Α9α10 Nicotinic Acetylcholine Receptor Research Articles

ChemInform Abstract: Discovery of Non‐Peptide, Small Molecule Antagonists of α9α10 Nicotinic Acetylcholine Receptors as Novel Analgesics for the Treatment of Neuropathic and Tonic Inflammatory Pain.

AbstractA series of azaaromatic quaternary ammonium analogues, e.g. (I) and (II), is discovered as potent and selective α9α10 nicotinic acetylcholine receptor (nAChR) antagonists.

Discovery of non-peptide, small molecule antagonists of α9α10 nicotinic acetylcholine receptors as novel analgesics for the treatment of neuropathic and tonic inflammatory pain

A series of azaaromatic quaternary ammonium analogs has been discovered as potent and selective α9α10 nicotinic acetylcholine receptor (nAChR) antagonists. The preliminary structure-activity relationships of these analogs suggest that increased rigidity in the linker units results in higher potency in inhibition of α9α10 nAChRs and greater selectivity over α7 nAChRs. These analogs represent a new class of analgesic for the treatment of neuropathic and tonic inflammatory pain.

Differential involvement of α4β2, α7 and α9α10 nicotinic acetylcholine receptors in B lymphocyte activation in vitro

Mouse B lymphocytes express several nicotinic acetylcholine receptor (nAChR) subtypes, their exact functions being not clearly understood. Here we show that α7 nAChR was present in about 60%, while α4β2 and α9(α10) nAChRs in about 10% and 20% of mouse spleen B lymphocytes, respectively; Balb/c and C57Bl/6 mice possessed different relative amounts of these nAChR subtypes. α4β2 and α7, but not α9(α10) nAChRs, were up-regulated upon B lymphocyte activation in vitro. Flow cytometry and sandwich ELISA studies demonstrated that α7 and α9(α10) nAChRs are coupled to CD40, whereas α4β2 nAChR is coupled to IgM. B lymphocytes of both α7 −/− and β2 −/− mice responded to anti-CD40 stronger than those of the wild-type mice, whereas the cells of β2 −/− mice responded to anti-IgM worse than those of the wild-type or α7 −/− mice. Inhibition of α7 and α9(α10) nAChRs with methyllicaconitine resulted in considerable augmentation of CD40-mediated B lymphocyte proliferation in cells of all genotypes; stimulation of α4β2 nAChRs with epibatidine increased the IgM-mediated proliferation of the wild-type and α7 −/−, but not β2 −/− cells. Inhibition of α9(α10) nAChRs with α-conotoxin PeAI exerted weak stimulating effect on CD40-mediated proliferation. This nAChR subtype was up-regulated in α7 −/− B-cells. α7 nAChRs were found recruited to immune synapses between human T and B lymphocytes, both of which produced acetylcholine. It is concluded that α7 nAChR fulfills inhibitory CD40-related mitogenic function, α4β2 nAChR produces a stimulatory IgM-related effect, while α9α10 nAChR is a “reserve” receptor, which partly compensates the absence of α7 nAChR in α7 −/− cells. Acetylcholine is an additional mediator to modulate activation of interacting T and B lymphocytes.

Effects of quercetin on α9α10 nicotinic acetylcholine receptor-mediated ion currents

Quercetin, one of the flavonoids, is a low molecular weight substance found in fruits and vegetables. Quercetin, like other flavonoids, has a wide range of neuropharmacological actions and antioxidant effects. The α9α10 nicotinic acetylcholine receptor is one of the numerous nicotinic acetylcholine receptors that exist as a heteropentameric form between efferent olivocochlear fibers and hair cells of the cochlea. In this study, we report the effects of quercetin on rat α9α10 nicotinic acetylcholine receptor-mediated ion currents using the two-electrode voltage-clamp technique. Treatment with acetylcholine evoked inward currents ( I ACh) in oocytes heterologously expressing the α9α10 nicotinic acetylcholine receptor. Quercetin blocked I ACh in concentration-dependent and reversible manners, and the blocking effect on I ACh was stronger with pre-application than co-application of quercetin. The half maximal inhibitory concentration (IC 50) of quercetin was 45.4 ± 10.1 μM. Quercetin-mediated I ACh inhibition was not affected by acetylcholine concentration and was independent of membrane-holding potential. Although the inhibitory effect of quercetin was significantly attenuated in the absence of extracellular Ca 2+, the action of quercetin was independent of extracellular Ca 2+ concentration, indicating that the presence of extracellular Ca 2+ might be needed for quercetin-related effects and might play an important role in quercetin-mediated regulation of the α9α10 nicotinic acetylcholine receptor. These results indicate that quercetin-mediated regulation of the α9α10 nicotinic acetylcholine receptor could provide a molecular basis for quercetin actions at the cellular level.

Analgesic α-conotoxins Vc1.1 and Rg1A inhibit N-type calcium channels in sensory neurons of α9 nicotinic receptor knockout mice

α-Conotoxins Vc1.1 and Rg1A are peptides from the venom of marine Conus snails that are currently in development as a treatment for neuropathic pain. We have reported previously that the α9α10 nicotinic acetylcholine receptor (nAChR) selective-conotoxins Vc1.1 and Rg1A potently and selectively inhibit high voltage-activated (HVA) N-type calcium channel currents in dissociated neurons from rat dorsal root ganglia (DRG). Our data indicated that Vc1.1 does not interact directly with N-type Ca2+ channels but inhibits them via GABAB receptor activation. The present study investigated Vc1.1 and Rg1A inhibition of N-type Ca2+ channels currents in DRG neurons of wild-type and α9 knockout (KO) mice to determine if the α9 nAChR was necessary for inhibition of the Ca2+ channel current. Application of Vc1.1 (100 nM) inhibited N-type Ca2+ channel currents to 69.2 ± 3.5% of control in DRG neurons isolated from wild-type mice. In {greater than or equal to >70% of DRG neurons isolated from the α9 KO mice, both Vc1.1 and Rg1A selectively inhibited N-type Ca2+ channel currents with an IC50 of 24.6 nM and 22.4 nM, respectively. The GABAB receptor antagonist CGP55845 (1 μM) antagonized the effect of Vc1.1 and Rg1A on the N-type Ca2+ channels in α9 KO mice. RT-PCR and western blot analysis confirmed the absence of the α9 nAChR in mice carrying a null mutation for the nAChR α9 gene. These results demonstrate that the inhibition of N-type Ca2+ channel channels by Vc1.1 and Rg1A is not mediated by the expression of α9α10 nAChRs in DRG neurons.

Scanning Mutagenesis of α-Conotoxin Vc1.1 Reveals Residues Crucial for Activity at the α9α10 Nicotinic Acetylcholine Receptor

Vc1.1 is a disulfide-rich peptide inhibitor of nicotinic acetylcholine receptors that has stimulated considerable interest in these receptors as potential therapeutic targets for the treatment of neuropathic pain. Here we present an extensive series of mutational studies in which all residues except the conserved cysteines were mutated separately to Ala, Asp, or Lys. The effect on acetylcholine (ACh)-evoked membrane currents at the alpha9alpha10 nicotinic acetylcholine receptor (nAChR), which has been implicated as a target in the alleviation of neuropathic pain, was then observed. The analogs were characterized by NMR spectroscopy to determine the effects of mutations on structure. The structural fold was found to be preserved in all peptides except where Pro was substituted. Electrophysiological studies showed that the key residues for functional activity are Asp(5)-Arg(7) and Asp(11)-Ile(15), because changes at these positions resulted in the loss of activity at the alpha9alpha10 nAChR. Interestingly, the S4K and N9A analogs were more potent than Vc1.1 itself. A second generation of mutants was synthesized, namely N9G, N9I, N9L, S4R, and S4K+N9A, all of which were more potent than Vc1.1 at both the rat alpha9alpha10 and the human alpha9/rat alpha10 hybrid receptor, providing a mechanistic insight into the key residues involved in eliciting the biological function of Vc1.1. The most potent analogs were also tested at the alpha3beta2, alpha3beta4, and alpha7 nAChR subtypes to determine their selectivity. All mutants tested were most selective for the alpha9alpha10 nAChR. These findings provide valuable insight into the interaction of Vc1.1 with the alpha9alpha10 nAChR subtype and will help in the further development of analogs of Vc1.1 as analgesic drugs.

Molecular modeling of the α9α10 nicotinic acetylcholine receptor subtype

This study reports the comparative molecular modeling, docking and dynamic simulations of human α9α10 nicotinic acetylcholine receptors complexed with acetylcholine, nicotine and α-conotoxin RgIA, using as templates the crystal structures of Aplysia californica and Lymnaea stagnalis acetylcholine binding proteins. The molecular dynamics simulations showed that Arg112 in the complementary α10(−) subunit, is a determinant for recognition in the site that binds small ligands. However, Glu195 in the principal α9(+), and Asp114 in the complementary α10(−) subunit, might confer the potency and selectivity to α-conotoxin RgIA when interacting with Arg7 and Arg9 of this ligand.

Selective inhibition of human heteromeric α9α10 nicotinic acetylcholine receptors at a low agonist concentration by low concentrations of ototoxic organic solvents

Ethylbenzene and para-xylene (p-xylene), but not the chemically closely related organic solvents ortho-xylene (o-xylene) and meta-xylene (m-xylene), are known to cause ototoxicity and irreversible hearing loss, though the underlying mechanisms are still unknown. In this study, effects of ethylbenzene and of p-, o-, and m-xylene on human heteromeric alpha9alpha10 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes were investigated using the two-electrode voltage clamp technique. ACh dose-dependently evoked an alpha9alpha10 nAChR-mediated ion current with an EC(50) of 137 microM. When ACh is applied at a low concentration (10 microM), the nAChR-mediated ion current is inhibited by a low concentration (10 microM) of ethylbenzene and p-xylene, but not by the same concentration of the non-ototoxic solvents. At a high solvent concentration (300 microM), all solvents cause inhibition of the ion currents evoked by 10 microM ACh. Ion currents evoked by a near maximum-effective concentration ACh (1mM) are inhibited by the selected organic solvents only at 300 microM. These results demonstrate that low concentrations of the known ototoxic solvents ethylbenzene and p-xylene inhibit alpha9alpha10 nAChR-mediated ion currents, whereas the structurally related, non-ototoxic solvents m-xylene and o-xylene do not, indicating that the alpha9alpha10 nAChR is a potential target for solvent-induced ototoxicity.

The three-dimensional structure of the analgesic α-conotoxin, RgIA

The α-conotoxin RgIA is a selective antagonist of the α9α10 nicotinic acetylcholine receptor and has been shown to be a potent analgesic and reduces nerve injury associated inflammation. RgIA was chemically synthesized and found to fold into two disulfide isomers, globular and ribbon. The native globular isomer inhibited ACh-evoked currents reversibly in oocytes expressing rat α9α10 nAChRs but the ribbon isomer was inactive. We determined the three-dimensional structure of RgIA using NMR methods to assist in elucidating the molecular role of RgIA in analgesia and inflammation.

Targeting the α9α10 nicotinic acetylcholine receptor to treat severe pain

The α9α10 nicotinic acetylcholine receptors (nAChRs) are recognized for their function in the hair cells of the inner ear; transcripts for a9 and/or a10 subunits have also been identified in a diverse range of other tissues , including immune cells. The functioning of α9α10 nAChRs in these latter tissues is unknown. However, a recent series of studies has provided evidence that blockade of the α9α10 nAChR can alleviate chronic pain resulting from overt peripheral nerve injury or inflammation and increase the functional recovery of damaged neurons. Systemic administration of α9α10 antagonists produces an acute analgesia; repeated daily administrations produces sustained and cumulative levels of analgesia across 7 days without the development of tolerance. Although the exact mechanism of action is unknown, antagonism of the α9α10 nAChRs reduces the number of immune cells present at the site of injury.

Inhibition of the α9α10 nicotinic cholinergic receptor by neramexane, an open channel blocker of N-methyl- d-aspartate receptors

In this study we report the effects of neramexane, a novel amino-alkyl-cyclohexane derivative that is a non-competitive N-methyl- d-aspartate (NMDA) receptor antagonist, on recombinant rat α9α10 nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes. We compared its effects with those of memantine, a well-studied pore blocker of NMDA receptors, currently used in therapeutics for the treatment of Alzheimer's disease. Our results indicate that both compounds block acetylcholine-evoked responses at micromolar concentrations with a rank order of potency of neramexane > memantine, P < 0.05. Block by neramexane of acetylcholine responses was not overcome at high concentrations of the agonist, indicative of a non-competitive inhibition. The lack of interaction of neramexane with the ligand binding domain was confirmed by radioligand binding experiments in transfected tsA201 cells. Moreover, block did not involve an increase in desensitization kinetics, it was independent of the resting potential of the membrane at low concentrations of neramexane and slightly voltage-dependent at concentrations higher than 1 μM. Finally, clinically-relevant concentrations of neramexane blocked native α9α10-containing nicotinic acetylcholine receptors of rat inner hair cells, thus demonstrating a possible in vivo relevance in potentially unexplored therapeutic areas.

Molecular mechanism for analgesia involving specific antagonism of α9α10 nicotinic acetylcholine receptors

alpha9alpha10 nicotinic acetylcholine receptors (nAChRs) have been identified in a variety of tissues including lymphocytes and dorsal root ganglia; except in the case of the auditory system, the function of alpha9alpha10 nAChRs is not known. Here we show that selective block (rather than stimulation) of alpha9alpha10 nAChRs is analgesic in an animal model of nerve injury pain. In addition, blockade of this nAChR subtype reduces the number of choline acetyltransferase-positive cells, macrophages, and lymphocytes at the site of injury. Chronic neuropathic pain is estimated to affect up to 8% of the world's population; the numerous analgesic compounds currently available are largely ineffective and act through a small number of pharmacological mechanisms. Our findings not only suggest a molecular mechanism for the treatment of neuropathic pain but also demonstrate the involvement of alpha9alpha10 nAChRs in the pathophysiology of peripheral nerve injury.

Ca 2+ permeability through rat cloned α9-containing nicotinic acetylcholine receptors

We investigated the functional properties of rat α9 and α9α10 nicotinic acetylcholine receptors (nAChRs) expressed by transient transfection in the rat GH4C1 cell line, using both Ca 2+ imaging and whole-cell recording. Acute applications of ACh generated short-delay fast-rising and quick-decaying Ca 2+ transients, suppressed in Ca 2+-free medium and invariably accompanied by the activation of whole-cell inward currents. The mean amplitude of ACh-induced currents was as small as −16 pA in α9 subunit cDNA-transfected GH4C1 cells (α9-GH4C1), while they were much larger (range: −150 to −300 pA) in α9α10 subunit cDNAs-transfected GH4C1 cells (α9α10-GH4C1). Currents were not activated by nicotine, were blocked by methyllycaconitine and were ACh concentration-dependent. Because the Ca 2+ permeability of α9-containing nAChRs has been estimated in immortalized cochlear UB/OC-2 mouse cells, we also characterized the ACh-induced responses in these cells. Unlike α9- and α9α10-GH4C1 cells, UB/OC-2 cells responded to ACh with both long-delay methyllycaconitine-insensitive whole-cell currents and long-lasting Ca 2+ transients, the latter being detected in the absence of Ca 2+ in the extracellular medium and being suppressed by the Ca 2+-ATPase inhibitor thapsigargin, known to deplete IP 3-sensitive stores. These results indicated the involvement of muscarinic nAChRs and the lack of functional ACh-gated receptor channels in UB/OC-2 cells. Thus, we measured the fractional Ca 2+ current ( P f, i.e. the percentage of total current carried by Ca 2+ ions) in α9α10-GH4C1, obtaining a P f value of 22 ± 4%; this is the largest value estimated to date for a ligand-gated receptor channel. The physiological role played by Ca 2+ entry through α9-containing nAChRs gated by ACh is discussed.

The α9α10 nicotinic acetylcholine receptor is permeable to and is modulated by divalent cations

The native cholinergic receptor that mediates synaptic transmission between olivocochlear fibers and outer hair cells of the cochlea is permeable to Ca 2+ and is thought to be composed of both the α9 and the α10 cholinergic nicotinic subunits. The aim of the present work was to study the permeability of the recombinant α9α10 nicotinic acetylcholine receptor to Ca 2+, Ba 2+ and Mg 2+ and its modulation by these divalent cations. Experiments were performed, by the two-electrode voltage-clamp technique, in Xenopus laevis oocytes injected with α9 and α10 cRNA. The relative divalent to monovalent cation permeability was high (∼10) for Ca 2+, Ba 2+ and Mg 2+. Currents evoked by acetylcholine (ACh) were potentiated by either Ca 2+ or Ba 2+ up to 500 μM but were blocked by higher concentrations of these cations. Potentiation by Ca 2+ was voltage-independent, whereas blockage was stronger at hyperpolarized than at depolarized potentials. Mg 2+ did not potentiate but it blocked ACh-evoked currents (IC 50=0.38 mM). In the absence of Ca 2+, the EC 50 for ACh was higher (48 μM) than that obtained with 1.8 mM Ca 2+ (14.3 μM), suggesting that potentiation by Ca 2+ involves changes in the apparent affinity of the α9α10 receptor for ACh.