ACh chloride (Cat# A6625), dimethyl sulfoxide (DMSO, Cat# D2650), LPS (E. coli 011:B4, Cat# L2630), macrophage colony-stimulating factor (M-CSF, Cat# SRP3110), mecamylamine hydrochloride (Mec, Cat# M9020), phorbol 12-myristate 13-acetate (PMA, Cat# P1585), PC (Cat# P0378), recombinant mouse interferon-γ (INF-γ, Cat# IF005), sodium bicarbonate (Cat# R8758), 3-aminobenzoate methanesulfonate (MS-222, Cat# E10521) and all chemicals for saline Ringer’s buffer preparations were purchased from Merck (Darmstadt, Germany) or from Millipore Sigma (St. Louis MO, United States). LPS from E. coli O26:B6 was purchased from Merck (Cat# L2654) or eBioscience (Thermo Fisher Scientific, Waltham MA, United States; Cat# L500497693). Gibco penicillin-streptomycin and L-glutamine were purchased from Thermo Fisher Scientific. BzATP [2′(3′)-O-(4-benzoyl-benzoyl)ATP trieethylammonium salt] was purchased from Jena Bioscience (Jena, Germany), EDTA from bioWORLD (Dublin, OH, United States) and recombinant human INF-γ from R&D Systems (Minneapolis, MN, United States).

pCF3-diEPP was synthesized as previously published (Quadri et al., 2017) and dissolved in DMSO. When appropriate, control experiments were performed with the corresponding concentrations of DMSO without pCF3-diEPP. The conopeptides [V11L, V16D]ArIB (specifically antagonizing α7 nACh (Whiteaker et al., 2008; Hone et al., 2009, 2010) and RgIA4 (antagonizing nAChRs composed of subunits α9 and α10 (Christensen et al., 2017; Romero et al., 2017; Grau et al., 2018) were produced and characterized as described previously (Innocent et al., 2008; Romero et al., 2017).

Male and female mice gene-deficient in Chrna7 (α7), Chrna9 (α9) or Chrna9/Chrna10 (α9/α10) as well as corresponding wild-type (WT) mice were used for isolation of bone marrow cells. The α9 (Chrna9tm1Bjmy MGI:5787807) and α10 (Chrna10tm1Bjmy MGI:5787808) knockout lines were generated by Genoway, Inc. (Lyon, France) and backcrossed to congenicity at the Boys Town National Research Hospital, Omaha, NE, United States (BTNRH) using C57Bl/6J mice from The Jackson Laboratory (JAX). The α7 animals were originally received from JAX and a colony maintained at BTNRH. The α7 animals were crossed with the α9 or α9/α10 to produce double and triple knockout lines. The animals used in the studies reported here were derived by crossing the double and triple knockout lines with C57Bl/6J mice to produce single α7 and α9 KOs and double α9/α10 KOs. This was done to produce backgrounds in the strains that were comparable. The use of the animals in the experiments reported here was approved by the BTNRH Institutional Animal Care Committee (protocol #16-04). Mice were bred and housed at BTNRH and shipped to the Laurentian University (Sudbury, Canada). Experimental animals received humane care according to NIH “Guide for the Care and Use of Laboratory Animals.” Animal experiments were reviewed and approved by the Laurentian University Animal Care Committee (file number 6013816). Genotypes were analyzed by PCR as described previously (St-Pierre et al., 2016; Morley et al., 2017).

Bone marrow cells were flushed from cleaned femoral and tibial bones using a 10 ml syringe and a 26–27-gauged needle. Debris was removed by passing the suspension through a 40 μm BD Falcon Cell Strainer (Becton, Dickinson (BD), Franklin Lakes, NJ, United States). Red blood cells were lysed by RBC Lysis buffer, as per the supplier’s protocol (BioLegend, San Diego, CA, United States). Cells were pelleted at 510 g for 10 min and 1 × 10⁶ cells/ml were resuspended in complete medium [RPMI 1640 medium (Cytiva, Marlborough, MA, United States) supplemented with 10% fetal calf serum (FCS; Life Technologies, Thermo Fisher Scientific, Cat# LS12484028), 50 U penicillin/ml, 50 μg streptomycin/ml and 2 mM L-glutamine] and 10 ng/ml M-CSF. 1 × 10⁶ cells per well were seeded in Costar 12-well plates (Corning, NY, United States) and cultured for 72 h. Thereafter, 1 ml of fresh complete medium was added supplemented with a final concentration of 10 ng/ml M-CSF, 10 ng/ml IFN-γ and 10 pg/ml LPS (E. coli O26:B6) to induce their differentiation into M1 macrophages. Cells were cultured for another period of 48 h. Then, the bone marrow-derived macrophages (BMDMs) were stimulated with higher concentrations of LPS (100 ng/ml) as described previously (Godin et al., 2020) in the absence and presence of PC (100 μM) or pCF3-diEPP (100 μM). Cell-free supernatant was harvested 6 h later and stored at −20°C for cytokine measurements.

For whole blood experiments, blood samples were obtained from healthy (self-reported) female and male non-smoking adult volunteers. Experiments were reviewed, approved, and performed in accordance with the policies outlined by the Laurentian University Research Ethics Board for Research Involving Human Participants (file number 6012214) and in accordance with the Helsinki Declaration. To prevent blood clotting, blood samples were collected in BD Vacutainer glass blood collection tubes containing sodium heparin (BD, Cat# B366489) and were individually shaken periodically during and after the blood draw. 200 μl of whole blood was added per well in Costar 24-well flat-bottom plates (Stemcell, Vancouver, Canada, Cat# 38017). Whole blood was then diluted in 700–800 μl of Sigma RPMI 1640 medium (Merck, Cat #R8758). Wells containing whole blood diluted in RPMI were pre-treated with 100 μM pCF3-diEPP or 0.1% DMSO for 1 h at 37°C. Immediately following pre-treatment, wells were stimulated with 100 ng/ml LPS (E. coli O26:B6) for 24 h (Bruchfeld et al., 2010). Controls wells were treated with an equivalent volume of Sigma RPMI 1640 medium. Following the 24 h of stimulation with LPS, cells were spun down (2,000 g, 5 min, RT). The cell-free supernatants were collected and stored at −20°C for later cytokine measurements.

Monocytic THP-1 cells (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were cultured in medium (Gibco™ RPMI 1640 medium (Thermo Fisher Scientific, Cat# 11530586) supplemented with 10% FCS (CellConcepts, Umkirch, Germany) and 2 mM L-glutamine). To investigate IL-1β release, monocytic cells were resuspended in FCS-free RPMI medium and transferred to 48-well plates (Greiner Bio-One, Frickenhausen, Germany; 0.5 × 10⁶ cells/0.5 ml and per well). Cells were primed for 5 h with 1 μg/ml LPS (E. coli O26:B6) as described previously (Hecker et al., 2015). After priming, the P2X7 receptor agonist BzATP (100 μM) was added for 40 min in presence or absence of different concentrations of cholinergic agonists (PC, pCF3-diEPP) and antagonists (mecamylamine, [V11L, V16D]ArIB and RgIA4). After cell treatment, cells were spun down (500 g, 8 min, 4°C), the supernatants were collected and stored at −20°C for later cytokine measurements.

In addition, THP-1 cells were differentiated into M1-like macrophages. Monocytic THP-1 cells were resuspended in complete medium supplemented with 50 U penicillin/ml and 50 μg streptomycin/ml and seeded in 12-well plates (Greiner; 0.3 × 10⁶ cells/ml and per well). Cells were differentiated into macrophages by 24 h incubation with 50 nM PMA followed by 24 h incubation in complete medium supplemented with 50 U penicillin/ml, 50 μg streptomycin/ml. Macrophages were then polarized to M1-like macrophages by incubation in fresh complete medium, supplemented with 50 U penicillin/ml, 50 μg streptomycin/ml, 10 ng/ml recombinant human IFN-γ and 10 ng/ml LPS (E. coli 011:B4) for 72 h. To investigate IL-1β release, the medium was replaced by fresh complete medium and the cells were stimulated with LPS (E. coli O26:B6) and BzATP as described for monocytic THP-1 cells.

Peripheral Blood Mononuclear Cells (PBMCs) were obtained from healthy (self-reported) female and male non-smoking adult volunteers. The study was approved by the ethics committee of the medical faculty Giessen, Germany (No. 90/18) and performed in accordance with the Helsinki Declaration. Blood was drawn into sterile syringes containing EDTA (1 mM per ml blood; bioWORLD, Dublin, OH, United States). LPS (E. coli O26:B6; 5 ng/ml) was added to blood samples shortly before PBMC isolation and PBMCs were separated using Leucosep gradients (Greiner) as described previously (Hecker et al., 2015). Thereafter, PBMCs were cultured for 3 h in 24-well plates (Greiner) at a density of 0.5 × 10⁶ cells/0.5 ml in Monocyte Attachment Medium (PromoCell, Heidelberg, Germany). Non-adherent cells were removed, and fresh Sigma RPMI 1640 medium was added. Stimulation with BzATP in the presence or absence of pCF3-diEPP was done as described for THP-1 cells. After cell treatment, cells were spun down (500 g, 8 min, 4°C), the supernatants were collected and stored at −20°C.

In samples obtained in the mouse BMDM experiments, Mouse IL-6 Flex Set (BD, Cat# 558301), Mouse TNF Flex Set (BD, Cat# 558299), Mouse IL-10 Flex Set (BD, Cat# 558300) and Mouse IL-1β Flex Set (BD, Cat# 560232) were used to measure cytokine concentrations with the BD Cytometric Bead Array technique per the supplier’s protocol. Human IL-1β Flex kit (BD, Cat# 558279), Human IL-6 Flex kit (BD, Cat# 558276), Human IL-10 Flex kit (BD, Cat# 558274), and Human TNF Flex kit (BD, Cat# 560112) were used to measure cytokine concentrations in the supernatants of human whole blood experiments according to the manufacturer’s protocol. The bead fluorescence in the samples was quantified by flow cytometry using a BD FACS Canto II flow cytometer in conjunction with BD FACS Diva Software (v6.1.3). Cytokine concentrations were interpreted using BD FCAP Array Software (v3.0).

IL-1β concentrations in supernatants obtained in experiments on THP-1 cells (monocytic, M1-like) and human PBMCs were measured using the Human IL-1 beta/IL-1F2 DuoSet enzyme-linked immunosorbent assay (ELISA) from R&D Systems. To estimate cell viability at the end of the cell culture experiments on human PBMCs, the non-radioactive cytotoxicity assay (Promega, Madison, WI, United States) was used to measure lactate dehydrogenase (LDH) activity in cell-free supernatants as indicated by the supplier. LDH values are given as percentage of the total LDH content of lysed control cells.

Plasmid DNA encoding the human CHRNA7 was obtained from Dr. J. Lindstrom (University of Pennsylvania, Philadelphia, PA, United States). The human resistance-to-cholinesterase 3 (RIC3) clone was obtained from Dr. M. Treinin (Hebrew University, Jerusalem, Israel) and co-injected with CHRNA7 to improve the level and speed of receptor expression without affecting their pharmacological properties (Halevi et al., 2003). Plasmid DNA encoding the human CHRNA9 and CHRNA10 as well as the human 43 kDa receptor-associated protein of the synapse (RAPSN) with codon optimization for expression in Xenopus laevis were obtained from Eurofins Genomics (Ebersberg, Germany), (Richter et al., 2016). After linearization and purification of the plasmid DNAs, RNAs were prepared using the mMessage mMachine in vitro RNA transcription kit (Ambion, Austin, TX, United States).

Frogs were maintained in the Animal Care Service facility of the University of Florida, and all procedures were approved by the University of Florida Institutional Animal Care and Use Committee (approval number 202002669). In brief, the animals were first anesthetized for 15–20 min in 1.5 l frog tank water containing 1 g of MS-222 buffered with sodium bicarbonate. Oocytes were obtained surgically from mature female Xenopus laevis (Nasco, Ft. Atkinson, WI, United States) and treated with 1.4 mg/ml type 1 collagenase (Worthington Biochemicals, Freehold, NJ, United States) for 2–4 h at RT in Ca²⁺-free Barth’s solution (88 mM NaCl, 1 mM KCl, 2.38 mM NaHCO₃, 0.82 mM MgSO₄, 15 mM HEPES, and 12 mg/l tetracycline, pH 7.6) to remove the ovarian tissue and the follicular layers. Stage V oocytes were injected with 4–6 ng CHRNA7 RNA and 2–3 ng RIC3 RNA (2:1 ratio) in 50 nl water, or with 12 ng CHRNA9 RNA and 3 ng RAPSN RNA, or along with 12 ng CHRNA10 RNA in 50 nl water. Oocytes were maintained in Barth’s solution containing 0.32 mM Ca(NO₃)₂ and 0.41 mM CaCl₂, and recordings were carried out 2–20 days after injection.

Two-electrode voltage-clamp experiments were conducted using OpusXpress 6000A (Molecular Devices, Union City, CA, United States) (Papke and Stokes, 2010). Both the voltage and current electrodes were filled with 3 M KCl. Oocytes were voltage-clamped at −60 mV at RT. The oocytes were perfused with Ringer’s solution (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl₂, 10 mM HEPES, 1 μM atropine, pH 7.2) at 2 ml/min. To evaluate the effects of experimental compounds, responses were compared to control ACh-evoked responses, defined as the average of two initial applications of 60 μM ACh made before test applications. Drug applications were 12 s in duration followed by 181 s washout periods. In some experiments after obtaining the control responses, the bath perfusion solution was switched to the alternative buffer B, and test solutions delivered during the buffer B perfusion were made up in buffer B solution.

The responses were calculated as both peak-current amplitudes and net charge, as previously described (Papke and Porter Papke, 2002). Data were collected at 50 Hz, filtered at 20 Hz, and analyzed by Clampfit (Molecular Devices) and Excel (Microsoft, Redmond, WA, United States). Data were expressed as means ± SEM from at least four oocytes for each experiment and plotted with Kaleidagraph 4.5.2 (Abelbeck Software, Reading, PA, United States). Each episode of data acquisition was a total of 210 s and included an initial 30 s period used to define the baseline for the drug-evoked responses. After 30 s, drugs were applied, and the following 120 s were defined as the drug response period for analysis. Data reported for α7 were net charged, while peak currents were used for α9 and α9/α10 responses since these receptors do not show the same concentration dependent desensitization that invalidates peak currents as measurements of α7 concentration-dependent responses (Papke and Porter Papke, 2002). Multi-cell averages were calculated for comparisons of complex responses. Averages of the normalized data were calculated for each of the 10,322 points in each of the 206.44 s traces (acquired at 50 Hz), as well as the standard errors for those averages.

Results obtained in the LPS- and BzATP-induced cytokine release experiments were analyzed using SPSS (Version 23, IBM, Armonk, NY, United States). The IC₅₀ value of pCF3-diEPP in human PBMCs was determined in GraphPad Prism (Version 6, GraphPad Software, San Diego, CA, United States) by fitting log-transformed concentration values and the original effect data. Paired data were analyzed first by the Friedman test followed by the Wilcoxon signed-rank test.

In two-electrode voltage-clamp experiments, the comparisons of results were made using one-way ANOVA or using t-tests between the pairs of experimental measurements. In cases, where multiple comparisons were made, a Bonferroni correction for multiple comparisons (Aickin and Gensler, 1996) was applied to correct for possible false positives. A value of p ≤ 0.05 was used to constitute a minimum level of significance. The statistics were calculated using an Excel template provided in Microsoft Office or ANOVA protocols in Kaleidagraph (4.5.2 Abelbeck Software, Reading, PA). Concentration-response relationships utilized data obtained over a range of concentration at roughly half log units. Average responses were fit to the Hill equation by the Levenberg-Marquardt algorithm in Kaleidagraph.

The number (n) of individual experiments is indicated in the Results section and the Figures. When primary cells were investigated, the n-number represents data obtained from the cells of individual mice or humans. In experiments with cell lines, the n-number refers to independent experiments, which were performed on different days with different cell passages. No outliers were excluded from the analyses. All two-electrode voltage-clamp experiments began with 8 oocytes voltage clamped and treated in parallel. Only cells, which lost voltage-clamp or otherwise failed to remain viable through the measurements, were excluded from the analyses but no viable cells. Data were visualized using Inkscape version 0.48.5 r10040 (Free and Open Source Software licensed under the GPL).

Agonists of nAChRs have been shown to inhibit the production and release of pro-inflammatory cytokines by mononuclear phagocytes (Kox et al., 2009). Here, we investigated in M1-like murine BMDMs whether the unconventional nAChR agonist PC (Hecker et al., 2015; Richter et al., 2016) and the α7 silent agonist pCF3-diEPP (Quadri et al., 2017) change the levels of IL-1β, IL-6, TNF-α and IL-10 in cell culture supernatants in response to treatment with LPS (1 μg/ml for 5 h; Figure 1). Throughout, IL-1β levels remained below the level of detection. LPS induced the release of IL-6 in the range of 1 to 1,092 pg/ml, TNF-α in the range of 1,012 to 12,672 pg/ml and IL-10 in the range of 13 to 561 pg/ml. The LPS-induced release of these cytokines was not affected in the presence or absence of PC (100 μM; Figures 1A,C,E). In contrast, pCF3-diEPP (100 μM) significantly reduced IL-6 (Figure 1B; p = 0.007; n = 5) and IL-10 concentrations (Figure 1F; p = 0.007; n = 5) but no effect was seen on TNF-α (Figure 1D). In BMDMs from mice deficient in nAChR subunits α7, α9 or α10 as well as in mice with a double-deficiency in nAChR subunits α9 and α10, essentially the same results were obtained (Figure 1). Only the pCF3-diEPP-mediated attenuation of the secretion of IL-6 was not seen in BMDMs from nAChR α7 gene-deficient mice (Figure 1). These results are in line with previous reports that pCF3-diEPP functions as a silent agonist at nAChR α7 (Quadri et al., 2017). The fact that PC does not inhibit the LPS-induced production and secretion of cytokines, is most probably due to its expected instability, which is further confirmed in experiments described below (Supplementary Figure 1). Therefore, PC was omitted from the experiments on human whole blood cells.

Human whole blood cells secreted IL-6 (in the range of 3 to 22,000 pg/ml), TNF-α (1 to 2,000 pg/ml), IL-1β (in the range of 3 to 5,600 pg/ml) and IL-10 (3 to 800 pg/ml), in response to LPS (100 ng/ml for 24 h; Figure 2). The LPS-induced release of IL-6 (p = 0.020), TNF-α (p = 0.003) and IL-1β (p = 0.0002) was significantly inhibited by pCF3-diEPP (Figures 2A–C), whereas no such effect was seen on IL-10 (p = 0.117; Figure 2D). In control experiments, in which LPS was omitted, pCF3-diEPP did not change the cytokine levels compared to supernatants of untreated cells (Figure 2).

We showed before that PC functions as an unconventional agonist at monocytic nAChRs containing subunits α7, α9 and α10 and as a potent inhibitor of the BzATP-induced maturation of IL-1β in monocytic U937 cells and human as well as murine peripheral mononuclear blood leukocytes (Hecker et al., 2015; Richter et al., 2016). To test if PC and pCF3-diEPP exert similar effects on monocytic THP-1 cells (Figure 3A) and THP-1-derived M1 macrophages (Figure 3B), cells were primed with LPS (1 μg/ml) for 5 h, followed by stimulation with the P2X7 receptor agonist BzATP (100 μM) for another 40 min in the presence or absence of PC (200 μM) or pCF3-diEPP (100 μM). Thereafter, the concentration of IL-1β was measured in cell culture supernatants by ELISA. As expected, untreated cells and cells primed with LPS did not release relevant amounts of IL-1β, whereas stimulation with BzATP resulted in elevated IL-1β levels released by monocytic THP-1 cells in the range of 19 to 122 pg/ml and by THP-1-derived M1-macrophages in the range of 53 to 314 pg/ml. In both monocytic cells and macrophage-like cells, PC and pCF3-diEPP significantly inhibited the BzATP-induced release of IL-1β. To examine the subtype(s) of nAChRs that underlie this effect, we used selective peptide antagonists beside the non-selective nAChR antagonist mecamylamine (Innocent et al., 2008). Homomeric α7, and α9-containing nAChRs can be difficult to pharmacologically differentiate. α-Bungarotoxin, for example, blocks both α7 and α9-containing nAChRs with low nM potency (Johnson et al., 1995; Briggs and McKenna, 1996; Elgoyhen et al., 2001). However, certain analogs of conotoxins have been developed that can effectively distinguish between these receptor subtypes (Grau et al., 2018). The inhibitory effect of PC and pCF3-diEPP on the BzATP-induced release of IL-1β was completely reverted by mecamylamine and blunted by the nAChRα7-specific conopeptide (Innocent et al., 2008; Whiteaker et al., 2008). [V11L, V16D]ArIB (500 nM) and by the α9α10 nAChR-specific conopeptide RgIA4 (200 nM), suggesting that nAChR subunits α7, α9 and/or α10 are involved in signaling (Figure 3A). In control experiments, in which mecamylamine, RgIA4 and [V11L, V16D]ArIB were given together with BzATP, the release of IL-1β was not significantly changed in monocytic THP-1 cells (Figure 3A). In THP-1-derived macrophage-like cells, however, a slightly reduced IL-1β concentration was measured in the presence of RgIA4, but not in the presence of [V11L, V16D]ArIB (Figure 3B). Further, PC, pCF3-diEPP, RgIA4, and [V11L, V16D]ArIB did not induce a relevant release of IL-1β in the absence of BzATP (Figures 3A,B).

To investigate the stability of PC and pCF3-diEPP in cell culture, PC (200 μM) and pCF3-diEPP (100 μM) were added to LPS-primed monocytic THP-1 cells 5, 15, and 30 min before stimulation with BzATP (100 μM). As shown in Supplementary Figure 1, the inhibitory effect of PC on the release of IL-1β was lost within 15 min, whereas pCF3-diEPP was effective throughout (Supplementary Figure 1).

We further tested if pCF3-diEPP inhibits the BzATP-induced release of IL-1β by freshly isolated, LPS-primed human PBMCs. These cells released IL-1β in response to BzATP (in the range of 499 to 1,931 pg/ml), which was concentration-dependently inhibited by pCF3-diEPP (Figure 4). Of note, the calculated half maximal inhibitory concentration (IC₅₀) value was as low as 64 fM (Figure 4). When different concentrations of pCF3-diEPP were added to LPS-primed PBMCs in control experiments without BzATP, IL-1β secretion remained at low levels (Supplementary Figure 2A). Cell viability as measured by LDH-release in LPS-primed and BzATP-stimulated PBMCs was not altered by different concentrations of pCF3-diEPP (Supplementary Figure 2B).

Previous experiments using 1 mM PC revealed an efficient PC-mediated reduction of choline-gated ion-currents at heterologously expressed α9α10 nAChRs (Richter et al., 2016). This prompted us to investigate the effects of PC and pCF3-diEPP on ACh-induced ion-currents in Xenopus laevis oocytes expressing human α7, α9 or α9α10 nAChRs in two-electrode voltage-clamp experiments. When ACh pulses (60 μM, 12 s, 2 ml/min) were applied to nAChR-expressing oocytes, repeatable ion-currents were induced (Figure 5). Concomitant application of PC (200 μM) had no effect at α7 nAChRs but provoked a slight reduction of the peak currents measured in oocytes expressing α9 or α9α10 nAChRs (Figure 5A). The concentration of 200 μM for PC was selected based on previous experiments (Richter et al., 2020) and on its potency to inhibit ATP-induced release of IL-1β as described above (Figures 3, 4). After wash-out of PC, ACh-induced peak currents increased most prominently in oocytes expressing α9α10 nAChRs, and to a lesser extent in oocytes expressing α9 or α7 nAChRs (Figure 5A). Of note, the ACh-induced ion currents in oocytes expressing α7, α9 or α9α10 nAChRs entirely vanished in the presence of 30 μM pCF3-diEPP (Figure 5B). When a concentration of 1 μM pCF3-diEPP was used in the same experimental setting, a minor attenuation of the ACh-induced peak currents was seen in oocytes expressing α7 nAChRs, whereas this concentration still fully inhibited ACh-gated currents at α9 or α9α10 nAChRs (Figure 5C). Interestingly, after wash-out of pCF3-diEPP, strongly increased peak currents induced by ACh were measured in oocytes expressing α9α10 nAChRs (Figures 5B,C). In contrast, such effects were not seen in oocytes expressing α7 nAChRs (Figures 5B,C). In oocytes expressing α9 nAChRs the responses to ACh after wash-out of pCF3-diEPP were reduced (Figures 5B,C).

As shown in Figure 6A, the application of 30 μM pCF3-diEPP to the bath solution initiated a current response in the α7, α9 and α9α10 nAChR-expressing cells during the initial 30 s periods normally used to define the baseline for drug-evoked responses (see section “Materials and Methods”). pCF3-diEPP has previously been characterized as a very weak partial agonist for α7 nAChRs effective at inducing large responses only when co-applied with a positive allosteric modulator such as PNU-120596 (Quadri et al., 2017). Therefore, not surprisingly, there was minimal response of the α7-expressing cells, when the bath was switched to a solution containing 30 μM pCF3-diEPP (Figure 6A, upper trace). However, we noted substantial responses to the cells expressing α9 alone or α9α10 nAChRs (Figure 6A, middle and lower traces), suggesting that pCF3-diEPP is an activator of these receptors. The ACh applications during the period of pCF3-diEPP perfusion were diminished and, at least initially, superimposed on an increased baseline current activated by pCF3-diEPP (Figure 6B). For the cells expressing α9 nAChRs alone, this shift in baseline, apparently representing a steady-state activation of the receptors, did not decline fully during the 210 s period of data acquisition (Figures 6A,B). These data indicate that the steady-state current of the α9 nAChR-expressing cells remained elevated through several of the acquisition periods that followed the switch to the bath containing 30 μM pCF3-diEPP.

We demonstrated before in Xenopus laevis oocytes heterologously expressing human α9α10 nAChR, that application of PC does not provoke ion-currents (Richter et al., 2016). Similarly, pCF3-diEPP functions as a very weak partial agonist in Xenopus laevis oocytes heterologously expressing α7 nAChRs (Quadri et al., 2017). Here, we performed two-electrode voltage-clamp measurements in Xenopus laevis oocytes heterologously expressing either human α9 nAChR or α9α10 nAChR. Of note, co-expression of α10 with α9 in the oocyte will favor the heteromeric composition of functional receptors compared to α9 alone (Elgoyhen et al., 2001; Sgard et al., 2002). Concentration-response studies of ACh or pCF3-diEPP were performed and the ion-current responses were normalized to those provoked by 60 μM ACh. ACh and pCF3-diEPP concentration-dependently stimulated ion-currents at α9 nAChRs (Figures 7A,B). The maximal ACh-induced ion-current (I_max) was set to 1 and for pCF3-diEPP a relative I_max of 0.36 ± 0.02 was determined. The half maximal effective concentration (EC₅₀) of ACh was 26.1 ± 4.2 μM, whereas that of pCF3-diEPP was 7.0 ± 1.9 μM. Similar ion-currents were induced at α9α10 nAChRs, with a relative I_max of 0.30 ± 0.01 for pCF3-diEPP, an EC₅₀ for ACh of 29.7 ± 9.9 μM and an EC₅₀ of 6.4 ± 0.9 μM for pCF3-diEPP (Figures 7C,D).