Analgesic α-conotoxins modulate GIRK1/2 channels via GABAB receptor activation and reduce neuroexcitability

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 GABAB receptor (GABABR) via a Pertussis toxin (PTX)-sensitive G protein. Recombinant co-expression of human GIRK1/2 subunits and GABABR in HEK293T cells resulted in a Ba2+-sensitive IKir potentiated 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 effects in 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.

GABA B Rs functionally couple to GIRK channels to attenuate nociceptive transmission (Blednov et al, 2003) analogous to the analgesic effects observed upon agonist activation of µopioid, α2 adrenergic, muscarinic or cannabinoid receptors (Blednov et al, 2003;Mitrovic et al, 2003;Nagi & Pineyro, 2014;Yudin & Rohacs, 2018), or by direct activation of GIRK channels (Lujan et al, 2014). GIRK channels are tetrameric assemblies of four distinct subunits, GIRK1, GIRK2, GIRK3 and GIRK4, of which only GIRK2 and GIRK4 channels form functional homotetramers whereas GIRK1 and GIRK3 are obligatory heterotetramers requiring other GIRK subunits to enable trafficking to the plasma membrane (Lujan et al, 2014;Lujan et al, 2015). All GIRK subunits, but in particular GIRK1 and GIRK2, are expressed in mammalian sensory neurons where they may couple with G protein-coupled receptors (GPCRs) including GABA B R (Marker et al, 2004;Kanjhan et al, 2005;Gao et al, 2007;Lyu et al, 2015). Direct binding of the G protein (G i/o ) Gβγ subunit to the GIRK channel has been shown to activate and modulate the inhibitory actions of several neurotransmitters (Hibino et al, 2010;Luscher & Slesinger, 2010;).

GIRK1/2 channels are not directly activated by Vc1.1
The involvement of GABA B R in the potentiation of GIRK1/2 and GIRK2 K + currents by analgesic -conotoxins and baclofen was investigated in HEK293T cells by expressing GIRK1/2 alone and using the selective GABA B R antagonist, CGP 55845 upon co-transfection with GABA B R. In the absence of GABA B R expression, application of either baclofen or Vc1.1 failed to potentiate GIRK1/2 K + currents indicating that Vc1.1 and baclofen potentiation of recombinant I Kir is GABA B R-dependent. Bath application of CGP 55845 (1 µM) did not affect the GIRK1/2 K + current but robustly and reversibly antagonized the potentiation of I Kir by both Vc1.1 (80%, n = 5) and baclofen (88%, n = 5), respectively ( Fig. 2A, B & I). In HEK293T cells, CGP 55845 antagonism of Vc1.1-and baclofen-induced potentiation of I Kir was reversible.
Taken together, these results highlight a requirement for GABA B R activation for the potentiation of GIRK1/2 K + currents by Vc1.1 and baclofen.

Vc1.1 potentiates inwardly rectifying K + currents and depresses excitability in mouse DRG neurons
All GIRK channel members have been identified in rat DRG neurons (Gao et al, 2007), however, the presence of GIRK1 and GIRK2 subunits in mouse DRG has been contested (Nockemann et al, 2013). The expression and co-localization of the GIRK1, GIRK2, and 9 GABA B R2 proteins in mouse DRG neurons was investigated using immunofluorescence and confocal microscopy. Fig. 3A shows images of double immunostainings in which positive immunoreactivity for GIRK1 (red) and GIRK2 (green) antibodies is demonstrated. The merged panel shows that these two GIRK subunits co-localize in ~81% of the DRG neurons (13 of 16 counted) present in the imaged field. Antibody validation experiments were performed in HEK293 cells transfected with GIRK1, GIRK2 and GABA B R2 (Fig. EV3). Double staining was also carried out to investigate GABA B R and GIRK channel co-localization in mouse DRG neurons which show abundant expression of GIRK2 and GABA B R2 (Fig. 3B). The fluorescent signals overlapped in 96% of the DRG neurons (96 of 100 counted) indicative of co-localization ( Fig. 3B merge). Due to antibody compatibility constraints, we consider immunodetection of GIRK2 and GABA B R2 as expression proxies, supported by previous studies showing that GIRK1 requires GIRK2 for trafficking to the plasma membrane (Hibino et al, 2010;Luscher & Slesinger, 2010) and that GABA B R can only function as R1/R2 heterodimers (Pinard et al, 2010;Cuny et al, 2012;Frangaj & Fan, 2018;Mao et al, 2020).
Accordingly, baclofen (100 µM) applied to the same neurons increased I Kir by 50.8% (I Kir = 25.8 ± 3.9 pA/pF; n = 15) consistent with GABA B R activation of GIRK channels in mouse DRG neurons (Fig. 3C, D; Table 1). Incubation of mouse DRG neurons with PTX (1 g/ml) for 24 hr inhibited both Vc1.1 and baclofen potentiation of I Kir (n = 5) consistent with our observation in recombinant human GABA B R and GIRK1/2 channels. Inhibition of I Kir by Tertiapin-Q (500 nM) was used as a reporter of GIRK channel activity (Kanjhan et al, 2005). Under these experimental conditions, Tertiapin-Q inhibited the whole-cell inward I K recorded in high external K + by 35.0 ± 4.3% whereas Ba 2+ inhibited 46.4 ± 4.0% inward I K in the same cell (n = 11, p = 0.001, paired t-test) (Fig. 3E). The co-localisation of GABA B R and GIRK channels in mouse DRG neurons and antagonism of Vc1.1-and baclofendependent potentiation of I Kir by Tertiapin-Q are both consistent with the modulation of native GIRK K + currents by -conotoxin Vc1.1 and baclofen via GABA B R in DRG neurons.
In contrast, spontaneous action potential firing was associated with a ~10 mV depolarization of the resting membrane potential from 65 ± 2 mV (control) to 55 ± 1 mV (n = 21; p < 0.0001) in the presence of 500 nM Tertiapin-Q (Fig. 5A, B; Table 2). Exposure of DRG neurons to Tertiapin-Q (500 nM) increased the input resistance more than ~1.5-fold from 326.0 ± 79.7 M to 510.7 ± 114.7 M (n = 14; p = 0.008) (Fig. 5C, Table 2). The rheobase was significantly reduced ~two-fold from 619 ± 122 pA to 376 ± 82 pA (n = 21; p = 0.0003) in the presence of 500 nM Tertiapin-Q (Fig. 5D, Table 2), and the number of action potentials elicited in response to a 25 pA depolarizing current step was increased more than three-fold (n = 21; p = 0.0011) (Fig. 5E, Table 2). In contrast, application of Vc1.1 in the presence of the GABA B R antagonist CGP 55845 did not alter passive or active membrane properties of mouse DRG neurons (n = 7; Fig. EV4A, B). Taken together, the results tabulated in Table 2 suggests that Vc1.1 and baclofen potentiation of GIRK-mediated K + currents via GABA B R activation acts to dampen DRG neuronal excitability, and thereby contribute to the reported anti-nociceptive activities in animal models of mechanical allodynia and chronic pain (Klimis et al, 2011;Castro et al, 2017).

Discussion
The major outcome of this study is the demonstration that analgesic α-conotoxin Vc1.1 modulates both GIRK1/2 and GIRK2 K + currents through G protein-coupled GABA B R in HEK293T cells and mouse DRG neurons. Vc1.1 and baclofen potentiate recombinant GIRK1/2 and GIRK2 K + currents only when co-expressed with GABA B R subunits R1 and R2. Inhibition of Vc1.1 potentiation of GIRK1/2 I Kir by the selective GABA B R antagonist, CGP 55845, and the lack of effect of Vc1.1 on GIRK-mediated I Kir in the absence of GABA B R expression, supports GABA B R as the primary target and not the GIRK channel itself. Our study also provides immunocytochemical support for the expression of GIRK channel protein in mouse DRG neurons.
GABA B receptor biology and pharmacology in pain processing has been studied extensively and the modulation of HVA N-type calcium channels and/or GIRK channels by GABA B R agonists, GABA and baclofen, is a recognized mechanism of action (Malcangio, 2018;Benke, 2020). Our findings propose another plausible mechanism of action through which analgesic α-conotoxin Vc1.1 regulates membrane excitability in sensory neurons. Disruption of GABA B R-GIRK channel association in recombinant GABA B R/GIRK complexes HEK293 cells and native DRG neurons precludes I Kir potentiation by baclofen and Vc1.1, consistent with previous studies supporting GIRK's involvement in anti-nociception in models of neuropathic 13 pain (Ippolito et al, 2005;Marker et al, 2005) and via satellite ganglion cells of the trigeminal ganglia (Takeda et al, 2015). Fluorescence resonance energy transfer (FRET) studies show that upon GPCR activation Gβγ preferentially binds to the GIRK channel whereas Gα binds to GABA B R (Fowler et al, 2007;Laviv et al, 2011;Richard-Lalonde et al, 2013). Agonist dependent and GPCR-mediated activation of GIRK channels is proposed to occur via direct Gβγ subunit interaction with the channel in a membrane-delimited fashion (Lujan et al, 2014;Yudin & Rohacs, 2018). The recruitment of Gβγ by the GIRK channel is essential in GPCR-mediated potentiation, whereas other prototypical agonists that activate GIRK channels in the absence of a GPCR or in a G protein-independent manner, do not require the Gβγ heterodimer (Jelacic et al, 2000;Wydeven 14 et al, 2014). Accordingly, our results provide evidence that Vc1.1 targeting of GABA B R modulates GIRK channels via activation of Gα i/o protein and Gβγ signaling as proposed in Fig. 6. α-Conotoxin Vc1.1 has been shown to act as an allosteric agonist as it does not bind to the canonical ligand binding site of GABA B R (Huynh et al, 2015), nevertheless, its precise binding site on the GABA B R remains to be identified (Sadeghi et al, 2017). In the present study, we show that Vc1.1 potentiates GIRK1/2 channels albeit with 10-to 100-fold lower potency than for inhibition of human and rat Ca v 2.2 channels Huynh et al, 2015;Hone et al, 2018). In a comparable manner, it was shown that even though GIRK and Cav2.2 channels are regulated by GABA B R by an analogous mechanism, the EC 50 for GIRK potentiation by baclofen is ~100-fold higher than for inhibition of HVA Ca 2+ channels in rat supraoptic neurons (Harayama et al, 2014).
A durable macromolecular complex between GIRK-G protein-GABA B R upon agonist binding (Padgett & Slesinger, 2010) that occurs through collision coupling (rather than a preexisting complex) has been proposed (Kahanovitch et al, 2017). It is conceivable that Vc1.1 targets the complex activating Gα i/o proteins and Gβγ via GABA B R to reversibly potentiate GIRK channel signaling. This is consistent with previous studies whereby agonist stimulation of Gα i/o -coupled GPCRs results in increased concentration of Gβγ heterodimers available to activate GIRK channels whereas the Gα i/o subunit enhances G protein-GPCR association in GPCR-GIRK complexes (Touhara & McKinnon, 2018).

Expression and modulation of GIRK channels in sensory neurons
GIRK channels are expressed in neurons of several regions of the central nervous system including the spinal cord (Ponce et al, 1996;Marker et al, 2006;Lujan & Aguado, 2015) supporting their role in modulating several neurological disorders or disease conditions 15 (Mayfield et al, 2015;Rifkin et al, 2017). Functional coupling to GABA B R has been described in rat (Gao et al, 2007) and human DRG neurons (Castro et al, 2017), yet the function of GIRK channels in mouse peripheral neurons has remained largely unexplored.
Functional recordings of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2/PIP2] activated GIRK currents in postnatal day zero (P0) mouse DRG neurons in response to neurotrophin-mediated cleavage of p75(NTR) have been reported (Coulson et al, 2008). Here, we show functional coupling between GIRK1/2 (and GIRK2) channels and GABA B R in mouse DRG neurons and heterologous expression systems evidenced by the potentiating actions of baclofen and Vc1.1 on I Kir . This is supported by co-localization of GIRK1, GIRK2, and GABA B R2 subunits in overlapping populations of small and medium diameter neurons of adult mouse DRG (Fig. 3A, B). In rat pyramidal neurons, GIRK channel density is higher in dendrites than in the soma (Takigawa & Alzheimer, 1999;Chen & Johnston, 2005) perhaps posing a limitation to our work as it relies on somatic DRG neuron I Kir recordings (within 24 hours of acute dissociation). The relatively low I Kir density in adult mouse DRG neurons implies that most processes and the dendritic pool of GIRK channels that may be associated are lost upon isolation. Future efforts will be directed to determine the relative abundance of GIRK channel isoforms at the mouse peripheral nerve terminals.

Neuroexcitability and implications for GIRK channel
Analgesic α-conotoxins inhibit α9α10 nAChRs directly and HVA calcium (Cav2.2 and Cav2.3) channels via GABA B R activation (Sadeghi et al, 2017;Kennedy et al, 2020). The GABA B R is abundantly expressed in the somatosensory (afferent) nervous system and its role in mitigating mechanical allodynia and chronic visceral hypersensitivity in animal models of neuropathic and visceral pain, respectively, has been demonstrated (Castro et al, 2017;Loeza-Alcocer et al, 2019). Furthermore, α-conotoxin Vc1.1 has been shown to reduce excitability in human DRG neurons via a GABA B R-mediated mechanism. In the present study, we demonstrate that analgesic α-conotoxins Vc1.1, RgIA and PeIA potentiate heteromeric and homomeric GIRK currents via GABA B R. However, the GABA B R agonists, baclofen and GABA, tested at maximally effective concentrations, were more efficacious than the analgesic α-conotoxins, Vc1.1, RgIA, and PeIA in potentiating of GIRK-mediated I Kir .
Hyper-excitability and ectopic firing are characteristic sensory neuron responses to nerve injury and chronic pain (Amir et al, 2005;Berta et al, 2017). Control of cell-surface expression and/or the biophysical properties of ion channels in sensory neurons is central to membrane excitability such that the activation of GIRK channels hyperpolarizes the neuron thus reducing excitability. Our findings are consistent with the functional expression of GIRK channels in mouse DRG neurons and their involvement in GABA B R-mediated anti-nociceptive activity in response to baclofen and -contotoxin Vc1.1. The modulation of HVA N-type (Cav2.2) calcium channels and/or GIRK channels by GABA B R agonists, GABA and baclofen, is recognized as analgesic mechanism of action. A new scenario involving diverse signaling mechanism(s) underlying α-conotoxin Vc1.1's analgesic effect is emerging. Synergistic action of Vc1.1 to inhibit Cav2.2/2.3 and potentiate GIRK channels reducing neuronal excitability likely contributes to its anti-nociceptive activity. Lastly, the precise molecular details of GIRK I Kir potentiation via GABA B R activation remains to be elucidated, therefore, further studies of sensory neuron GIRK channels and their coupling to GPCRs will follow.

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HEK293T cells were transiently co-transfected with plasmid cDNAs encoding human GIRK 1 (KCNJ3 or Kir3.1) and/or GIRK 2 (KCNJ6 or Kir3.2) (both pcDNA3 based and were kindly provided by Dr Paul Slesinger, Mt Sinai, New York, USA) and human GABA B1 and GABA B2 subunits (OriGene Technologies, Inc, Rockville, MD USA) using Lipofectamine 2000 (ThermoFisher Scientific, Australia). Cells were seeded in 12-well plates the day before transfection and 2 g of each plasmid DNA with 0.2 g of green fluorescent protein (GFP) were transfected following the manufacturer's protocol of Lipofectamine. Cells were then replated on 12 mm glass coverslips 48-72 hours after transfection for subsequent patch clamp studies.

Isolation and culture of rodent dorsal root ganglion (DRG)
All animal procedures were conducted in accordance with the University of Wollongong Animal Ethics Committee (AE16/10) guidelines and regulations. Adult C57BL/6 mice (8-10 weeks old) were purchased from Australian BioResources (Moss Vale, NSW, Australia) and housed in individually ventilated cages with a 12 h light/dark cycle; food pellets and water were available ad libitum.
Mice were euthanized by isoflurane inhalation followed by rapid decapitation. A laminectomy exposed the DRGs in the thoracic and lumbar regions. DRGs were harvested and transferred to ice cold (4 o C) Hanks Buffered Saline Solution (HBSS), free of Ca 2+ and Mg 2+ .
DRGs were then trimmed, removing central and peripheral nerve processes, digested in HBSS containing Collagenase type II (3 mg/ml; Worthington Biomedical Corp., Lakewood, NJ, USA) and Dispase (4 mg/ml; GIBCO, Australia). The DRGs were incubated in this enzyme mix at 37 0 C in 5% CO 2 in a humidified incubator for 40 min. The ganglia were then rinsed three to four times in warm (37 o C) F12/Glutamax (Invitrogen) media supplemented with 10% heat-inactivated foetal bovine serum (FBS; GIBCO, ThermoFisher Scientific, Australia) and 1% penicillin/streptomycin. The ganglia were dispersed by mechanical trituration with progressively smaller fire polished glass Pasteur Pipettes. The supernatant was filtered through a 160 m nylon mesh (Millipore Australia Pty Ltd, North Ryde, NSW) to remove undigested material. The dissociated DRG neurons were then plated onto poly-D-lysine coated 12 mm cover glass (Sigma-Aldrich, Australia). Neurons were left to attach for ~3 hours at 37C after which media was added and then incubated overnight and used within 24 hrs.
Whole-cell GIRK channel K + currents were recorded from isolated DRG neurons superfused with high K + extracellular solution containing (in mM): 120 NaCl, 20 KCl, 2 CaCl 2 , 1 MgCl 2 , 10 HEPES and 10 Glucose, pH 7.4 with NaOH (~320 mOsmol.kg -1 ). Patch pipettes were filled with the same intracellular solution as above. Currents were elicited using a similar voltage protocol to that used for HEK293 cells (ramp from 100 mV to +40 mV, at a frequency of 0.1 Hz, from a holding potential of 40 mV and sampled at 10 kHz). Recordings were electronically compensated for cell capacitance and series resistance to ~80%. All solutions including the pharmacological agents were superfused using a Peristaltic pump at an exchange speed of 1 ml/min. The volume of the experimental chamber was ~500 µl. All experiments were carried out at room temperature (21-23 o C).

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Dissociated DRG neurons plated on poly-D-lysine coated glass coverslips were washed twice with HBSS and fixed using Zamboni's fixative solution containing 1.6% formaldehyde (Australian Biostain Pty Ltd, Traralgon, VIC, Australia) for 15 min. Cells were permeabilized with 0.1% Triton X-100 for 10 min. Non-specific antibody binding was reduced by incubating the cells for 1 hour in HBSS based blocking solution containing 5% goat serum, 5% BSA and 0.1% Triton X-100. All primary and secondary antibodies were diluted in blocking solution to the working concentration. For DRG neuron double staining of GIRK1 and GIRK2 channels, both anti-rabbit polyclonal GIRK1 (1:100 dilution) and anti-guinea pig polyclonal GIRK2 (1:100 dilution) antibodies were added simultaneously and incubated at 4 o C overnight. Following three 5 min washes with blocking solution, the samples were incubated with the secondary antibody, a donkey anti-rabbit IgG (1:500) 1 hr, in the dark, at room temperature (RT). Triplicate washes with blocking solution preceded addition of the goat anti-guinea pig IgG secondary antibody (1:500) which again was incubated 1 hr at RT, in the dark. For GIRK2 and GABA B R2 double staining, a mixture of the primary antibodies rabbit polyclonal GIRK2 (1:100) and rabbit monoclonal GABA B R2 (1:400) were incubated at 4 o C overnight, washed and followed by sequential incubations with donkey anti-rabbit IgG secondary antibody (1:500, 1 hr, RT), wash and goat anti-rabbit IgG secondary (1:500, 1 hr, RT). Control experiments for single and double staining procedures were performed by omitting the primary antibodies. Nuclei were counterstained with DAPI (1:5000, 5 min, RT), washed, covered with mounting media (Dako North America Inc., Carpinteria, CA, USA), sealed and stored at 4 o C. Images were recorded with a Leica SP8 confocal microscope using 40x oil immersion objective. Images were analysed using ImageJ (Java, NIH) and LAS X softwares (Leica, Macquarie Park, NSW, Australia). where n is the Hill coefficient, EC 50 is the half-maximal response.

Data analysis and statistics
In current clamp experiments, all DRG neurons accepted for analysis had a RMP more negative than 40 mV. The whole-cell input resistance (R i ) was measured in the absence and presence of various modulators by calculating the slope of the linear fit of hyperpolarizing responses to current steps from 5 to 40 pA in 5 pA increments. Rheobase threshold was measured by applying a series of depolarizing current steps (500 ms) in 5 pA increments to determine which first evoked action potential discharge. The number of action potentials was counted at a current injection of 2x rheobase in both control conditions and in the presence of modulators or pharmacological agents.
Statistical significance was determined between two groups by using paired t-test or Student's t-test. Multiple group comparisons were done by one-way ANOVA with Tukey's test.
All results are presented as mean ± SEM and n, number of observations. Statistical significance is shown by asterisk *indicating p < 0.05, **indicating p < 0.005 and ***indicating p < 0.001 and ****indicating p < 0.0001.

F
Corresponding diary plot to (E) showing peak K + current amplitude at 100 mV as a function of time in response to bath application of Vc1.1 (blue), baclofen (red) or Ba 2+ (grey).  B Corresponding dairy plot of K + current amplitude at 100 mV (A) as a function of time.
C Representative K + currents elicited from cells co-expressing GABA B R and GIRK1/2 channels with 24 hr pre-treatment with 1 µg/ml Pertussis toxin (PTX).
D Corresponding diary plots of peak K + currents at 100 mV (C) as a function of time. I Bar graph of I Kir density recorded at 100 mV in response to 1 µM Vc1.1 (29.5 ± 1.52 pA/pF, blue) or 100 µM baclofen (68.4 ± 5 pA/pF, red) in HEK293T cells co-expressing GIRK1/2 channels and GABA B R (control). Potentiation of I Kir density by Vc1.1 and baclofen was significantly reduced by 1 µM CGP 55845. Following pre-treatment with PTX (1 µg/ml) or inclusion of either GDP-β-S (500 µM) or Gβγ scavenger, GRK2i (10 µM), in the intracellular pipette solution, I Kir density was similarly attenuated compared to peak current density measured for Vc1.1 or baclofen in control condition. Data are expressed as mean ± SEM and statistical significance, **** p < 0.0001 vs control; one-way ANOVA followed by Tukey's post hoc test; number of experiments is given in parentheses. A Immunolabeling of mouse DRG neurons and visual inspection by confocal microscopy revealed both GIRK1 and GIRK2 channels express and show co-localization with each other using antibodies directed against GIRK1 and GIRK2 channels.  and number of APs, p = 0.0474 (iv) in response to 500 ms depolarizing current step in small to medium diameter neurons of adult mice DRG. Data represented as mean ± SEM; Paired t-test.
D Peak K + current amplitude plotted as a function of time during sequential application of ImI, Vc1.1, baclofen, and Ba 2+ .

E
Quantification of the effect of reduced (linear) Vc1.1 and ImI compared to globular Vc1.1 and baclofen. Data represent mean ± SEM. Statistical significance, **** p < 0.0001; oneway ANOVA. Number of experiments is given in parentheses.  Number of experiments is given in parentheses.