Dbeta3, an atypical nicotinic acetylcholine receptor subunit from Drosophila : molecular cloning, heterologous expression and coassembly

Insect nicotinic acetylcholine receptors (nAChRs) play a central role in mediating neuronal synaptic transmission and are the target sites for the increasingly important group of neonicotinoid insecticides. Six nicotinic acetylcholine receptor (nAChR) subunits (four alpha-type and two beta-type) have been cloned previously from the model insect species Drosophila melanogaster. Despite extensive efforts, it has not been possible to generate functional recombinant nAChRs by heterologous expression of any combination of these six subunits. It has, however, been possible to express functional hybrid receptors when Drosophila alpha subunits are co-expressed with vertebrate beta subunits. This has led to the assumption that successful heterologous expression might require an, as yet, uncloned beta-type insect subunit. Examination of the recently completed Drosophila genomic sequence data has identified a novel putative nAChR beta-type subunit. Here we report the molecular cloning, heterologous expression and characterization of this putative Drosophila nAChR subunit (Dbeta3). Phylogenetic comparisons with other ligand-gated ion channel subunit sequences support its classification as a nAChR subunit but show it to be a distantly related member of this neurotransmitter receptor subunit family. Evidence that the Dbeta3 subunit is able to coassemble with other Drosophila nAChR subunits and contribute to recombinant nAChRs has been obtained by both radioligand binding and coimmunoprecipitation studies in transfected Drosophila S2 cells.     

A role for Leu247 residue within transmembrane domain 2 in ginsenoside-mediated α7 nicotinic acetylcholine receptor regulation

Nicotinic acetylcholine receptors (nAChRs) play important roles in nervous system functions and are involved in a variety of diseases. We previously demonstrated that ginsenosides, the active ingredients of Panax ginseng, inhibit subsets of nAChR channel currents, but not α7, expressed in Xenopus laevis oocytes. Mutation of the highly conserved Leu247 to Thr247 in the transmembrane domain 2 (TM2) channel pore region of α7 nAChR induces alterations in channel gating properties and converts α7 nAChR antagonists into agonists. In the present study, we assessed how point mutations in the Leu247 residue leading to various amino acids affect 20(S)-ginsenoside Rg₃ (Rg₃) activity against the α7 nAChR. Mutation of L247 to L247A, L247D, L247E, L247I, L247S, and L247T, but not L247K, rendered mutant receptors sensitive to Rg₃. We further characterized Rg₃ regulation of L247T receptors. We found that Rg₃ inhibition of mutant α7 nAChR channel currents was reversible and concentration-dependent. Rg₃ inhibition was strongly voltage-dependent and noncompetitive manner. These results indicate that the interaction between Rg₃ and mutant receptors might differ from its interaction with the wild-type receptor. To identify differences in Rg₃ interactions between wild-type and L247T receptors, we utilized docked modeling. This modeling revealed that Rg₃ forms hydrogen bonds with amino acids, such as Ser240 of subunit I and Thr244 of subunit II and V at the channel pore, whereas Rg₃ localizes at the interface of the two wild-type receptor subunits. These results indicate that mutation of Leu247 to Thr247 induces conformational changes in the wild-type receptor and provides a binding pocket for Rg₃ at the channel pore.     

Key Residues in the Nicotinic Acetylcholine Receptor β2 Subunit Contribute to α-Conotoxin LvIA Binding

α-Conotoxin LvIA (α-CTx LvIA) is a small peptide from the venom of the carnivorous marine gastropod Conus lividus and is the most selective inhibitor of α3β2 nicotinic acetylcholine receptors (nAChRs) known to date. It can distinguish the α3β2 nAChR subtype from the α6β2* (* indicates the other subunit) and α3β4 nAChR subtypes. In this study, we performed mutational studies to assess the influence of residues of the β2 subunit versus those of the β4 subunit on the binding of α-CTx LvIA. Although two β2 mutations, α3β2[F119Q] and α3β2[T59K], strongly enhanced the affinity of LvIA, the β2 mutation α3β2[V111I] substantially reduced the binding of LvIA. Increased activity of LvIA was also observed when the β2-T59L mutant was combined with the α3 subunit. There were no significant difference in inhibition of α3β2[T59I], α3β2[Q34A], and α3β2[K79A] nAChRs when compared with wild-type α3β2 nAChR. α-CTx LvIA displayed slower off-rate kinetics at α3β2[F119Q] and α3β2[T59K] than at the wild-type receptor, with the latter mutant having the most pronounced effect. Taken together, these data provide evidence that the β2 subunit contributes to α-CTx LvIA binding and selectivity. The results demonstrate that Val¹¹¹ is critical and facilitates LvIA binding; this position has not previously been identified as important to binding of other 4/7 framework α-conotoxins. Thr⁵⁹ and Phe¹¹⁹ of the β2 subunit appear to interfere with LvIA binding, and their replacement by the corresponding residues of the β4 subunit leads to increased affinity.     

A spinosyn-sensitive Drosophila melanogaster nicotinic acetylcholine receptor identified through chemically induced target site resistance,resistance gene identification,and heterologous expression

Strains of Drosophila melanogaster with resistance to the insecticides spinosyn A, spinosad, and spinetoram were produced by chemical mutagenesis. These spinosyn-resistant strains were not cross-resistant to other insecticides. The two strains that were initially characterized were subsequently found to have mutations in the gene encoding the nicotinic acetylcholine receptor (nAChR) subunit Dα6. Subsequently, additional spinosyn-resistant alleles were generated by chemical mutagenesis and were also found to have mutations in the gene encoding Dα6, providing convincing evidence that Dα6 is a target site for the spinosyns in D. melanogaster. Although a spinosyn-sensitive receptor could not be generated in Xenopus laevis oocytes simply by expressing Dα6 alone, co-expression of Dα6 with an additional nAChR subunit, Dα5, and the chaperone protein ric-3 resulted in an acetylcholine- and spinosyn-sensitive receptor with the pharmacological properties anticipated for a native nAChR.     

Molecular characterisation of nicotinic acetylcholine receptor subunits from the cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae)

As part of a program to monitor the susceptibility of cat flea populations to the insecticide imidacloprid we have examined the cat flea nicotinic acetylcholine receptor, the target site protein of the neonicotinoid group of insecticides. Seven nAChR subunits (six alpha-type and one beta-type) were identified in cat flea using a degenerate PCR-based strategy. Five of these were expressed in vitro by creating chimeras containing the N-terminal ligand-binding domain of the cat flea subunits and the C-terminal region of the Drosophila Dalpha2 (SAD) subunit. Two of the five chimeric subunits, Cfalpha1/Dalpha2 and Cfalpha3/Dalpha2, when co-expressed with rat beta2 in Drosophila S2 cells, showed high-affinity binding of both epibatidine (Kd=1.6+/-0.6 and 0.13+/-0.06nM, respectively), and imidacloprid (Ki=142+/-34 and 28.7+/-2.4nM, respectively). It is likely therefore that Cfalpha1 and Cfalpha3 contribute to nAChR populations in vivo that are sensitive to imidacloprid. The identification of cat flea nAChR subunits that have a high affinity for imidacloprid presents candidate genes in which to look for resistance-associated mutations if target-site resistance to imidacloprid arises in domestic pet flea populations.     

Inflammation has synergistic effect with nicotine in periodontitis by up‐regulating the expression of α7 nAChR via phosphorylated GSK‐3β

Periodontitis is the leading cause of adult tooth loss, and those who smoke are at an increased risk of developing periodontitis. α7 nicotinic acetylcholine receptor (α7 nAChR) is proposed to mediate the potential synergistic effect of nicotine and inflammation in smoking‐related periodontitis. However, this has not been experimentally demonstrated. We isolated and cultured human periodontal ligament stem cells (PDLSCs) from healthy and inflamed tissues. PDLSCs were treated with either inflammatory factors or nicotine. We measured expression of genes that are associated with osteogenic differentiation and osteoclast formation using RT‐qPCR and Western blot analyses. Besides, immunohistochemical staining, micro‐CT analysis and tartaric acid phosphatase staining were used to measure α7 nAChR expression and function. Inflammation up‐regulated α7 nAChR expression in both periodontal ligament tissues and PDLSCs. The up‐regulated α7 nAChR contributed to the synergistic effect of nicotine and inflammation, leading to a decreased capability of osteogenic differentiation and increased capability of osteoclast formation‐induction of PDLSCs. Moreover, the inflammation‐induced up‐regulation of α7 nAChR was partially dependent on the level of phosphorylated GSK‐3β. This study provides experimental evidence for the pathological development of smoking‐related periodontitis and sheds new light on developing inflammation and α7 nAChR‐targeted therapeutics to treat and prevent the disease.     

Identification of a multigene family encoding putative beta-glucan-binding proteins in Medicago truncatula

Branched 1,6-1,3-beta-glucans from Phytophthora sojae cell walls represent pathogen-associated molecular patterns (PAMPs) that have been shown to mediate the activation of plant defence reactions in many legumes. In soybean, a receptor protein complex containing a high affinity beta-glucan-binding protein (GBP) was identified and investigated in detail. In the model legume Medicago truncatula, used for functional genomic studies of various plant-microbe interactions, a high-affinity beta-glucan-binding site was characterized biochemically. However, to date, none of the genes encoding GBPs from M. truncatula have been described. Here, we report the identification of four full-length clones encoding putative beta-glucan-binding proteins from M. truncatula, MtGBP1, 2, 3, and 4, composing a multigene family encoding GBP-related proteins in this plant. Differences in expression patterns as well as in regulation on treatment with two different biotic elicitors are demonstrated for the members of the GBP family and for a selection of defence-related genes.     

Insights into Distinct Modulation of α7 and α7β2 Nicotinic Acetylcholine Receptors by the Volatile Anesthetic Isoflurane

Nicotinic acetylcholine receptors (nAChRs) are targets of general anesthetics, but functional sensitivity to anesthetic inhibition varies dramatically among different subtypes of nAChRs. Potential causes underlying different functional responses to anesthetics remain elusive. Here we show that in contrast to the α7 nAChR, the α7β2 nAChR is highly susceptible to inhibition by the volatile anesthetic isoflurane in electrophysiology measurements. Isoflurane-binding sites in β2 and α7 were found at the extracellular and intracellular end of their respective transmembrane domains using NMR. Functional relevance of the identified β2 site was validated via point mutations and subsequent functional measurements. Consistent with their functional responses to isoflurane, β2 but not α7 showed pronounced dynamics changes, particularly for the channel gate residue Leu-249(9′). These results suggest that anesthetic binding alone is not sufficient to generate functional impact; only those sites that can modulate channel dynamics upon anesthetic binding will produce functional effects.     

Pharmacological profiles of recombinant and native insect nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are targets for insect-selective neonicotinoid insecticides exemplified by imidacloprid (IMI) and mammalian-selective nicotinoids including nicotine and epibatidine (EPI). Despite their importance, insect nAChRs are poorly understood compared with their vertebrate counterparts. This study characterizes the [(3)H]IMI, [(3)H]EPI, and [(3)H]alpha-bungarotoxin (alpha-BGT) binding sites in hybrid nAChRs consisting of Drosophila melanogaster (fruit fly) or Myzus persicae (peach-potato aphid) alpha2 coassembled with rat beta2 subunits (Dalpha2/Rbeta2 and Mpalpha2/Rbeta2) and compares them with native insect and vertebrate alpha4beta2nAChRs. [(3)H]IMI and [(3)H]EPI bind to Dalpha2/Rbeta2 and Mpalpha2/Rbeta2 hybrids but [(3)H]alpha-BGT does not. In native Drosophila receptors, [(3)H]EPI has a single high-affinity binding site that is independent from that for [(3)H]IMI and, interestingly, overlaps the [(3)H]alpha-BGT site. In the Mpalpha2/Rbeta2 hybrid, [(3)H]IMI and [(3)H]EPI bind to the same site and have similar pharmacological profiles. On considering both neonicotinoids and nicotinoids, the Dalpha2/Rbeta2 and Mpalpha2/Rbeta2 receptors display intermediate pharmacological profiles between those of native insect and vertebrate alpha4beta2 receptors, limiting the use of these hybrid receptors for predictive toxicology. These findings are consistent with the agonist binding site being located at the nAChR subunit interface and indicate that both alpha and beta subunits influence the pharmacological properties of insect nAChRs.     

α6β2*‐subtype nicotinic acetylcholine receptors are more sensitive than α4β2*‐subtype receptors to regulation by chronic nicotine administration

Nicotinic acetylcholine receptors (nAChR) of the α6β2* subtype (where *indicates the possible presence of additional subunits) are prominently expressed on dopaminergic neurons. Because of this, their role in tobacco use and nicotine dependence has received much attention. Previous studies have demonstrated that α6β2*‐nAChR are down‐regulated following chronic nicotine exposure (unlike other subtypes that have been investigated – most prominently α4β2* nAChR). This study examines, for the first time, effects across a comprehensive chronic nicotine dose range. Chronic nicotine dose–responses and quantitative ligand‐binding autoradiography were used to define nicotine sensitivity of changes in α4β2*‐nAChR and α6β2*‐nAChR expression. α6β2*‐nAChR down‐regulation by chronic nicotine exposure in dopaminergic and optic‐tract nuclei was ≈three‐fold more sensitive than up‐regulation of α4β2*‐nAChR. In contrast, nAChR‐mediated [³H]‐dopamine release from dopamine‐terminal region synaptosomal preparations changed only in response to chronic treatment with high nicotine doses, whereas dopaminergic parameters (transporter expression and activity, dopamine receptor expression) were largely unchanged. Functional measures in olfactory tubercle preparations were made for the first time; both nAChR expression levels and nAChR‐mediated functional measures changed differently between striatum and olfactory tubercles. These results show that functional changes measured using synaptosomal [³H]‐DA release are primarily owing to changes in nAChR, rather than in dopaminergic, function.

     

Functional expression and sites of gene transcription of a novel α subunit of the GABAA receptor in rat brain

Two α subunits of the gabaa receptor in rat brain have been identified by molecular cloning. The deduced polypeptide sequences share major characteristics with other chemically gated ion channel proteins. One polypeptide represents the rat homologue of the α3 subunit previously cloned from bovine brain [14], while the other polypeptide is a yet unknown subunit, termed α5. When coexpressed with the β1 subunit in Xenopus oocytes the receptors containing the α5 subunit revealed a higher sensitivity to GABA than receptors expressed from α1 + β1 subunits or α3 + β1 subunits (K_a = 1 μM, 13 μM and 14 μM, respectively). The α5 subunit was expressed only in a few brain areas such as cerebral cortex, hippocampal formation and olfactory bulb granular layer as shown by in situ hybridization histochemistry. Since the mRNA of the α5 subunit was colocalized with the αl and α3 subunits only in cerebral cortex and in the hippocampal formation the α5 subunit may be part of distinct GABA_A receptors in neuronal populations within the olfactory bulb.     

Evolutionary trade‐offs of insecticide resistance — The fitness costs associated with target‐site mutations in the nAChR of Drosophila melanogaster

The evolution of resistance to drugs and pesticides poses a major threat to human health and food security. Neonicotinoids are highly effective insecticides used to control agricultural pests. They target the insect nicotinic acetylcholine receptor and mutations of the receptor that confer resistance have been slow to develop, with only one field‐evolved mutation being reported to date. This is an arginine‐to‐threonine substitution at position 81 of the nAChR_β1 subunit in neonicotinoid‐resistant aphids. To validate the role of R81T in neonicotinoid resistance and to test whether it may confer any significant fitness costs to insects, CRISPR/Cas9 was used to introduce an analogous mutation in the genome of Drosophila melanogaster. Flies carrying R81T showed an increased tolerance (resistance) to neonicotinoid insecticides, accompanied by a significant reduction in fitness. In comparison, flies carrying a deletion of the whole nAChR_α6 subunit, the target site of spinosyns, showed an increased tolerance to this class of insecticides but presented almost no fitness deficits.     

Mutations in the M1 region of the nicotinic acetylcholine receptor alter the sensitivity to inhibition by quinacrine

Summary 1. Site directed mutagenesis was used to alter the structure ofTorpedo californica nicotinic acetylcholine receptor (nAChR) and to identify amino acid residues which contribute to noncompetitive inhibition by quinacrine. Mutant receptors were expressed inXenopus laevis oocytes injected within vitro synthesized mRNA and the whole cell currents induced by acetylcholine (ACh) were recorded by two electrode voltage clamp.2. A series of mutations of a highly conserved Arg at position 209 of the subunit ofTorpedo californica nAChR revealed that positively charged amino acids are required for functional receptor expression. Mutation of Arg to Lys (R209K) or His (R209H) at position 209 shifted the EC₅₀ for ACh slightly from 5µM to 12µM and increased the normalized maximal channel activity 8.5-and 3.2-fold, respectively.3. These mutations altered the sensitivity of nAChR to noncompetitive inhibition by quinacrine. The extent of inhibition of ion channel function by quinacrine was decreased as pH increased in both wild type and mutant nAChR suggesting that the doubly charged form of quinacrine was responsible for the inhibition.4. Further mutations at different positions of the subunit suggest the contribution of Pro and Tyr residues at positions 211 and 213 to quinacrine inhibition whereas mutationsI210A andL212A did not have any effects. None of these mutations changed the sensitivity of nAChR to inhibition by a different noncompetitive inhibitor, chlorpromazine.5. These findings support a hypothesis that the quinacrine binding site is located in the lumen of the ion channel. In addition, the quantitative effect of point mutations at alternate positions on the sensitivity of quinacrine inhibition suggests that the secondary structure at the beginning of M1 region might be sheet structure.     

Distinct functional roles for the M4 α-helix from each homologous subunit in the heteropentameric ligand-gated ion channel nAChR

The outermost lipid-exposed α-helix (M4) in each of the homologous α, β, δ, and γ/ε subunits of the muscle nicotinic acetylcholine receptor (nAChR) has previously been proposed to act as a lipid sensor. However, the mechanism by which this sensor would function is not clear. To explore how the M4 α-helix from each subunit in human adult muscle nAChR influences function, and thus explore its putative role in lipid sensing, we functionally characterized alanine mutations at every residue in αM4, βM4, δM4, and εM4, along with both alanine and deletion mutations in the post-M4 region of each subunit. Although no critical interactions involving residues on M4 or in post-M4 were identified, we found that numerous mutations at the M4–M1/M3 interface altered the agonist-induced response. In addition, homologous mutations in M4 in different subunits were found to have different effects on channel function. The functional effects of multiple mutations either along M4 in one subunit or at homologous positions of M4 in different subunits were also found to be additive. Finally, when characterized in both Xenopus oocytes and human embryonic kidney 293T cells, select αM4 mutations displayed cell-specific phenotypes, possibly because of the different membrane lipid environments. Collectively, our data suggest different functional roles for the M4 α-helix in each heteromeric nAChR subunit and predict that lipid sensing involving M4 occurs primarily through the cumulative interactions at the M4–M1/M3 interface, as opposed to the alteration of specific interactions that are critical to channel function.     

Trafficking of cholinesterases and neuroligins mutant proteins. An association with autism

Autism encompasses a wide spectrum of disorders arising during brain development. Recent studies reported that sequence polymorphisms in neuroligin-3 (NLGN3) and neuroligin-4 (NLGN4) genes have been linked to autism spectrum disorders indicating neuroligin genes as candidate targets in brain disorders. We have characterized a single mutation found in two affected brothers that substituted Arg451 to Cys in NL3. Our data show that the exposed Cys causes retention of the protein in the endoplasmic reticulum (ER) when expressed in HEK-293 cells. To examine whether the introduction of a Cys in the C-terminal region of other alpha/beta-hydrolase fold proteins could promote the same cellular phenotype, we made homologous mutations in acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) and found a similar processing deficiency and intracellular retention (De Jaco et al., J Biol Chem. 2006, 281:9667-76). NL3, AChE and BChE mutant proteins are recognized as misfolded in the ER, and degraded via the proteasome pathway. A 2D electrophoresis coupled with mass spectrometry based approach was used to analyze proteins co-immunoprecipitating with NL3 and show differential expression of factors interacting with wild type and mutant NL3. We identified several proteins belonging to distinct ER resident chaperones families, including calnexin, responsible for playing a role in the folding steps of the AChE and NLs.     

Intrinsic ligand binding properties of the human and bovine α,-interferon receptors

The Type I interferon receptor (IFN-αR) interacts with all IFN-αs, IFN-β and IFN-ω, and seems to be a multisubunit receptor. To investigate the role of a cloned receptor subunit (IFN-αR1), we have examined the intrinsic ligand binding properties of the bovine and human IFN-αR1 polypeptides expressed in Xenopus laevis oocytes. Albeit with different efficiencies, Xenopus oocytes expressing either the human or bovine IFN-αR1 polypeptide exhibit significant binding and formation of crosslinked complexes with human IFN-αA and IFN-αB. Thus, the IFN-αR1 polypeptide most likely plays a direct role in ligand binding.     

Stimulation of peripheral blood mononuclear cells with lipopolysaccharide induces expression of the plasma protein alpha2-macroglobulin

The human alpha(2)-macroglobulin gene is approximately 48 kb in size and consists of 36 exons, which encode the 180 kDa subunit of this large tetrameric protein. In this investigation, a procedure of sequencing human alpha(2)-macroglobulin mRNA, using mRNA from lipopolysaccharide-stimulated peripheral blood mononuclear cells as template in RT-PCR, was developed. Incubation of peripheral blood mononuclear cell populations with lipopolysaccharide induced alpha(2)-macroglobulin mRNA expression reaching levels detectable by RT-PCR. Extracted human alpha(2)-macroglobulin mRNA was used to determine the nucleotide sequence of a 500 bp DNA segment encoding the most C-terminal, receptor-binding part of the protein, using alpha(2)-macroglobulin specific primers. The sequence obtained matched the earlier published sequence of human alpha(2)-macroglobulin, except for three point mutations, i.e., cytosine for guanine, cytosine for thymidine and thymidine for adenine substitutions at positions 4369, 4423, and 4511, respectively. None of these alterations, however, affect the amino acid sequence of the protein. In conclusion, we demonstrate a new, improved, approach to sequence human alpha(2)-macroglobulin mRNA by overexpressing the protein in peripheral blood mononuclear cells. This procedure may be useful in the search for mutations in alpha(2)-macroglobulin, examining its role in the pathogenesis of human diseases.