ACh-induced Currents Research Articles

The human alpha7 nicotinic acetylcholine receptor is a host target for the rabies virus glycoprotein.

The rabies virus enters the nervous system by interacting with several molecular targets on host cells to modify behavior and trigger receptor-mediated endocytosis of the virion by poorly understood mechanisms. The rabies virus glycoprotein (RVG) interacts with the muscle acetylcholine receptor and the neuronal α4β2 subtype of the nicotinic acetylcholine receptor (nAChR) family by the putative neurotoxin-like motif. Given that the neurotoxin-like motif is highly homologous to the α7 nAChR subtype selective snake toxin α-bungarotoxin (αBTX), other nAChR subtypes are likely involved. The purpose of this study is to determine the activity of the RVG neurotoxin-like motif on nAChR subtypes that are expressed in brain regions involved in rabid animal behavior. nAChRs were expressed in Xenopus laevis oocytes, and two-electrode voltage clamp electrophysiology was used to collect concentration-response data to measure the functional effects. The RVG peptide preferentially and completely inhibits α7 nAChR ACh-induced currents by a competitive antagonist mechanism. Tested heteromeric nAChRs are also inhibited, but to a lesser extent than the α7 subtype. Residues of the RVG peptide with high sequence homology to αBTX and other neurotoxins were substituted with alanine. Altered RVG neurotoxin-like peptides showed that residues phenylalanine 192, arginine 196, and arginine 199 are important determinants of RVG peptide apparent potency on α7 nAChRs, while serine 195 is not. The evaluation of the rabies ectodomain reaffirmed the observations made with the RVG peptide, illustrating a significant inhibitory impact on α7 nAChR with potency in the nanomolar range. In a mammalian cell culture model of neurons, we confirm that the RVG peptide binds preferentially to cells expressing the α7 nAChR. Defining the activity of the RVG peptide on nAChRs expands our understanding of basic mechanisms in host-pathogen interactions that result in neurological disorders.

Desformylflustrabromine (dFBr), a positive allosteric modulator of the α4β2 nicotinic receptor modulates the hypnotic response to ethanol.

Binge drinking can increase an individual's risk of developing alcohol use disorder (AUD). Ethanol targets multiple neurotransmitter systems; however, not much is known about its effects on the cholinergic system. Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels, the heteromeric α4β2 nAChR being a commonly expressed subtype. Desformylflustrabromine (dFBr), a positive allosteric modulator (PAM), increases the efficacy of α4β2 nAChR invitro and has previously been shown to have translational potential. In this study, we investigated whether dFBr modulates the hypnotic response to ethanol. Ethanol-induced loss of righting reflex (LORR) duration was measured in the presence and absence of dFBr. The β2 nAChR selective antagonist dihydro-β-erythroidine (DHβE) was used to study the involvement of the β2 subunit. Additionally, we used a crosslinking-based western blot assay to estimate changes in total versus intracellular α4 nAChR protein in thalamic tissue of rats treated with vehicle, dFBr, ethanol, or ethanol and dFBr. Lastly, using Xenopus oocyte two-electrode voltage clamp (TEVC) studies, we determined the effects of ethanol and dFBr on α4β2 nAChR. Pretreatment with 6mg/kg dFBr reduced ethanol-induced LORR duration as compared to rats treated with ethanol alone. LORR studies with DHβE suggest that dFBr reduced ethanol-induced LORR duration via the β2 nAChR subunit. Crosslinking-based western analyses revealed that ethanol caused early increases in total and presumably surface thalamic α4 nAChR subunit protein levels. This ethanol-induced α4 nAChR upregulation was significantly reduced in rats pretreated with 6mg/kg dFBr. In TEVC studies, ethanol potentiated ACh-induced currents in α4β2 nAChR, while it slightly reduced dFBr potentiation of maximal ACh currents. Our results suggest that thalamic nAChRs containing the α4 subunit are rapidly upregulated by a single intoxicating dose of ethanol. Furthermore, dFBr, an α4β2 nAChR-selective PAM, significantly attenuates the hypnotic response to ethanol via actions on β2 nAChR. Overall, these results indicate that dFBr represents an option to reverse ethanol intoxication.

Defective Tmprss3-Associated Hair Cell Degeneration in Inner Ear Organoids.

SummaryMutations in the gene encoding the type II transmembrane protease 3 (TMPRSS3) cause human hearing loss, although the underlying mechanisms that result in TMPRSS3-related hearing loss are still unclear. We combined the use of stem cell-derived inner ear organoids with single-cell RNA sequencing to investigate the role of TMPRSS3. Defective Tmprss3 leads to hair cell apoptosis without altering the development of hair cells and the formation of the mechanotransduction apparatus. Prior to degeneration, Tmprss3-KO hair cells demonstrate reduced numbers of BK channels and lower expressions of genes encoding calcium ion-binding proteins, suggesting a disruption in intracellular homeostasis. A proteolytically active TMPRSS3 was detected on cell membranes in addition to ER of cells in inner ear organoids. Our in vitro model recapitulated salient features of genetically associated inner ear abnormalities and will serve as a powerful tool for studying inner ear disorders.

Muscarinic Acetylcholine Receptor-2 in the Cerebellar Fastigial Nucleus is Involved in Acetylcholine-Mediated Blood Pressure Regulation in Rats

Our previous investigations have revealed that microinjection of acetylcholine (ACh) into the cerebellar fastigial nucleus (FN) induces a systemic blood pressure (BP) depressor response and that the muscarinic ACh (mACh) receptor contributes substantially to this process. This study was determined to explore the role of muscarinic ACh receptor-2 (M2 receptor) in ACh-mediated BP regulation and its underlying electrophysiological mechanism. ACh mixed with methoctramine hydrate (MCT, a selective M2 receptor antagonist) and normal saline (0.9% NaCl) were separately microinjected into the FN of anaesthetized rats, and the mean arterial pressure during the reactive time (the duration for BP to return to its basal level) was calculated; the responses of cholinergic reagents to the inward currents in FN neurons were also examined via patch-clamp recording in vitro; in addition, the receptor expression in the FN was confirmed by immunohistochemistry. The results demonstrated that MCT effectively blocked the ACh-mediated BP depressor response and the ACh-induced inward currents in FN neurons; moreover, M2 receptors were unequivocally distributed in the FN. Taken together, the results indicate that M2 receptors in the FN may be involved in ACh-mediated BP down-regulation by tuning neuronal excitation.

Chronic Menthol Does Not Change Stoichiometry or Functional Plasma Membrane Levels of Mouse α3β4-Containing Nicotinic Acetylcholine Receptors.

Heteromeric α3β4 nicotinic acetylcholine (ACh) receptors (nAChRs) are pentameric ligand-gated cation channels that include at least two α3 and two β4 subunits. They have functions in peripheral tissue and peripheral and central nervous systems. We examined the effects of chronic treatment with menthol, a major flavor additive in tobacco cigarettes and electronic nicotine delivery systems, on mouse α3β4 nAChRs transiently transfected into neuroblastoma-2a cells. Chronic menthol treatment at 500 nM, near the estimated menthol concentration in the brain following cigarette smoking, altered neither the [ACh]-response relationship nor Zn2+ sensitivity of ACh-evoked currents, suggesting that menthol does not change α3β4 nAChR subunit stoichiometry. Chronic menthol treatment failed to change the current density (peak current amplitude/cell capacitance) of 100 μM ACh-evoked currents. Chronic menthol treatment accelerated desensitization of 100 and 200 μM ACh-evoked currents. Chronic nicotine treatment (250 μM) decreased ACh-induced currents, and we found no additional effect of including chronic menthol. These data contrast with previously reported, marked effects of chronic menthol on β2* nAChRs studied in the same expression system. Mechanistically, the data support the emerging interpretation that both chronic menthol and chronic nicotine act on nAChRs in the early exocytotic pathway, and that this pathway does not present a rate-limiting step to the export of α3β4 nAChRs; these nAChRs include endoplasmic reticulum (ER) export motifs but not ER retention motifs. Previous reports show that smoking mentholated cigarettes enhances tobacco addiction; but our results show that this effect is unlikely to arise via menthol actions on α3β4 nAChRs.

TLR7 agonist attenuates acetylcholine-induced, Ca2+ -dependent ionic currents in swine tracheal submucosal gland cells.

What is the central question of this study? Does Toll-like receptor 7 (TLR7) have any direct effects on Ca2+ -dependent physiological function of tracheal submucosal gland cells? What is the main finding and its importance? TLR7 is co-localized with SERCA2 in tracheal submucosal gland cells and causes a rapid attenuation of acetylcholine (ACh)-induced, Ca2+ -dependent ionic currents through the activation of SERCA2-dependent Ca2+ clearance. TLR7 is abundantly expressed in the airways of both swine and healthy human subjects, but is significantly downregulated in chronic obstructive pulmonary disease (COPD) airways. These findings suggest that a dysfunction of TLR7 in COPD removes the brake on ACh-induced serous secretion during viral infections, resulting in prolonged airway hypersecretion, and that it is one of the triggers of COPD exacerbations. Airway surface fluids are mainly secreted from submucosal glands (SMGs) and play important roles in the defence of airways via the activation of mucociliary transport. Toll-like receptor 7 (TLR7) recognizes and eliminates single stranded RNA (ssRNA) viruses through the induction of innate immunity. However, there is no obvious connection between TLR7 and mucus secretion, aside from TLR7 recognizing ssRNA viruses, which are often associated with airway hypersecretion in chronic obstructive pulmonary disease (COPD). Here, we investigated whether TLR7 has any direct effects on the Ca2+ -dependent physiological function of tracheal SMG cells. Patch-clamp analyses revealed that TLR7 ligand inhibited the acetylcholine (ACh)-induced ionic currents in isolated tracheal SMG cells. Intracellular calcium assays and pharmacological analyses revealed that TLR7 attenuated the transient rises in the intracellular calcium concentration evoked by ACh by activating sarco/endoplasmic reticulum Ca2+ -ATPase 2 (SERCA2). Immunofluorescence staining and immunohistochemical staining revealed that TLR7 was co-localized with SERCA2. These findings suggest that the activation of TLR7 during viral infections contributes to the rapid attenuation of ACh-induced ionic currents through an increase in SERCA2-dependent Ca2+ clearance in healthy airway SMG cells. Our study also revealed that TLR7 expression was significantly downregulated in COPD airways. Based on these findings, we speculate that a dysfunction of TLR7 may not only have an adverse effect on the elimination of these viruses but also remove the brake on ACh-induced serous secretion, resulting in prolonged hypersecretion and acting as one of the triggers of COPD exacerbations.

Prorocentrolide-A from Cultured Prorocentrum lima Dinoflagellates Collected in Japan Blocks Sub-Types of Nicotinic Acetylcholine Receptors.

Prorocentrolides are members of the cyclic imine phycotoxins family. Their chemical structure includes a 26-membered carbo-macrocycle and a 28-membered macrocyclic lactone arranged around a hexahydroisoquinoline that incorporates the characteristic cyclic imine group. Six prorocentrolides are already known. However, their mode of action remains undetermined. The aim of the present work was to explore whether prorocentrolide-A acts on nicotinic acetylcholine receptors (nAChRs), using competition-binding assays and electrophysiological techniques. Prorocentrolide-A displaced [125I]α-bungarotoxin binding to Torpedo membranes, expressing the muscle-type (α12β1γδ) nAChR, and in HEK-293 cells, expressing the chimeric chick neuronal α7-5HT3 nAChR. Functional studies revealed that prorocentrolide-A had no agonist action on nAChRs, but inhibited ACh-induced currents in Xenopus oocytes that had incorporated the muscle-type α12β1γδ nAChR to their membranes, or that expressed the human α7 nAChR, as revealed by voltage-clamp recordings. Molecular docking calculations showed the absence of the characteristic hydrogen bond between the iminium group of prorocentrolide-A and the backbone carbonyl group of Trp147 in the receptor, explaining its weaker affinity as compared to all other cyclic imine toxins. In conclusion, this is the first study to show that prorocentrolide-A acts on both muscle and neuronal nAChRs, but with higher affinity on the muscle-type nAChR.

LY2087101 and dFBr share transmembrane binding sites in the (\u03b14)3(\u03b22)2 Nicotinic Acetylcholine Receptor

Positive allosteric modulators (PAMs) of nicotinic acetylcholine receptors (nAChRs) have potential therapeutic application in neuropathologies associated with decrease in function or loss of nAChRs. In this study, we characterize the pharmacological interactions of the nAChRs PAM, LY2087101, with the α4β2 nAChR using mutational and computational analyses. LY2087101 potentiated ACh-induced currents of low-sensitivity (α4)3(β2)2 and high-sensitivity (α4)2(β2)3 nAChRs with similar potencies albeit to a different maximum potentiation (potentiation Imax = ~840 and 450%, respectively). Amino acid substitutions within the α4 subunit transmembrane domain [e.g. α4Leu256 and α4Leu260 within the transmembrane helix 1 (TM1); α4Phe316 within the TM3; and α4Gly613 within TM4] significantly reduced LY2087101 potentiation of (α4)3(β2)2 nAChR. The locations of these amino acid residues and LY2087101 computational docking analyses identify two LY2087101 binding sites: an intrasubunit binding site within the transmembrane helix bundle of α4 subunit at the level of α4Leu260/α4Phe316 and intersubunit binding site at the α4:α4 subunit interface at the level of α4Leu256/α4Ile315 with both sites extending toward the extracellular end of the transmembrane domain. We also show that desformylflustrabromine (dFBr) binds to these two sites identified for LY2087101. These results provide structural information that are pertinent to structure-based design of nAChR allosteric modulators.

Effects of the antidepressant mirtazapine and zinc on nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) and zinc are associated with regulation of mood and related disorders. In addition, several antidepressants inhibit muscle and neuronal nAChRs and zinc potentiates inhibitory actions of them. Moreover, mirtazapine (a noradrenergic, serotonergic and histaminergic antidepressant) inhibits muscarinic AChRs and its effects on nAChRs are unknown. Therefore, we studied the modulation of muscle α1β1γd nAChRs expressed in oocytes and native α7-containing nAChRs in hippocampal interneurons by mirtazapine and/or zinc, using voltage-clamp techniques. The currents elicited by ACh in oocytes (at -60 mV) were similarly inhibited by mirtazapine in the absence and presence of 100 μM zinc (IC50 ∼15 μM); however, the ACh-induced currents were stronger inhibited with 20 and 50 μM mirtazapine in the presence of zinc. Furthermore, the potentiation of ACh-induced current by zinc in the presence of 5 μM mirtazapine was 1.48 ± 0.06, and with 50 μM mirtazapine zinc potentiation did not occur. Interestingly, in stratum radiatum interneurons (at -70 mV), 20 μM mirtazapine showed less inhibition of the current elicited by choline (Ch) than at 10 μM (0.81 ± 0.02 and 0.74 ± 0.02 of the Ch-induced current, respectively). Finally, the inhibitory effects of mirtazapine depended on membrane potential: 0.81 ± 0.02 and 0.56 ± 0.05 of the control Ch-induced current at -70 and -20 mV, respectively. These results indicate that mirtazapine interacts with muscle and neuronal nAChRs, possibly into the ion channel; that zinc may increase the sensitivity of nAChRs to mirtazapine; and that mirtazapine decreases the sensitivity of nAChRs to zinc.

Neonicotinoid insecticides differently modulate acetycholine-induced currents on mammalian α7 nicotinic acetylcholine receptors.

Neonicotinoid insecticides are described as poor agonists of mammalian nicotinic ACh receptors. In this paper, we show that their effects on mammalian nicotinic receptors differ between compounds. Two-electrode voltage-clamp electrophysiology was used to characterize the pharmacology of three neonicotinoid insecticides on nicotinic α7 receptors expressed in Xenopus oocytes. Single and combined application of clothianidin, acetamiprid and thiamethoxam were tested. Two neonicotinoid insecticides, clothianidin and acetamiprid, were partial agonists of mammalian neuronal α7 nicotinic receptors, whereas another neonicotinoid insecticide, thiamethoxam, which is converted to clothianidin in insect and plant tissues, had no effect. Pretreatment with clothianidin and acetamiprid (10μM) ACh significantly enhanced the subsequent currents evoked by ACh (100μM ) whereas pretreatment with thiamethoxam (10μM) reduced ACh-induced current amplitudes.A combination of the three neonicotinoids decreased the ACh-evoked currents. The present findings suggest that neonicotinoid insecticides differ markedly in their direct effects on mammalian α7 nicotinic ACh receptors and can also modulate ACh-induced currents. Furthermore, our data indicate a previously unknown modulation of mammalian α7 nicotinic receptors by a combination of clothianidin, acetamiprid and thiamethoxam. This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc.

Effects of triterpenoid Alisol-F on human 5-hydroxytryptamine 3A and α3β4 nicotinic acetylcholine receptor channel activity

Alisma Rhizome is a known tradition medication, which has been used for its diuretic, hypolipidemic, anti-diabetic, and anti-inflammatory purposes for thousands of years. The primary compounds of Alisma Rhizome are protostane type triterpenes, such as Alisols A, B or C. We previously demonstrated that Alisol derivatives (Alisols A, B, and C) have inhibitory effects on 5-hydroxytryptamine 3A (5-HT3A) currents1. In this study, we tested the effects of a new triterpene, Alisol-F, on human 5-HT3A and α3β4 nicotinic acetylcholine (nACh) receptor channel currents by using Xenopus oocytes expressing these channels. Co-application of Alisol-F inhibited 5-HT3A and α3β4 nACh receptor-mediated inward peak currents. The inhibitory effect of Alisol-F on 5-HT and ACh-induced inward peak currents occurred in a reversible and concentration- dependent manner. The half maximal inhibitory concentrations (IC50) of Alisol-F were 79.4 ± 11.0 and 21.2 ±6.0 μM for the 5-HT3A and α3β4 nACh receptors, respectively. In addition, the inhibition of I 5-HT and I ACh by Alisol-F occurred noncompetitive and voltage insensitive manner. Taken together, these results show that Alisol-F may regulate 5-HT3A and α3β4 nACh receptors channel expressed in Xenopus oocytes.

1,3-diphenylpropan-1-ones as allosteric modulators of α7 nACh receptors with analgesic and antioxidant properties.

Nicotine acethylcholine receptors (nAChRs) play critical roles in cognitive processes, neuroprotection and inflammation. According to their substituents, 1,3-diphenylpropan-1-one derivatives act as α7 nAChRs negative allosteric modulators (NAM, OMe) or Type I positive allosteric modulators (PAMs, OH). Compounds 7 and 31 were the most effective (989 and 666% enhancement of ACh-induced currents) and potent (EC50: 12.9 and 6.85 μM) PAMs. They exhibited strong radical scavenging values. Compound 31, selective over other neuronal nAChR subtypes and with acceptable pharmacokinetic profile, showed antinociceptive effects in a model of inflammatory pain. Compound 31 is a novel, potent and selective α7 nAChR PAM, displaying antioxidant and analgesic activities. The 1,3-diphenylpropan-1-one scaffold could be the base toward more advanced type I PAMs for the treatment of nAChR-mediated diseases.

Propofol and AZD3043 Inhibit Adult Muscle and Neuronal Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes.

Propofol is a widely used general anaesthetic with muscle relaxant properties. Similarly as propofol, the new general anaesthetic AZD3043 targets the GABAA receptor for its anaesthetic effects, but the interaction with nicotinic acetylcholine receptors (nAChRs) has not been investigated. Notably, there is a gap of knowledge about the interaction between propofol and the nAChRs found in the adult neuromuscular junction. The objective was to evaluate whether propofol or AZD3043 interact with the α1β1δε, α3β2, or α7 nAChR subtypes that can be found in the neuromuscular junction and if there are any differences in affinity for those subtypes between propofol and AZD3043. Human nAChR subtypes α1β1δε, α3β2, and α7 were expressed into Xenopus oocytes and studied with an automated voltage-clamp. Propofol and AZD3043 inhibited ACh-induced currents in all of the nAChRs studied with inhibitory concentrations higher than those needed for general anaesthesia. AZD3043 was a more potent inhibitor at the adult muscle nAChR subtype compared to propofol. Propofol and AZD3043 inhibit nAChR subtypes that can be found in the adult NMJ in concentrations higher than needed for general anaesthesia. This finding needs to be evaluated in an in vitro nerve-muscle preparation and suggests one possible explanation for the muscle relaxant effect of propofol seen during higher doses.

Interaction of the Positive Allosteric Modulator LY2087101 with α4β2 Nicotinic Acetylcholine Receptor

LY2087101 ([2-[(4-Fluorophenyl)amino]-4-methyl-5-thiazolyl]-3-thienylmethanone) was identified as a positive allosteric modulator (PAM) of nicotinic acetylcholine receptors (nAChR) in a high-throughput screen at Eli Lilly and Company. LY2087101 potentiated α7 and α4β2 nAChRs but not α3β2 nAChR (Broad et al. 2008, J. Pharmacol. Exp. Ther., 318: 1108). Mutational analyses and computer docking simulation with α7 nAChR predicted that PNU120596 and LY2087101 bind to a common site within the transmembrane domain (Young et al. 2008, PNAS 105:14686). In this study, we use mutational analyses and two-electrode voltage-clamp recording from Xenopus oocytes to begin pharmacological characterization of LY208101 interaction with α4β2 nAChR and to identify the molecular determinants of LY2087101 selectivity for α4β2 vs. α3β4 nAChRs. LY2078101 potentiated ACh-induced currents of low ACh sensitivity (α4)3(β2)2 nAChR and high ACh sensitivity (α4)2(β2)3 nAChR with similar potency. However, the maximum potentiation at 10 μM LY2087101 was higher in (α4)3(β2)2 than (α4)2(β2)3 nAChR. Mutational analyses of amino acids contributing to extracellular subunits interface (e.g. α4His114, α4Gln122, and α4Thr124, known selectivity determinants for the (α4)3(β2)2 nAChR selective PAM NS9283; Olsen et al 2013, JBC 288: 35977) had no effect on LY2087101 potentiation. Within the transmembrane domain, mutation at α4Ser285 but not α4Thr261 within the M1 helix significantly reduced LY2087101 potentiation of α4β2 nAChR α4Ser285 and α4Thr261 are equivalent to α7Ser222 and α7Ala225, respectively; two important positions for potentiation of α7 nAChR by LY2087101 and PNU120596. α4Thr261 projects to intra-subunit pocket within the α4 transmembrane helix bundle whereas α4Ser285 is exposed to intra-subunit pocket and inter-subunit space. Ongoing mutational analyses within the M1 transmembrane domain will define the contribution of intra-subunit vs. inter-subunit binding sites on LY2087101 potentiation of α4β2 nAChR.

Smoking-Relevant Nicotine Concentration Attenuates the Unfolded Protein Response in Dopaminergic Neurons.

Parkinson's disease (PD) cannot yet be cured or prevented. However, many retrospective epidemiological studies reveal that PD is diagnosed less frequently in tobacco users. Existing programs attempting to develop nicotinic drugs that might exert this apparent neuroprotective effect are asking whether agonists, antagonists, partial agonists, or channel blockers show the most promise. The underlying logic resembles the previous development of varenicline for smoking cessation. We studied whether, and how, nicotine produces neuroprotective effects in cultured dopaminergic neurons, an experimentally tractable, mechanistically revealing neuronal system. We show that nicotine, operating via nicotinic receptors, does protect these neurons against endoplasmic reticulum stress. However, the mechanism is probably "inside-out": pharmacological chaperoning in the endoplasmic reticulum. This cellular-level insight could help to guide neuroprotective strategies.

The prototoxin LYPD6B modulates heteromeric α3β4-containing nicotinic acetylcholine receptors, but not α7 homomers.

Prototoxins are a diverse family of membrane-tethered molecules expressed in the nervous system that modulate nicotinic cholinergic signaling, but their functions and specificity have yet to be completely explored. We tested the selectivity and efficacy of leukocyte antigen, PLAUR (plasminogen activator, urokinase receptor) domain-containing (LYPD)-6B on α3β4-, α3α5β4-, and α7-containing nicotinic acetylcholine receptors (nAChRs). To constrain stoichiometry, fusion proteins encoding concatemers of human α3, β4, and α5 (D and N variants) subunits were expressed in Xenopus laevis oocytes and tested with or without LYPD6B. We used the 2-electrode voltage-clamp method to quantify responses to acetylcholine (ACh): agonist sensitivity (EC50), maximal agonist-induced current (Imax), and time constant (τ) of desensitization. For β4-α3-α3-β4-α3 and β4-α3-β4-α3-α3, LYPD6B decreased EC50 from 631 to 79 μM, reduced Imax by at least 59%, and decreased τ. For β4-α3-α5D-β4-α3 and β4-α3-β4-α-α5D, LYPD6B decreased Imax by 63 and 32%, respectively. Thus, LYPD6B acted only on (α3)3(β4)2 and (α3)2(α5D)(β4)2 and did not affect the properties of (α3)2(β4)3, α7, or (α3)2(α5N)(β4)2 nAChRs. Therefore, LYPD6B acts as a mixed modulator that enhances the sensitivity of (α3)3(β4)2 nAChRs to ACh while reducing ACh-induced whole-cell currents. LYPD6B also negatively modulates α3β4 nAChRs that include the α5D common human variant, but not the N variant associated with nicotine dependence.

Distinctive effects of nicotinic receptor intracellular-loop mutations associated with nocturnal frontal lobe epilepsy

Previously characterized nicotinic acetylcholine receptor (nAChR) autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE)-associated mutations are found in α2, α4 and β2 subunit transmembrane (TM) domains. They predominantly increase ACh potency and, for β2-subunit mutants, increase macroscopic currents. Two recently-identified mutations, α4(R336H) and β2(V337G), located in the intracellular cytoplasmic loop (C2) have been associated with non-familial NFLE. Effects of these mutations on α4β2-nAChR function and expression were studied for the first time, using two-electrode voltage clamp recordings in Xenopus laevis oocytes. Biased-ratio preparations elucidated the mutations' effects at alternate isoforms: high-sensitivity [HS; (α4)2(β2)3] or low-sensitivity [LS; (α4)3(β2)2] via 1:10 or 30:1 [α4:β2] cRNA injection ratios, respectively. An unbiased (1:1 [α4:β2] cRNA) injection ratio was also used to study potential shifts in isoform expression. α4(R336H)-containing receptors showed significant increases in maximal ACh-induced currents (Imax) in all preparations (140% increase compared to wild type control). β2(V337G)-containing receptors significantly increased Imax in the LS-favoring preparation (20% increase compared to control). Expression of either mutation consistently produced enrichment of HS-isoform expression in all preparations. α4β2-nAChR harboring either NFLE mutant subunit showed unchanged ACh, sazetidine-A, nicotine, cytisine and mecamylamine potency. However, both mutant subunits enhanced partial agonist efficacies in the LS-biased preparation. Using β2-subunit-specific [125I]mAb 295 immunolabeling, nAChR cell-surface expression was determined. Antibody binding studies revealed that the β2(V337G) mutation tended to reduce cell-surface expression, and function per receptor was significantly increased by either NFLE mutant subunit in HS-favoring preparations. These findings identify both common and differing features between TM- and C2-domain AD/NFLE-associated mutations. As we discuss, the shared features may be particularly salient to AD/NFLE etiology.

Luminal acetylcholine does not affect the activity of the CFTR in tracheal epithelia of pigs

Fluid homeostasis mediated by the airway epithelium is required for proper lung function, and the CFTR (cystic fibrosis transmembrane conductance regulator) Cl− channel is crucial for these processes. Luminal acetylcholine (ACh) acts as an auto-/paracrine mediator to activate Cl− channels in airway epithelia and evidence exists showing that nicotinic ACh receptors activate CFTR in murine airway epithelia. The present study investigated whether or not luminal ACh regulates CFTR activity in airway epithelia of pigs, an emerging model for investigations of human airway disease and cystic fibrosis (CF) in particular. Transepithelial ion currents of freshly dissected pig tracheal preparations were measured with Ussing chambers. Application of luminal ACh (100μM) induced an increase of the short-circuit current (ISC). The ACh effect was mimicked by muscarine and pilocarpine (100μM each) and was sensitive to muscarinic receptor antagonists (atropine, 4-DAMP, pirenzepine). No changes of the ISC were observed by nicotine (100μM) and ACh responses were not affected by nicotine or mecamylamine (25μM). Luminal application of IBMX (I, 100μM) and forskolin (F, 10μM), increase the ISC and the I/F-induced current were decreased by the CFTR inhibitor GlyH-101 (GlyH, 50μM) indicating increased CFTR activity by I/F. In contrast, GlyH did not affect the ACh-induced current, indicating that the ACh response does not involve the activation of the CFTR. Results from this study suggest that luminal ACh does not regulate the activity of the CFTR in tracheal epithelia of pigs which opposes observation from studies using mice airway epithelium.

Muscarinic inhibition of nicotinic transmission in rat sympathetic neurons and adrenal chromaffin cells.

Little is known about the interactions between nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Here we report that methacholine (MCh), a selective agonist of mAChRs, inhibited up to 80% of nicotine-induced nAChR currents in sympathetic superior cervical ganglion neurons and adrenal chromaffin cells. The muscarine-induced inhibition (MiI) substantially reduced ACh-induced membrane currents through nAChRs and quantal neurotransmitter release. The MiI was time- and temperature-dependent. The slow recovery of nAChR current after washout of MCh, as well as the high value of Q10 (3.2), suggested, instead of a direct open-channel blockade, an intracellular metabotropic process. The effects of GTP-γ-S, GDP-β-S and pertussis toxin suggested that MiI was mediated by G-protein signalling. Inhibitors of protein kinase C (bisindolymaleimide-Bis), protein kinase A (H89) and PIP2 depletion attenuated the MiI, indicating that a second messenger pathway is involved in this process. Taken together, these data suggest that mAChRs negatively modulated nAChRs via a G-protein-mediated second messenger pathway. The time dependence suggests that MiI may provide a novel mechanism for post-synaptic adaptation in all cells/neurons and synapses expressing both types of AChRs.

Desformylflustrabromine Modulates α4β2 Neuronal Nicotinic Acetylcholine Receptor High- and Low-Sensitivity Isoforms at Allosteric Clefts Containing the β2 Subunit.

Alterations in expression patterns of α4β2 nicotinic acetylcholine receptors have been demonstrated to alter cholinergic neurotransmission and are implicated in neurologic disorders, including autism, nicotine addiction, Alzheimer's disease, and Parkinson's disease. Positive allosteric modulators (PAMs) represent promising new leads in the development of therapeutic agents for the treatment of these disorders. This study investigates the involvement of the β2-containing subunit interfaces of α4β2 receptors in the modulation of acetylcholine (ACh)-induced responses by the PAM desformylflustrabromine (dFBr). Eight amino acids on the principal face of the β2 subunit were mutated to alanine to explore the involvement of this region in the potentiation of ACh-induced currents by dFBr. ACh-induced responses obtained from wild-type and mutant α4β2 receptors expressed in Xenopus laevis oocytes were recorded in the presence and absence of dFBr using two-electrode voltage clamp electrophysiology. Wild-type and mutant receptors were expressed in both high and low ACh sensitivity isoforms by using biased injection ratios of 1:5 or 5:1 α4 to β2 complementary RNA. Mutations were made in the B, C, and A loops of the principal face of the β2 subunit, which are regions not involved in the binding of ACh. Mutant β2(Y120A) significantly eliminated dFBr potency in both isoform preparations. Several other mutations altered dFBr potentiation levels in both preparations. Our findings support the involvement of the principal face of the β2 subunit in dFBr modulation of ACh-induced responses. Findings from this study will aid in the improved design of dFBr-like PAMs for potential therapeutic use.