Alkaloid ligands enable function of homomeric human α10 nicotinic acetylcholine receptors

Abstract

In the nervous system, nicotinic acetylcholine receptors (nAChRs) rapidly transduce a chemical signal into one that is electrical via ligand-gated ion flux through the central channel of the receptor. However, some nAChR subunits are expressed by non-excitable cells where signal transduction apparently occurs through non-ionic mechanisms. One such nAChR subunit, α10, is present in a discreet subset of immune cells and has been implicated in pathologies including cancer, neuropathic pain, and chronic inflammation. Longstanding convention holds that human α10 subunits require co-assembly with α9 subunits for function. Here we assessed whether cholinergic ligands can enable or uncover ionic functions from homomeric α10 nAChRs. Xenopus laevis oocytes expressing human α10 subunits were exposed to a panel of ligands and examined for receptor activation using voltage-clamp electrophysiology. Functional expression of human α10 nAChRs was achieved by exposing the oocytes to the alkaloids strychnine, brucine, or methyllycaconitine. Furthermore, acute exposure to the alkaloid ligands significantly enhanced ionic responses. Acetylcholine-gated currents mediated by α10 nAChRs were potently inhibited by the snake toxins α-bungarotoxin and α-cobratoxin but not by α-conotoxins that target α9 and α9α10 nAChRs. Our findings indicate that human α10 homomers are expressed in oocytes and exposure to certain ligands can enable ionic functions. To our knowledge, this is the first demonstration that human α10 subunits can assemble as functional homomeric nAChRs. These findings have potential implications for receptor regulatory-mechanisms and will enable structural, functional, and further pharmacological characterization of human α10 nAChRs.

Introduction

Nicotinic acetylcholine receptors (nAChRs) are composed of five individual subunits and form an ion channel that can be gated by acetylcholine (ACh) and related ligands (Dani 2015). Most heteromeric nAChRs, such as the α4β2 subtype, are composed of α and β subunits, whereas homomeric subtypes including α7 and α9 nAChRs are composed of a single gene product. Since the discovery of the CHRNA10 gene approximately 20 years ago, attempts to heterologously express mammalian α10 subunits as functional homomeric receptors have consistently failed in both mammalian and non-mammalian expression systems (Elgoyhen, Vetter et al., 2001; Lustig, Peng et al., 2001; Sgard, Charpantier et al., 2002). By contrast, chick and frog α10 subunits do form functional homopentamers (Lipovsek, Fierro et al., 2014; Marcovich, Moglie et al., 2020). Furthermore, mammalian α9 subunits also form functional receptors in oocytes and mammalians cell lines although expression levels are generally low (Elgoyhen, Johnson et al., 1994; Filchakova and McIntosh 2013). These observations led to the conclusion that mammalian α10 subunits do not form functional receptors in the absence of α9 subunits but form α9α10 heteromers when the two subunits are expressed together.

The distribution of α10 subunits in mammalian organisms has a very restricted expression pattern and is limited to tissues outside the central nervous system (Elgoyhen, Vetter et al., 2001; Morley, Whiteaker et al., 2018). In humans, the presence of mRNA for CHRNA10 or immunohistochemical evidence of α10 subunits has been demonstrated in pituitary and cochlear tissue (Sgard, Charpantier et al., 2002), immune cells, including monocytes (Richter, Mathes et al., 2016) and lymphocytes (Lustig, Peng et al., 2001; Peng, Ferris et al., 2004), and certain epithelial tissues such as urothelium (Bschleipfer, Schukowski et al., 2007) and skin (Kurzen, Berger et al., 2004). CHRNA10 mRNA has also been detected in human breast and lung cancer-derived cell lines (Lee, Huang et al., 2010; Mucchietto, Fasoli et al., 2018). In rodents, mRNA for CHRNA10 has been detected in pituitary (Hone, Rueda-Ruzafa et al., 2020), hair cells of the cochlea (Elgoyhen, Vetter et al., 2001), dorsal root ganglion neurons (Hone, Meyer et al., 2012), and several immune cell types (Kawashima, Yoshikawa et al., 2007; Mikulski, Hartmann et al., 2010).

The functional role of α10 subunits outside the auditory system is mostly unknown, but studies using subtype-selective ligands have implicated α10-containing nAChRs in a number of pathophysiological processes. Several α9α10-targeting ligands have been shown to inhibit the release of the proinflammatory cytokine interleukin-1β from human U937 monocytes (Richter, Mathes et al., 2016; Zakrzewicz, Richter et al., 2017). In rodent models of nerve injury (Vincler, Wittenauer et al., 2006), inflammatory bowel disease (AlSharari, Toma et al., 2020), and chemotherapeutic-induced neuropathy (Pacini, Micheli et al., 2016; Gajewiak, Christensen et al., 2021) administration of antagonist ligands of α9α10 nAChRs has been shown to reduce signs and symptoms of disease. α9α10 antagonists have also been shown to accelerate functional recovery of damaged nerves in models of neuropathic pain (Satkunanathan, Livett et al., 2005). Thus, development of a non-opioid based analgesic that targets α9α10 nAChRs is an active area of research.

Mammalian α10 subunits have long been thought to require α9 subunits for functional expression, and therefore ligand-receptor interactions have usually been examined in the context of α9α10 heteromers (Perez, Cassels et al., 2009; Azam and McIntosh 2012; Yu, Kompella et al., 2013; Indurthi, Pera et al., 2014; Zouridakis, Papakyriakou et al., 2019). Consequently, information concerning the interaction of ligands with human α10 subunits is significantly limited. Plant alkaloids including strychnine (STR) and methyllycaconitine (MLA) are known ligands of nAChRs containing α9 or α10 subunits, and MLA has been shown to promote functional expression of human α9α10 heteromers in Xenopus oocytes and human embryonic kidney cells (Gu, Knowland et al., 2020). Strychnine and the related compound brucine (BRU) are the principal alkaloids found in the seeds of Strychnos nux-vomica, the strychnine tree (Guo, Wang et al., 2018; Lu, Huang et al., 2020). Interestingly, the seeds of this tree are widely consumed as alternative medicine in India and southeast Asia where the tree is endemic. Internet sales of nux-vomica seeds and extracts known as Kuchla are now promoting wider use for indications that include pain, gastrointestinal disorders, and erectile dysfunction (Ades, Alteri et al., 2009; Akbar, Khan et al., 2010; Lu, Huang et al., 2020). Here we demonstrate that, when exposed to strychnos alkaloids or the larkspur alkaloid MLA, human α10 subunits form functional nAChRs in oocytes. The information presented in this report will facilitate the study of a nAChR subunit implicated in a number of human disease states and will allow, for the first time, structural, pharmacological and functional characterization of homomeric human α10 nAChRs.

Methods

Peptide synthesis

α-Conopeptide synthesis was performed using Fmoc solid-phase synthesis techniques and described in detail elsewhere (Hone, Fisher et al., 2019). The masses of the peptides were verified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Correct folding of the peptides and purity were determined by reverse-phase high-performance liquid chromatography and purity was ≥95% for all.

Oocyte electrophysiology

Protocols (No. 17-07020) for obtaining oocytes from Xenopus laevis frogs were approved by the University of Utah’s Institutional Animal Care and Use Committee. Frogs were purchased from Xenopus1 (Dexter, MI, United States) and maintained by university personnel in an AAALAC accredited facility. Oocytes were obtained from frogs that were anesthetized with 0.4% wt/vol Tricaine-S (Thermo Fisher Scientific, Waltham, MA, United States) and sacrificed after removal of the ovarian lobes. Methods for preparation of cRNA constructs for expression of nAChRs in oocytes have been previously described (Hone, Talley et al., 2018). Clones for human α9 and α10 subunits were obtained from L.R. Lustig (University of California San Francisco, San Francisco, CA, United States) and were subsequently subcloned into a pSGEM vector that contained an alfalfa mosaic virus sequence (Filchakova and McIntosh 2013). Stage IV-V oocytes were generally injected with 50 ng of cRNAs encoding human nAChR subunits and subjected to two-electrode voltage-clamp electrophysiology 3 to 5 days after injection. However, injection of 2 ng of α10 subunit cRNA yielded currents that were −740 ± 245 nA (n = 13) indicating that small amounts of cRNA are sufficient to induce robust expression of α10 nAChRs. The effects of ACh, choline, nicotine, α-Bgtx, MLA, STR, and BRU on the functional expression of α10 nAChRs were assessed 3 days after injection of 50 ng of cRNA. For these experiments, the oocytes were removed from the incubation solution, placed in the recording chamber and, after voltage-clamping the membrane, were stimulated with 1 mM ACh while continuously perfused with frog saline only. The terms incubation and treated are used interchangeably throughout the manuscript to indicate the presence of ligand in the saline solution used to maintain the oocytes while in the incubator.

For the assessment of ligand activity, the oocyte membranes were clamped at a holding potential of −70 mV and continuously perfused with saline (control solution). The oocytes were pulsed with ACh (1 mM) for 2 s, once per minute, until a stable baseline-response was observed, then the control solution was switched to one containing the ligand of interest and the ACh responses monitored for changes in amplitude. The ACh responses in the presence of the ligand were normalized to the average of three responses in control solution. Ligands were applied in this manner for concentrations up to 1 µM. For concentrations >1 μM, ligands were applied in a static bath for 5 min and the current amplitudes were normalized to the ACh response after a 5 min bath application of control solution. For the concentration-response experiments using STR, BRU, or MLA, the ligands were perfusion applied at all concentrations. The estimated IC₅₀ value for inhibition of ACh-evoked currents by STR and α-Bgtx were obtained by non-linear regression using a four-parameter logistic equation Y=Bottom + (Top-Bottom)/(1 + 10^((LogEC₅₀-X)*HillSlope)). The estimated EC₅₀ value for activation of α10 nAChRs by ACh was obtained according to the following procedures: the oocytes were stimulated with 2 s pulses of ACh (1 mM) every 60 s, and after a steady-state baseline was observed, the oocytes were then stimulated with ascending concentrations of ACh. The estimated maximal response for activation by ACh in each cell was determined using a four-parameter logistic equation and the responses to all concentrations of ACh, choline, and nicotine were then normalized to this value and shown as a percent response. The estimated plateau value for experiments examining the decay of ACh-evoked currents was determined using a one-phase exponential equation. For experiments that assessed the permeability of α10 channels to Ca²⁺ and the contribution of endogenous calcium-activated chloride channels to the observed responses to ACh, frog saline was used where CaCl₂ was replaced with an equimolar concentration of BaCl₂. For experiments examining the desensitization kinetics of α10-containing nAChRs, sibling oocytes were injected with 25 ng of cRNA for α10 subunits or cRNA for α9 and α10 subunits (25 ng each). Actinomycin-D (1 µM) was also used in some experiments to prevent RNA transcription and inhibit the expression of endogenous chloride channels. The oocytes were incubated in actinomycin-D for 36 h prior to assessment with TEVC electrophysiology.

Statistical analysis

All statistical analyses were performed using Prism 9 (RRID: SCR_002798) (GraphPad Software, San Diego, CA, United States). The error bars for all experiments indicate the SD of the data and are provided to assess variance of the data. The data were analyzed for normality using a Shapiro-Wilk test. Significant differences in the ACh responses after incubation with saline or saline containing nAChR ligands were determined using a Kruskal–Wallis test with a post hoc Dunn’s multiple comparisons test. A paired student’s t-test was used to determine differences in current amplitudes obtained from α10 nAChR expressing oocytes in the presence of Ca²⁺ compared to Ba²⁺. An unpaired student’s t-test was used to determine differences in α10 nAChR current amplitudes obtained in the presence 10 nM compared to 1 μM MLA, 100 nM BRU compared to 10 μM BRU, or 10 nM STR compared to 1 μM STR. A paired student’s t-test was also used to determine differences in current amplitudes for α9 nAChR expressing oocytes incubated in saline and reassessed 24 h later after incubation in saline containing STR. To evaluate the inhibitory effects of antagonist ligands on α10 nAChR, a one-sample t-test was used and the values compared to a hypothetical response mean of 100% except in the case of Vc1.1 where a non-parametric Wilcoxon signed-rank test was used. Results were considered significant if p < 0.05*, p < 0.01**, p < 0.001***, or p < 0.0001****.

Materials

Acetylcholine chloride and methyllycaconitine citrate were obtained from Tocris (Minneapolis, MN, United States). Strychnine hydrochloride, dimethoxystrychnine sulfate hydrate (brucine), atropine sulfate monohydrate (-)-nicotine hydrogen tartrate, sodium chloride, potassium chloride, calcium chloride dihydrate, magnesium chloride hexahydrate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), actinomycin-D, α-Bgtx from Bungurus multicinctus, α-cobratoxin from Naja kaouthia, were obtained from Sigma-Aldrich (St. Louis, MO, United States).

Results

Strychnine, BRU, and MLA enable ionic functions of human α10 nAChRs

Certain nAChR ligands, most notably the tobacco alkaloid nicotine, have been shown to promote functional receptor expression in different cell types (Srinivasan, Pantoja et al., 2011; Henderson and Lester 2015). To determine if expression of human α10 nAChRs could be facilitated by exposure to alkaloids or other nicotinic ligands, we expressed α10 subunits in oocytes and exposed them to STR (20 μM), BRU (20 μM), acetylcholine (ACh; 5 mM), choline (5 mM), nicotine (20 μM), α-bungarotoxin (α-Bgtx; 1 μM), or MLA (20 μM) for 3 days. The oocytes were then stimulated with ACh (1 mM) under voltage-clamp conditions and assessed for functional responses. Oocytes injected with water and incubated in saline did not respond to ACh (Figures 1A–C). Very small or no ACh-evoked currents were observed in oocytes injected with cRNA for α10 subunits (−3.6 ± 2.8 nA; n = 32) (Figures 1A,B,D), but relatively large α10 nAChR currents were observed after incubating the oocytes in STR, BRU, or MLA (Figures 1A,E–G). Oocytes incubated with STR responded to ACh with large amplitude currents (−738 ± 545 nA; n = 28), but control oocytes injected with water and treated with STR did not respond to ACh (−1.6 ± 0.7 nA; n = 7). Current amplitudes recorded from oocytes incubated in BRU were −302 ± 225 nA (n = 13), and those from oocytes incubated in MLA were −813 ± 641 nA (n = 16). Small gains in current amplitudes were observed when the oocytes were incubated with ACh (−17 ± 11 nA; n = 13) or α-Bgtx (−31 ± 21 nA; n = 9), but only currents from those treated with α-Bgtx were significantly different than saline controls (Figures 1A,B,H,I). Currents recorded from oocytes incubated with choline (−7.2 ± 4.2 nA; n = 10) or nicotine (−6.0 ± 6.2 nA; n = 15) were not statistically different in amplitude than those from controls (Figures 1A,B,J,K).

FIGURE 1

Xenopus oocytes express endogenous calcium-activated chloride channels, and Ca²⁺ entering the cell through calcium-permeable channels can contribute to currents in response to exogenously applied ligands (Barish 1983; Miledi and Parker 1984). To determine if these chloride channels contributed to the observed ACh-evoked currents, we conducted experiments by replacing external calcium (1.8 mM) with equimolar barium, an ion that does not activate chloride channels. Currents recorded in barium were smaller than those recorded in calcium (Figures 2A–C) indicating that activation of the ion channels formed from α10 subunits have less of an effect on Xenopus calcium-activated chloride channels compared to activation of mammalian α9-containing subtypes (Lipovsek, Fierro et al., 2014; Marcovich, Moglie et al., 2020). To further assess the potential contribution of chloride-channel activation to the ACh-evoked current amplitudes, we incubated oocytes exogenously expressing α10 nAChR subunits in actinomycin-D to inhibit RNA transcription and prevent expression of endogenous receptors and ion channels. The current amplitudes from oocytes treated with actinomycin-D and perfused with saline containing calcium were no different than those from sibling oocytes not exposed to actinomycin-D and perfused with barium saline (Figures 2B,C). Together, these results indicate that ACh evokes current through exogenously expressed α10 homomers both in the presence and in the absence of endogenous calcium-activated chloride channels.

FIGURE 2

Strychnine, BRU, and MLA enhance functionality of α10 nAChRs but act as antagonists at high concentrations

During our initial experiments, we observed that currents from oocytes incubated with STR, BRU, or MLA tended to decrease in amplitude over time, but there were differences in the extent to which the currents decayed (Figures 3A–D). Currents from oocytes incubated with MLA decayed to approximately 12% of the initial response to ACh. By contrast, currents from oocytes incubated with STR decayed to 74% of initial amplitudes. Interestingly, currents recorded from oocytes incubated with the STR analog BRU decayed to 15%, a value much smaller than that obtained with STR. To further investigate the enhancing effects of STR on the functionality of α10 nAChRs, we perfused STR-naïve oocytes expressing human α10 nAChRs with STR for 30 min and monitored the current amplitudes in response to ACh. Remarkably, at the end of this perfusion the current amplitudes were enhanced by 518 ± 411% (n = 7) (Figure 4A). Next, we continuously perfused oocytes with STR, BRU, or MLA over a range of concentrations and compared the responses to those recorded prior to ligand perfusion. Oocytes were incubated for 3–4 days in saline containing MLA and consistent with the observations observed during initial experiments, the ACh-evoked currents decayed overtime when perfused with saline alone. Once a stable baseline was achieved, the oocytes were then perfused with increasing concentrations of MLA. Methyllycaconitine increased the amplitudes of ACh-evoked currents at concentrations up to 1 µM but was inhibitory at higher concentrations (Figure 4B). Similar results were obtained with BRU (Figure 4C). Current amplitudes recorded from oocytes pre-incubated with MLA were also enhanced by perfusion with STR (Figure 4D), but by contrast, those from oocytes pre-treated with STR were not enhanced by acute perfusion with STR (Figure 4E).

FIGURE 3

FIGURE 4

Previous reports indicated that STR is an antagonist of mammalian α9-containing nAChRs (Elgoyhen, Johnson et al., 1994; Rothlin, Katz et al., 1999; Baker, Zwart et al., 2004) an effect similar to the results presented in Figure 4E for human α10 nAChRs. Therefore, we also sought to determine the effects of STR on human subtypes closely related to α10 nAChRs including α9 homomers and α9α10 heteromers. Acute STR (20 μM) perfusion of oocytes expressing α9 homomers almost completely inhibited ACh-evoked responses (Figure 5A). By contrast, those from oocytes expressing α10 nAChRs were inhibited by only ∼50% at the same concentration (Figure 5B). Acute perfusion with STR inhibited the ACh-evoked responses in oocytes (incubated in saline) expressing α9 and α10 subunits by ∼99% (Figure 5C).

FIGURE 5

Human α9 nAChR function is abolished by STR but enhanced by choline

The differential results obtained with STR on α9, α10, and α9 plus α10 expressing oocytes prompted us to examine STR effects on homomeric α9 nAChRs. Oocytes injected with cRNA for α9 subunits were incubated in saline or saline containing choline (5 mM) or STR (20 µM). In contrast to the results obtained for α10 nAChRs, choline enhanced functional expression of α9 homomers (Figures 6A,B,D). Strikingly, oocytes incubated in STR did not respond to ACh and no responses were detected even after a 20 min perfusion with saline (Figures 6C,D).

FIGURE 6

Snake α-neurotoxins are potent antagonists of homomeric α10 nAChRs

The snake toxin α-Bgtx, isolated from the venom of Bungarus multicinctus, has been shown to inhibit homomeric human and rat α9 nAChRs and α9α10 heteromers (Elgoyhen, Johnson et al., 1994; Elgoyhen, Vetter et al., 2001; Sgard, Charpantier et al., 2002). Other potent antagonists of mammalian α9-containing nAChRs include α-conotoxins (α-Ctxs) PeIA, Vc1.1, and RgIA (Johnson, Martinez et al., 1995; Satkunanathan, Livett et al., 2005; Ellison, Haberlandt et al., 2006). To assess the activity of α-Bgtx on human α10 nAChRs, we obtained a concentration-response curve for inhibition of ACh-evoked responses in oocytes that had been incubated in MLA for 4 days (Figures 7A,B). α-Bungarotoxin inhibited α10 nAChRs with a potency (IC₅₀ 21 nM) similar to that previously reported for human α9α10 nAChRs (Sgard, Charpantier et al., 2002). We also assessed a panel of α-Ctxs, atropine, and nicotine that are known antagonists of α9α10 nAChRs as well as a second snake peptide from the cobra Naja kaokouthia (Figure 7C). Notably, none of the α-Ctxs, atropine, or nicotine showed substantial antagonist effects on ACh-evoked responses mediated by α10 nAChRs at the concentrations used in this study. However, like α-Bgtx, α-Cbtx nearly eliminated responses to ACh. The alkaloids MLA, STR, and atropine significantly enhanced responses to ACh under these experimental conditions. Lastly, we tested a recently described analog of α-Ctx RgIA that showed pM potency for inhibition of human α9α10 nAChRs (Gajewiak, Christensen et al., 2021). This analog, RgIA-5474, completely inhibited ACh-evoked responses mediated by α9 and α9α10 nAChRs (Figures 8A,C) but had little to no effect on responses from oocytes expressing homomeric α10 nAChRs (Figure 8B).

FIGURE 7

FIGURE 8

Acetylcholine and choline, but not nicotine, are agonists of human α10 nAChRs

Acetylcholine and choline are agonists of α9 and α9α10 nAChRs, but the canonical nicotinic agonist nicotine has been shown to antagonize α9α10 nAChRs expressed in rat cochlear hair cells and human receptors expressed in Xenopus oocytes (Elgoyhen, Vetter et al., 2001; Sgard, Charpantier et al., 2002). To assess the activities of choline and nicotine, first we obtained a concentration-response curve for activation of α10 nAChRs by ACh then the oocytes were stimulated with choline followed by nicotine. Choline evoked small amplitude currents, relative to those evoked by ACh, but only at a concentration of 10 mM (Figures 9A–C). Nicotine however, failed to evoke current responses up to a concentration of 1 mM (Figures 9A–D). These results suggest that physiologically ACh, and not choline, would likely be the primary agonist of homomeric α10 nAChRs.

FIGURE 9

Human α10 nAChRs desensitize to ACh faster than α9α10 nAChRs

To examine the desensitization kinetics of α10 nAChRs, oocytes expressing α10 subunits were exposed to 15 s pulses of an EC₅₀ concentration of ACh, and the current amplitude at the end of the ACh pulse was compared to the peak current value. For comparison, sibling oocytes expressing α9α10 nAChRs were also examined with 15 s pulses of EC₅₀ ACh (Sgard, Charpantier et al., 2002). The ACh-evoked currents were examined in the presence of calcium and also in barium to eliminate the calcium-activated chloride component of the observed response to ACh. Similar to the results presented in Figure 2, peak ACh-evoked currents from α10 nAChRs were reduced in the presence of barium compared to calcium (Figures 10A,B). The responses at the end of the ACh pulse were also smaller compared to peak values as a result of desensitization to the agonist. By contrast with α10 nAChRs, α9α10 currents were much smaller in the presence of barium compared to calcium (Figures 10C,D). Furthermore, α9α10 nAChRs desensitized more slowly than α10 nAChRs.

FIGURE 10

Discussion

Under previously tested experimental conditions, mammalian α10 subunits showed no ionic responses to agonists (Elgoyhen, Vetter et al., 2001; Sgard, Charpantier et al., 2002; Fucile, Sucapane et al., 2006; Marcovich, Moglie et al., 2020), and this led to the widely held view that mammalian α10 subunits do not form homomeric receptors and require α9 subunits for function. In this work, we demonstrate that human α10 subunits form homopentameric receptors and display properties distinct from those of the closely related α9 and α9α10 subtypes. For example, the ion channel formed by α10 subunits appeared to show marked differences in permeability to divalent cations compared to α9α10 heteromers (Figure 10). The calcium permeability of α9 homomers and α9α10 heteromers has been measured and shown to be exceptionally high among ligand-gated ion channels (Katz, Verbitsky et al., 2000; Sgard, Charpantier et al., 2002; Fucile, Sucapane et al., 2006). Evolutionary analysis of α9 and α10 subunits revealed that the high calcium permeability of mammalian α9-containing nAChRs is due to three non-synonymous amino-acid substitutions in the α9 subunit (Lipovsek, Fierro et al., 2014). These substitutions are not present in mammalian α10 subunits precluding the formation of a homomeric ion channel with high calcium permeability. Furthermore, human α10 nAChRs are unique in their sensitivity to known ligands of α9 and α9α10 nAChRs. We tested several α-Ctxs, that inhibit human and rodent α9 homomers and α9α10 heteromers including Vc1.1, PeIA, RgIA and its analog RgIA-5474 (Jakubowski, Keays et al., 2004; McIntosh, Plazas et al., 2005; Ellison, Haberlandt et al., 2006; Gajewiak, Christensen et al., 2021), and none of these α-Ctxs showed potent inhibitory effects on α10 nAChRs (Figures 7, 8). We also tested the muscarinic receptor antagonist atropine as well as nicotine and found a similar lack of inhibitory activity (Figure 7). In fact, α10 currents from oocytes exposed to atropine were significantly larger than baseline controls suggesting that this alkaloid may also enhance function of α10 nAChRs. Evolutionary analysis of chick and mammalian α10 subunits concluded that the low potency and partial efficacy of choline for activation of mammalian α9α10 nAChRs can be attributed to non-conserved amino-acid residues in the α10 subunit (Moglie, Marcovich et al., 2021). The low efficacy of choline for activation of human α10 nAChRs is consistent with these observations (Figure 9). By contrast, choline is a full agonist of chick α10 nAChRs again due to non-conserved amino-acid residues in the ACh-binding pocket (Moglie, Marcovich et al., 2021).

There are substantial similarities in the amino-acid sequences of mammalian α10 and α9 subunits, and the latter have previously been shown to form functional homomers (Elgoyhen, Johnson et al., 1994; Sgard, Charpantier et al., 2002; Fucile, Sucapane et al., 2006; Filchakova and McIntosh 2013). Mammalian α10 subunits are also quite similar in sequence to chick α10 which also assemble as functional homomers (Marcovich, Moglie et al., 2020). Despite these similarities, functional mammalian α10 homopentamers have not been previously reported. Several lines of evidence suggest that there are features of mammalian α10 and α9 subunits that impair function when expressed as homomers. Firstly, human α9α10 heteromers express well in oocytes, but, by contrast, α9 homomers express poorly or not at all (Filchakova and McIntosh 2013). The fact that when the two subunits are expressed together results in increased function compared to α9 or α10 alone suggests that α9α10 heteromers might be assembled more efficiently. However, binding studies using ¹²⁵I-α-Bgtx demonstrated that human α9 homomers are assembled and inserted in the oocyte membrane at levels similar to α9α10 heteromers, but very few of them show canonical ionic function (Sgard, Charpantier et al., 2002). Alternatively, human α9 and α10 subunits when expressed together might result in a larger fraction of the receptors expressed in a conformation that can be gated by agonists for ionic functions. Secondly, chimeric constructs containing the extracellular ligand-binding domain of the mammalian α10 subunit and the transmembrane and cytoplasmic domains of the serotonin 5-HT₃ receptor have been shown to express as homomers in oocytes and human embryonic kidney cells (Baker, Zwart et al., 2004) suggesting that some element of the transmembrane or cytoplasmic domains may impair function. Such impairments have been shown for human nAChRs containing α6 subunits which express poorly, or not at all, in oocytes and cell lines (Kuryatov, Olale et al., 2000; Evans, Bose et al., 2003). Poor expression of α6-containing nAChRs was overcome by replacing the transmembrane and cytoplasmic regions with those of the closely related α3 subunit (Kuryatov, Olale et al., 2000; McIntosh, Azam et al., 2004). Enhanced expression of α6-containing nAChRs was also found when the first cytoplasmic loop of α6 was replaced with that of α3 (Jensen, Hoestgaard-Jensen et al., 2013; Ley, Kuryatov et al., 2014). Collectively, expression studies of α6, α9, and α10 indicate that structural elements of some nAChR subunits can potentially inhibit expression of receptors in a state that can be gated by agonists for ionic function.

Receptor function may also be dependent on or be regulated by other cellular proteins as demonstrated for the lynx1 prototoxin which modulates the expression and function of several nAChR subtypes (Ibanez-Tallon, Miwa et al., 2002; George, Bloy et al., 2017; Parker, O'Neill et al., 2017; Miwa 2021). Recently it was shown that certain transmembrane proteins found in native cells associate with α6 and α9 subunits and enable heterologous expression of functional nAChRs (Gu, Matta et al., 2019; Gu, Knowland et al., 2020). In the case of α9-containing nAChRs, transmembrane protein of the inner ear (TMIE) was found to synergize with choline acetyltransferase to enable functional expression of human α9 homomers and α9α10 heteromers in human embryonic kidney cells. However, although choline acetyltransferase alone facilitated surface expression of α9α10 heteromers, ionic function required TMIE. Interestingly, several members of an orphan family of transmembrane proteins (TMEM) unrelated to TMIE were shown to facilitate ionic functions of human α9α10 heteromers and appeared to show α10-dependent effects. Speculatively, α10 homomers may also require an endogenous protein for ionic functions. Lastly, Gu et al., also reported that ionic responses in cells transfected with α9α10 plus TMIE were enhanced by incubation with ACh, MLA, or α-Bgtx suggesting that external ligand-binding can also enhance function. Thus, an endogenous protein or ligand might be used by certain cells to respond to environmental conditions in order to modulate the functional properties of α10 nAChRs according to cellular needs.

Here we show that ionic responses mediated by human α10 nAChRs were obtained by incubation with the membrane impermeant ligand α-Bgtx, as well as STR, BRU, or MLA (Figure 1). Although the conditions used in this study are not physiological, they may have pathophysiological relevance. Strychnine and BRU are regularly ingested as non-traditional medicine in the form of kuchula seeds, and concentrations of BRU and STR in the nM range showed activity on α10 nAChRs (Figures 4C,D). Strychnine also affected the activity of α9 homomers and α9α10 heteromers (Figures 5, 6). Therefore, multiple systems that express α9 and α10 subunits could be affected by ingestion of kuchula seeds. We also show that human α9 nAChR responses, but not those of α10, could be enhanced by incubation with choline (Figures 1, 6) which may correlate with ligand activity; choline is an agonist of α9 homomers with µM potency (Sgard, Charpantier et al., 2002) but only partially activates α10 nAChRs at 10 mM (Figure 9). This discovery may facilitate studies that examine the pharmacological properties of homomeric human α9 nAChRs, studies that have historically been difficult to conduct. Exposure to STR produced differential effects depending on the nAChR subtype and ligand-incubation procedures. Ionic function of α10 nAChRs was enabled by acute STR exposure which produced progressively larger currents over the course of 30 min in oocytes that had been previously incubated in saline only (Figure 4A). By contrast, acute STR perfusion of oocytes expressing α10 nAChRs and pre-treated with STR resulted in inhibition of ACh-evoked currents (Figure 5). Strychnine inhibition was also observed for α9 homomers and α9α10 heteromers. These results may indicate that STR interacts with at an external site on the receptor. Alternatively, STR and other membrane permeant alkaloids might cross the cell membrane and modulate ionic functions or promote receptor insertion into the plasma membrane. An allosteric ligand-binding site has been proposed for the α9 subunit (Zorrilla de San Martin, Ballestero et al., 2007) and the closely related α7 nAChR (Bertrand, Bertrand et al., 2008; daCosta, Free et al., 2011). Future studies are needed to determine if the effects of alkaloid ligands on α10 nAChR function are consistent with interaction with a positive allosteric modulatory site. It is noteworthy that ligands that bind to the allosteric site of α7 nAChRs produce differential effects on receptor function. For example, the allosteric modulator NS6740 is capable of inducing two different conformational states of α7 nAChRs, one of which is a prolonged non-activatable state (Papke, Bagdas et al., 2015). Similarly, STR abolished function of human α9 nAChRs, an effect that lasted for the duration of the experiment (Figure 6) in stark contrast to results obtained for α10 nAChRs. In this way, different nAChR subtypes might be selectively recruited from mixed populations via ligand binding. Indeed, STR incubation of oocytes co-expressing α9 and α10 subunits produced a population of receptors that was resistant to inhibition by acute exposure to STR or RgIA-5474 (data not shown). These results are reminiscent of studies of the lynx1 protein that was shown to change the functional properties and expression patterns of human α3β4-containing nAChRs (George, Bloy et al., 2017).

The quiescent nature of human α10 nAChRs expressed in oocytes may occur in native cells. To our knowledge, ionic currents mediated by native α10-containing nAChRs have thus far only been recorded in hair cells of rodent inner ear (Elgoyhen, Johnson et al., 1994; McIntosh, Plazas et al., 2005; Ellison, Haberlandt et al., 2006; Lipovsek, Marcovich et al., 2021). However, α10 subunits have been implicated in non-ionic nAChR responses in immune cells (Peng, Ferris et al., 2004; Mikulski, Hartmann et al., 2010; Mishra, Rir-sima-ah et al., 2010; Richter, Mathes et al., 2016). These immune cells studies provide evidence that some nAChRs function in a metabotropic-like state rather than one with ionic functions. Immune cell nAChRs include those assembled from α7, α9, or α10 subunits and form part of a cholinergic anti-inflammatory system (Tracey 2007; Fujii, Mashimo et al., 2017; Alen 2022). The present study suggests the possibility that some nAChRs might be expressed in a quiescent state, but canonical ion channel functions can be induced by certain ligands. Indeed, there is precedent for native cell expression of quiescent nAChRs that can be pharmacologically converted to an ionic conformation by allosteric modulators. Rat adrenal chromaffin cells abundantly express α7 nAChRs, but a large fraction of the receptor population is evidently present in a non-activatable state (Hone, Rueda-Ruzafa et al., 2020); see also (Higgins and Berg 1988). In fact, in some chromaffin cells α7-mediated responses could only be observed in the presence of the allosteric modulator PNU-120596. Similar results have been found for bovine and human adrenal chromaffin cells (del Barrio, Egea et al., 2011; Perez-Alvarez, Hernandez-Vivanco et al., 2012).

In human epidermis, expression of α10 and α9 subunits often do not parallel each other, and therefore it is possible that homomeric receptors are present in these tissues (Kurzen, Wessler et al., 2007). In dorsal root ganglion neurons molecular biology assays consistently detect mRNA and protein for α10 but, in some cases, not for α9 subunits though ionic currents attributable to α10-containing nAChRs have not been identified (Haberberger, Bernardini et al., 2004; Rau, Johnson et al., 2005; Bschleipfer, Nandigama et al., 2012; Hone, Meyer et al., 2012; Zhang, Hartung et al., 2019). Thus, it remains unknown whether α10 nAChRs expressed in the absence of α9 subunits would function in an ionic or in a metabotropic-like state. The discovery that ligand-binding can enable the ionic functions of α10 nAChRs should greatly facilitate the development of α10 homomer-specific ligands. Such ligands can then be used to resolve questions concerning the native expression of α10-containing nAChRs and their roles in human physiology and pathology.

References

• 1

  AdesT.AlteriR.GanslerT. (2009). Strychnous nux-Vomica. American cancer society's complete guide to complementary and alternative cancer therapies. 2nd ed. American Cancer Society, 504–507.

  • Google Scholar

• 2

  AkbarS.KhanS. A.MasoodA.IqbalM. (2010). Use of strychnos nux-vomica (azraqi) seeds in unani system of medicine: Role of detoxification. Afr. J. Tradit. Complement. Altern. Med.7 (4), 286–290. 10.4314/ajtcam.v7i4.56689

  • CrossRef
  • Google Scholar

• 3

  AlenN. V. (2022). The cholinergic anti-inflammatory pathway in humans: State-of-the-art review and future directions. Neurosci. Biobehav. Rev.136, 104622. 10.1016/j.neubiorev.2022.104622

  • CrossRef
  • Google Scholar

• 4

  AlSharariS. D.TomaW.MahmoodH. M.Michael McIntoshJ.Imad DamajM. (2020). The α9α10 nicotinic acetylcholine receptors antagonist α-conotoxin RgIA reverses colitis signs in murine dextran sodium sulfate model.Eur. J. Pharmacol.883, 173320. 10.1016/j.ejphar.2020.173320

  • CrossRef
  • Google Scholar

• 5

  AzamL.McIntoshJ. M. (2012). Molecular basis for the differential sensitivity of rat and human α9α10 nAChRs to α-conotoxin RgIA. J. Neurochem.122 (6), 1137–1144. 10.1111/j.1471-4159.2012.07867.x

  • CrossRef
  • Google Scholar

• 6

  BakerE. R.ZwartR.SherE.MillarN. S. (2004). Pharmacological properties of alpha 9 alpha 10 nicotinic acetylcholine receptors revealed by heterologous expression of subunit chimeras. Mol. Pharmacol.65 (2), 453–460. 10.1124/mol.65.2.453

  • CrossRef
  • Google Scholar

• 7

  BarishM. E. (1983). A transient calcium-dependent chloride current in the immature Xenopus oocyte. J. Physiol.342, 309–325. 10.1113/jphysiol.1983.sp014852

  • CrossRef
  • Google Scholar

• 8

  BertrandD.BertrandS.CassarS.GubbinsE.LiJ.GopalakrishnanM. (2008). Positive allosteric modulation of the alpha7 nicotinic acetylcholine receptor: Ligand interactions with distinct binding sites and evidence for a prominent role of the M2-M3 segment. Mol. Pharmacol.74 (5), 1407–1416. 10.1124/mol.107.042820

  • CrossRef
  • Google Scholar

• 9

  BschleipferT.NandigamaR.MoellerS.IlligC.WeidnerW.KummerW. (2012). Bladder outlet obstruction influences mRNA expression of cholinergic receptors on sensory neurons in mice. Life Sci.91 (21-22), 1077–1081. 10.1016/j.lfs.2012.05.007

  • CrossRef
  • Google Scholar

• 10

  BschleipferT.SchukowskiK.WeidnerW.GrandoS. A.SchwantesU.KummerW.et al (2007). Expression and distribution of cholinergic receptors in the human urothelium. Life Sci.80 (24-25), 2303–2307. 10.1016/j.lfs.2007.01.053

  • CrossRef
  • Google Scholar

• 11

  daCostaC. J.FreeC. R.CorradiJ.BouzatC.SineS. M. (2011). Single-channel and structural foundations of neuronal α7 acetylcholine receptor potentiation.J. Neurosci.31 (39), 13870–13879. 10.1523/JNEUROSCI.2652-11.2011

  • CrossRef
  • Google Scholar

• 12

  DaniJ. A. (2015). Neuronal nicotinic acetylcholine receptor structure and function and response to nicotine. Int. Rev. Neurobiol.124, 3–19. 10.1016/bs.irn.2015.07.001

  • CrossRef
  • Google Scholar

• 13

  del BarrioL.EgeaJ.LeonR.RomeroA.RuizA.MonteroM.et al (2011). Calcium signalling mediated through α7 and non-α7 nAChR stimulation is differentially regulated in bovine chromaffin cells to induce catecholamine release. Br. J. Pharmacol.162 (1), 94–110. 10.1111/j.1476-5381.2010.01034.x

  • CrossRef
  • Google Scholar

• 14

  ElgoyhenA. B.JohnsonD. S.BoulterJ.VetterD. E.HeinemannS. (1994). Alpha 9: An acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells. Cell79 (4), 705–715. 10.1016/0092-8674(94)90555-x

  • CrossRef
  • Google Scholar

• 15

  ElgoyhenA. B.VetterD. E.KatzE.RothlinC. V.HeinemannS. F.BoulterJ. (2001). alpha10: a determinant of nicotinic cholinergic receptor function in mammalian vestibular and cochlear mechanosensory hair cells. Proc. Natl. Acad. Sci. U. S. A.98 (6), 3501–3506. 10.1073/pnas.051622798

  • CrossRef
  • Google Scholar

• 16

  EllisonM.HaberlandtC.Gomez-CasatiM. E.WatkinsM.ElgoyhenA. B.McIntoshJ. M.et al (2006). Alpha-RgIA: A novel conotoxin that specifically and potently blocks the alpha9alpha10 nAChR. Biochemistry45 (5), 1511–1517. 10.1021/bi0520129

  • CrossRef
  • Google Scholar

• 17

  EvansN. M.BoseS.BenedettiG.ZwartR.PearsonK. H.McPhieG. I.et al (2003). Expression and functional characterisation of a human chimeric nicotinic receptor with alpha6beta4 properties. Eur. J. Pharmacol.466 (1-2), 31–39. 10.1016/s0014-2999(03)01540-1

  • CrossRef
  • Google Scholar

• 18

  FilchakovaO.McIntoshJ. M. (2013). Functional expression of human α9* nicotinic acetylcholine receptors in X. laevis oocytes is dependent on the α9 subunit 5' UTR. PLoS One8 (5), e64655. 10.1371/journal.pone.0064655

  • CrossRef
  • Google Scholar

• 19

  FucileS.SucapaneA.EusebiF. (2006). Ca2+ permeability through rat cloned alpha9-containing nicotinic acetylcholine receptors. Cell Calcium39 (4), 349–355. 10.1016/j.ceca.2005.12.002

  • CrossRef
  • Google Scholar

• 20

  FujiiT.MashimoM.MoriwakiY.MisawaH.OnoS.HoriguchiK.et al (2017). Expression and function of the cholinergic system in immune cells. Front. Immunol.8, 1085. 10.3389/fimmu.2017.01085

  • CrossRef
  • Google Scholar

• 21

  GajewiakJ.ChristensenS. B.DowellC.HararahF.FisherF.HuynhP. N.et al (2021). Selective penicillamine substitution enables development of a potent analgesic peptide that acts through a non-opioid-based mechanism. J. Med. Chem.64 (13), 9271–9278. 10.1021/acs.jmedchem.1c00512

  • CrossRef
  • Google Scholar

• 22

  GeorgeA. A.BloyA.MiwaJ. M.LindstromJ. M.LukasR. J.WhiteakerP. (2017). Isoform-specific mechanisms of α3β4*-nicotinic acetylcholine receptor modulation by the prototoxin lynx1. FASEB J.31 (4), 1398–1420. 10.1096/fj.201600733R

  • CrossRef
  • Google Scholar

• 23

  GuS.KnowlandD.MattaJ. A.O'CarrollM. L.DaviniW. B.DharaM.et al (2020). Hair cell α9α10 nicotinic acetylcholine receptor functional expression regulated by ligand binding and deafness gene products. Proc. Natl. Acad. Sci. U. S. A.117 (39), 24534–24544. 10.1073/pnas.2013762117

  • CrossRef
  • Google Scholar

• 24

  GuS.MattaJ. A.DaviniW. B.DaweG. B.LordB.BredtD. S. (2019). α6-Containing nicotinic acetylcholine receptor reconstitution involves mechanistically distinct accessory components. Cell Rep.26 (4), 866–874. 10.1016/j.celrep.2018.12.103

  • CrossRef
  • Google Scholar

• 25

  GuoR.WangT.ZhouG.XuM.YuX.ZhangX.et al (2018). Botany, phytochemistry, Pharmacology and toxicity of strychnos nux-vomica L.: A review. Am. J. Chin. Med.46 (1), 1–23. 10.1142/S0192415X18500015

  • CrossRef
  • Google Scholar

• 26

  HaberbergerR. V.BernardiniN.KressM.HartmannP.LipsK. S.KummerW. (2004). Nicotinic acetylcholine receptor subtypes in nociceptive dorsal root ganglion neurons of the adult rat. Auton. Neurosci.113 (1-2), 32–42. 10.1016/j.autneu.2004.05.008

  • CrossRef
  • Google Scholar

• 27

  HendersonB. J.LesterH. A. (2015). Inside-out neuropharmacology of nicotinic drugs. Neuropharmacology96, 178–193. 10.1016/j.neuropharm.2015.01.022

  • CrossRef
  • Google Scholar

• 28

  HigginsL. S.BergD. K. (1988). A desensitized form of neuronal acetylcholine receptor detected by 3H- nicotine binding on bovine adrenal chromaffin cells [published erratum appears in J Neurosci 1988 Dec;8(12):preceding 4415]. J. Neurosci.8 (4), 1436–1446. 10.1523/jneurosci.08-04-01436.1988

  • CrossRef
  • Google Scholar

• 29

  HoneA. J.FisherF.ChristensenS.GajewiakJ.LarkinD.WhiteakerP.et al (2019). PeIA-5466: A novel peptide antagonist containing non-natural amino acids that selectively targets α3β2 nicotinic acetylcholine receptors.J. Med. Chem.62 (13), 6262–6275. 10.1021/acs.jmedchem.9b00566

  • CrossRef
  • Google Scholar

• 30

  HoneA. J.MeyerE. L.McIntyreM.McIntoshJ. M. (2012). Nicotinic acetylcholine receptors in dorsal root ganglion neurons include the α6β4* subtype.FASEB J.26 (2), 917–926. 10.1096/fj.11-195883

  • CrossRef
  • Google Scholar

• 31

  HoneA. J.Rueda-RuzafaL.GordonT. J.GajewiakJ.ChristensenS.DyhringT.et al (2020). Expression of α3β2β4 nicotinic acetylcholine receptors by rat adrenal chromaffin cells determined using novel conopeptide antagonists.J. Neurochem.154 (2), 158–176. 10.1111/jnc.14966

  • CrossRef
  • Google Scholar

• 32

  HoneA. J.TalleyT. T.BobangoJ.Huidobro MeloC.HararahF.GajewiakJ.et al (2018). Molecular determinants of α-conotoxin potency for inhibition of human and rat α6β4 nicotinic acetylcholine receptors.J. Biol. Chem.293 (46), 17838–17852. 10.1074/jbc.RA118.005649

  • CrossRef
  • Google Scholar

• 33

  Ibanez-TallonI.MiwaJ. M.WangH. L.AdamsN. C.CrabtreeG. W.SineS. M.et al (2002). Novel modulation of neuronal nicotinic acetylcholine receptors by association with the endogenous prototoxin lynx1. Neuron33 (6), 893–903. 10.1016/s0896-6273(02)00632-3

  • CrossRef
  • Google Scholar

• 34

  IndurthiD. C.PeraE.KimH. L.ChuC.McLeodM. D.McIntoshJ. M.et al (2014). Presence of multiple binding sites on α9α10 nAChR receptors alludes to stoichiometric-dependent action of the α-conotoxin, Vc1.1.Biochem. Pharmacol.89 (1), 131–140. 10.1016/j.bcp.2014.02.002

  • CrossRef
  • Google Scholar

• 35

  JakubowskiJ. A.KeaysD. A.KelleyW. P.SandallD. W.BinghamJ. P.LivettB. G.et al (2004). Determining sequences and post-translational modifications of novel conotoxins in Conus victoriae using cDNA sequencing and mass spectrometry. J. Mass Spectrom.39 (5), 548–557. 10.1002/jms.624

  • CrossRef
  • Google Scholar

• 36

  JensenA. B.Hoestgaard-JensenK.JensenA. A. (2013). Elucidation of molecular impediments in the α6 subunit for in vitro expression of functional α6β4* nicotinic acetylcholine receptors. J. Biol. Chem.288 (47), 33708–33721. 10.1074/jbc.M113.509356

  • CrossRef
  • Google Scholar

• 37

  JohnsonD. S.MartinezJ.ElgoyhenA. B.HeinemannS. F.McIntoshJ. M. (1995). alpha-Conotoxin ImI exhibits subtype-specific nicotinic acetylcholine receptor blockade: preferential inhibition of homomeric alpha 7 and alpha 9 receptors. Mol. Pharmacol.48 (2), 194–199.

  • Google Scholar

• 38

  KatzE.VerbitskyM.RothlinC. V.VetterD. E.HeinemannS. F.ElgoyhenA. B. (2000). High calcium permeability and calcium block of the alpha9 nicotinic acetylcholine receptor. Hear. Res.141 (1-2), 117–128. 10.1016/s0378-5955(99)00214-2

  • CrossRef
  • Google Scholar

• 39

  KawashimaK.YoshikawaK.FujiiY. X.MoriwakiY.MisawaH. (2007). Expression and function of genes encoding cholinergic components in murine immune cells. Life Sci.80 (24-25), 2314–2319. 10.1016/j.lfs.2007.02.036

  • CrossRef
  • Google Scholar

• 40

  KuryatovA.OlaleF.CooperJ.ChoiC.LindstromJ. (2000). Human alpha6 AChR subtypes: Subunit composition, assembly, and pharmacological responses. Neuropharmacology39 (13), 2570–2590. 10.1016/s0028-3908(00)00144-1

  • CrossRef
  • Google Scholar

• 41

  KurzenH.BergerH.JagerC.HartschuhW.NaherH.GratchevA.et al (2004). Phenotypical and molecular profiling of the extraneuronal cholinergic system of the skin. J. Invest. Dermatol.123 (5), 937–949. 10.1111/j.0022-202X.2004.23425.x

  • CrossRef
  • Google Scholar

• 42

  KurzenH.WesslerI.KirkpatrickC. J.KawashimaK.GrandoS. A. (2007). The non-neuronal cholinergic system of human skin. Horm. Metab. Res.39 (2), 125–135. 10.1055/s-2007-961816

  • CrossRef
  • Google Scholar

• 43

  LeeC. H.HuangC. S.ChenC. S.TuS. H.WangY. J.ChangY. J.et al (2010). Overexpression and activation of the alpha9-nicotinic receptor during tumorigenesis in human breast epithelial cells. J. Natl. Cancer Inst.102 (17), 1322–1335. 10.1093/jnci/djq300

  • CrossRef
  • Google Scholar

• 44

  LeyC. K.KuryatovA.WangJ.LindstromJ. M. (2014). Efficient expression of functional (α6β2)2β3 AChRs in Xenopus oocytes from free subunits using slightly modified α6 subunits.PLoS One9 (7), e103244. 10.1371/journal.pone.0103244

  • CrossRef
  • Google Scholar

• 45

  LipovsekM.FierroA.PerezE. G.BoffiJ. C.MillarN. S.FuchsP. A.et al (2014). Tracking the molecular evolution of calcium permeability in a nicotinic acetylcholine receptor. Mol. Biol. Evol.31 (12), 3250–3265. 10.1093/molbev/msu258

  • CrossRef
  • Google Scholar

• 46

  LipovsekM.MarcovichI.ElgoyhenA. B. (2021). The hair cell α9α10 nicotinic acetylcholine receptor: Odd cousin in an old family.Front. Cell. Neurosci.15, 785265. 10.3389/fncel.2021.785265

  • CrossRef
  • Google Scholar

• 47

  LuL.HuangR.WuY.JinJ. M.ChenH. Z.ZhangL. J.et al (2020). Brucine: A review of phytochemistry, Pharmacology, and toxicology. Front. Pharmacol.11, 377. 10.3389/fphar.2020.00377

  • CrossRef
  • Google Scholar

• 48

  LustigL. R.PengH.HielH.YamamotoT.FuchsP. A. (2001). Molecular cloning and mapping of the human nicotinic acetylcholine receptor alpha10 (CHRNA10). Genomics73 (3), 272–283. 10.1006/geno.2000.6503

  • CrossRef
  • Google Scholar

• 49

  MarcovichI.MoglieM. J.Carpaneto FreixasA. E.TrigilaA. P.FranchiniL. F.PlazasP. V.et al (2020). Distinct evolutionary trajectories of neuronal and hair cell nicotinic acetylcholine receptors. Mol. Biol. Evol.37 (4), 1070–1089. 10.1093/molbev/msz290

  • CrossRef
  • Google Scholar

• 50

  McIntoshJ. M.AzamL.StaheliS.DowellC.LindstromJ. M.KuryatovA.et al (2004). Analogs of alpha-conotoxin MII are selective for alpha6-containing nicotinic acetylcholine receptors. Mol. Pharmacol.65 (4), 944–952. 10.1124/mol.65.4.944

  • CrossRef
  • Google Scholar

• 51

  McIntoshJ. M.PlazasP. V.WatkinsM.Gomez-CasatiM. E.OliveraB. M.ElgoyhenA. B. (2005). A novel alpha-conotoxin, PeIA, cloned from Conus pergrandis, discriminates between rat alpha9alpha10 and alpha7 nicotinic cholinergic receptors. J. Biol. Chem.280 (34), 30107–30112. 10.1074/jbc.M504102200

  • CrossRef
  • Google Scholar

• 52

  MikulskiZ.HartmannP.JositschG.ZaslonaZ.LipsK. S.PfeilU.et al (2010). Nicotinic receptors on rat alveolar macrophages dampen ATP-induced increase in cytosolic calcium concentration. Respir. Res.11, 133. 10.1186/1465-9921-11-133

  • CrossRef
  • Google Scholar

• 53

  MilediR.ParkerI. (1984). Chloride current induced by injection of calcium into Xenopus oocytes. J. Physiol.357, 173–183. 10.1113/jphysiol.1984.sp015495

  • CrossRef
  • Google Scholar

• 54

  MishraN. C.Rir-sima-ahJ.BoydR. T.SinghS. P.GundavarapuS.LangleyR. J.et al (2010). Nicotine inhibits Fc epsilon RI-induced cysteinyl leukotrienes and cytokine production without affecting mast cell degranulation through alpha 7/alpha 9/alpha 10-nicotinic receptors. J. Immunol.185 (1), 588–596. 10.4049/jimmunol.0902227

  • CrossRef
  • Google Scholar

• 55

  MiwaJ. M. (2021). Lynx1 prototoxins: Critical accessory proteins of neuronal nicotinic acetylcholine receptors. Curr. Opin. Pharmacol.56, 46–51. 10.1016/j.coph.2020.09.016

  • CrossRef
  • Google Scholar

• 56

  MoglieM. J.MarcovichI.CorradiJ.Carpaneto FreixasA. E.GallinoS.PlazasP. V.et al (2021). Loss of choline agonism in the inner ear hair cell nicotinic acetylcholine receptor linked to the α10 subunit. Front. Mol. Neurosci.14, 639720. 10.3389/fnmol.2021.639720

  • CrossRef
  • Google Scholar

• 57

  MorleyB. J.WhiteakerP.ElgoyhenA. B. (2018). Commentary: Nicotinic acetylcholine receptor α9 and α10 subunits are expressed in the brain of mice. Front. Cell. Neurosci.12, 104. 10.3389/fncel.2018.00104

  • CrossRef
  • Google Scholar

• 58

  MucchiettoV.FasoliF.PucciS.MorettiM.BenfanteR.MaroliA.et al (2018). α9- and α7-containing receptors mediate the pro-proliferative effects of nicotine in the A549 adenocarcinoma cell line.Br. J. Pharmacol.175 (11), 1957–1972. 10.1111/bph.13954

  • CrossRef
  • Google Scholar

• 59

  PaciniA.MicheliL.MarescaM.BrancaJ. J.McIntoshJ. M.GhelardiniC.et al (2016). The α9α10 nicotinic receptor antagonist α-conotoxin RgIA prevents neuropathic pain induced by oxaliplatin treatment.Exp. Neurol.282, 37–48. 10.1016/j.expneurol.2016.04.022

  • CrossRef
  • Google Scholar

• 60

  PapkeR. L.BagdasD.KulkarniA. R.GouldT.AlSharariS. D.ThakurG. A.et al (2015). The analgesic-like properties of the alpha7 nAChR silent agonist NS6740 is associated with non-conducting conformations of the receptor. Neuropharmacology91, 34–42. 10.1016/j.neuropharm.2014.12.002

  • CrossRef
  • Google Scholar

• 61

  ParkerR. L.O'NeillH. C.HenleyB. M.WagemanC. R.DrenanR. M.MarksM. J.et al (2017). Deletion of lynx1 reduces the function of α6* nicotinic receptors. PLoS One12 (12), e0188715. 10.1371/journal.pone.0188715

  • CrossRef
  • Google Scholar

• 62

  PengH.FerrisR. L.MatthewsT.HielH.Lopez-AlbaiteroA.LustigL. R. (2004). Characterization of the human nicotinic acetylcholine receptor subunit alpha (alpha) 9 (CHRNA9) and alpha (alpha) 10 (CHRNA10) in lymphocytes. Life Sci.76 (3), 263–280. 10.1016/j.lfs.2004.05.031

  • CrossRef
  • Google Scholar

• 63

  PerezE. G.CasselsB. K.Zapata-TorresG. (2009). Molecular modeling of the alpha9alpha10 nicotinic acetylcholine receptor subtype. Bioorg. Med. Chem. Lett.19 (1), 251–254. 10.1016/j.bmcl.2008.10.094

  • CrossRef
  • Google Scholar

• 64

  Perez-AlvarezA.Hernandez-VivancoA.GregorioS. A.TaberneroA.McIntoshJ. M.AlbillosA. (2012). Pharmacological characterization of native α7 nicotinic ACh receptors and their contribution to depolarization-elicited exocytosis in human chromaffin cells. Br. J. Pharmacol.165 (4), 908–921. 10.1111/j.1476-5381.2011.01596.x

  • CrossRef
  • Google Scholar

• 65

  RauK. K.JohnsonR. D.CooperB. Y. (2005). Nicotinic AChR in subclassified capsaicin-sensitive and -insensitive nociceptors of the rat DRG. J. Neurophysiol.93 (3), 1358–1371. 10.1152/jn.00591.2004

  • CrossRef
  • Google Scholar

• 66

  RichterK.MathesV.FroniusM.AlthausM.HeckerA.Krasteva-ChristG.et al (2016). Phosphocholine - an agonist of metabotropic but not of ionotropic functions of α9-containing nicotinic acetylcholine receptors. Sci. Rep.6, 28660. 10.1038/srep28660

  • CrossRef
  • Google Scholar

• 67

  RothlinC. V.KatzE.VerbitskyM.ElgoyhenA. B. (1999). The alpha9 nicotinic acetylcholine receptor shares pharmacological properties with type A gamma-aminobutyric acid, glycine, and type 3 serotonin receptors. Mol. Pharmacol.55 (2), 248–254. 10.1124/mol.55.2.248

  • CrossRef
  • Google Scholar

• 68

  SatkunanathanN.LivettB.GaylerK.SandallD.DownJ.KhalilZ. (2005). Alpha-conotoxin Vc1.1 alleviates neuropathic pain and accelerates functional recovery of injured neurones. Brain Res.1059 (2), 149–158. 10.1016/j.brainres.2005.08.009

  • CrossRef
  • Google Scholar

• 69

  SgardF.CharpantierE.BertrandS.WalkerN.CaputD.GrahamD.et al (2002). A novel human nicotinic receptor subunit, alpha10, that confers functionality to the alpha9-subunit. Mol. Pharmacol.61 (1), 150–159. 10.1124/mol.61.1.150

  • CrossRef
  • Google Scholar

• 70

  SrinivasanR.PantojaR.MossF. J.MackeyE. D.SonC. D.MiwaJ.et al (2011). Nicotine up-regulates alpha4beta2 nicotinic receptors and ER exit sites via stoichiometry-dependent chaperoning. J. Gen. Physiol.137 (1), 59–79. 10.1085/jgp.201010532

  • CrossRef
  • Google Scholar

• 71

  TraceyK. J. (2007). Physiology and immunology of the cholinergic antiinflammatory pathway. J. Clin. Invest.117 (2), 289–296. 10.1172/JCI30555

  • CrossRef
  • Google Scholar

• 72

  VinclerM.WittenauerS.ParkerR.EllisonM.OliveraB. M.McIntoshJ. M. (2006). Molecular mechanism for analgesia involving specific antagonism of alpha9alpha10 nicotinic acetylcholine receptors. Proc. Natl. Acad. Sci. U. S. A.103 (47), 17880–17884. 10.1073/pnas.0608715103

  • CrossRef
  • Google Scholar

• 73

  YuR.KompellaS. N.AdamsD. J.CraikD. J.KaasQ. (2013). Determination of the α-conotoxin Vc1.1 binding site on the α9α10 nicotinic acetylcholine receptor. J. Med. Chem.56 (9), 3557–3567. 10.1021/jm400041h

  • CrossRef
  • Google Scholar

• 74

  ZakrzewiczA.RichterK.AgneA.WilkerS.SiebersK.FinkB.et al (2017). Canonical and novel non-canonical cholinergic agonists inhibit ATP-induced release of monocytic interleukin-1β via different combinations of nicotinic acetylcholine receptor subunits α7, α9 and α10.Front. Cell. Neurosci.11, 189. 10.3389/fncel.2017.00189

  • CrossRef
  • Google Scholar

• 75

  ZhangX.HartungJ. E.FriedmanR. L.KoerberH. R.BelferI.GoldM. S. (2019). Nicotine evoked currents in human primary sensory neurons. J. Pain20 (7), 810–818. 10.1016/j.jpain.2019.01.005

  • CrossRef
  • Google Scholar

• 76

  Zorrilla de San MartinJ.BallesteroJ.KatzE.ElgoyhenA. B.FuchsP. A. (2007). Ryanodine is a positive modulator of acetylcholine receptor gating in cochlear hair cells. J. Assoc. Res. Otolaryngol.8 (4), 474–483. 10.1007/s10162-007-0090-y

  • CrossRef
  • Google Scholar

• 77

  ZouridakisM.PapakyriakouA.IvanovI. A.KasheverovI. E.TsetlinV.TzartosS.et al (2019). Crystal structure of the monomeric extracellular domain of α9 nicotinic receptor subunit in complex with α-conotoxin RgIA: Molecular dynamics insights into RgIA binding to α9α10 nicotinic receptors. Front. Pharmacol.10, 474. 10.3389/fphar.2019.00474

  • CrossRef
  • Google Scholar