxin (PTX)-sensitive G protein. Recombinant co-expression of human GIRK1/2 subunits and GABABR in HEK293T cells resulted in a Ba2+-sensitive IKirpotentiated by baclofen and Vc1.1 which was inhibited by PTX, intracellular GDP-β-S, or the GABABR-selective antagonist CGP 55845. In adult mouse DRG neurons, GABABR-dependent GIRK channel potentiation by Vc1.1 and baclofen hyperpolarizes the cell resting membrane potential with concomitant reduction of excitability consistent with Vc1.1 and baclofen analgesic effectsin vivo. This study provides new insight into Vc1.1 as an allosteric agonist for GABABR-mediated potentiation of GIRK channels and may aid in the development of novel non-opioid treatments for chronic pain.