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N S Millar

Publications and source records attributed to N S Millar.

At least 19 recordsLinked to original sources
PubMed2003

Assembly and subunit diversity of nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) are a diverse family of neurotransmitter-gated ion channels which contain five transmembrane subunits arranged around a central pore. Distinct receptor subtypes are expressed at the vertebrate skeletal neuromuscular junction, in mechanosensory cells and within the central and peripheral nervous systems. A total of 17 nAChR subunits (alpha1-alpha10, beta1-beta4, gamma, delta and epsilon ) have been identified in vertebrate species, which can co-assemble to generate a wide variety of nAChRs. Nicotinic receptors also constitute an abundant and diverse family of receptors in invertebrates. As a consequence of studies which have been conducted with both native and recombinant nAChRs, the subunit composition of nAChRs and the rules governing subunit co-assembly are becoming clearer. In this paper the extent of nAChR subunit diversity and evidence for receptor subunit composition is reviewed.

Animals↗
PubMed2001

Inefficient cell-surface expression of hybrid complexes formed by the co-assembly of neuronal nicotinic acetylcholine receptor and serotonin receptor subunits.

Previous studies have demonstrated that relatively low levels of alpha4beta2 neuronal nicotinic acetylcholine receptors (nAChRs) are expressed on the cell surface of transfected mammalian cell lines but that surface expression levels can be dramatically up-regulated by co-expression of these subunits with chimeric subunits containing the N-terminal portion of the neuronal nAChR alpha4 or beta2 subunits together with the C-terminal domain of the 5-HT(3A) subunit. Recent work has also suggested that the nAChR alpha4 subunit can co-assemble in a "promiscuous" manner with the serotonin receptor 5-HT(3A) subunit to form functional hybrid receptors. In this study we have examined whether co-assembly of either alpha4 or beta2 with 5-HT(3A) itself (rather than with the alpha4/5-HT(3A) or beta2/5-HT(3A) subunit chimeras) can also facilitate cell surface expression of alpha4 and beta2 subunits in transfected mammalian cells. Evidence has been obtained by immunoprecipitation, cell-surface antibody binding and radioligand binding which indicates that the 5-HT(3A) can co-assemble with both the alpha4 and beta2 nAChR subunits. We conclude, however, that co-assembly of 5-HT(3A) with either alpha4 or beta2 does not result in efficient cell surface expression of the nAChR subunits and that co-assembled hybrid (nAChR subunit + 5-HT(3)R subunit) receptor complexes are largely retained within the cell.

Cell Line↗
PubMed2000

Cloning, heterologous expression and co-assembly of Mpbeta1, a nicotinic acetylcholine receptor subunit from the aphid Myzus persicae.

Nicotinic acetylcholine receptors (nAChRs) play a major role in excitatory synaptic transmission in insects and are also the target site for chloronicotinyl insecticides such as imidacloprid. Here we report the cloning and characterization of a novel nAChR beta subunit, Mpbeta1, from the aphid Myzus persicae, an economically important pest species. Sequence analysis has identified an open reading frame of 509 amino acids with features typical of nAChR subunits. The Mpbeta1 gene is expressed as a single major transcript of 4.6 kb, considerably larger than the predicted length of the Mpbeta1 open reading frame (1527 bp). By heterologous expression in Drosophila S2 cells, the Mpbeta1 subunit has been shown to co-assemble with the previously cloned nAChR subunits Mpalpha1 and Mpalpha2. In contrast, no co-assembly of Mpbeta1 could be detected with either Mpalpha3 or Mpalpha4. With the aim of gaining a clearer insight into the influence of subunit composition upon assembly, the ability of M. persicae nAChR subunits to co-assemble with vertebrate nAChR subunits has also been examined.

Animals↗
PubMed2000

The influence of nicotinic receptor subunit composition upon agonist, alpha-bungarotoxin and insecticide (imidacloprid) binding affinity.

A series of cell lines stably expressing recombinant nicotinic acetylcholine receptors (nAChRs) has been established by transfection of mammalian (rat) and insect (Drosophila) nicotinic subunit cDNAs. By equilibrium radioligand binding, we have examined the influence of individual subunits upon the affinity of two nicotinic agonists (epibatidine and methylcarbamylcholine), an antagonist (the snake neurotoxin, alpha-bungarotoxin) and a recently developed chloronicotinyl insecticide (imidacloprid). Imidacloprid bound with very low affinity to the rat alpha4/beta2 nAChR but did so with high affinity to hybrid nAChRs containing Drosophila alpha subunits co-assembled with rat beta2. Of the subunit combinations examined, imidacloprid showed highest affinity binding to nAChRs containing the recently identified Drosophila alpha subunit, D alpha3, co-assembled with beta2. In contrast, no specific binding of imidacloprid was detected when D alpha3 was co-expressed with the mammalian neuronal beta4 subunit, or with the muscle-type (gamma or delta) subunits. However, despite the absence of imidacloprid binding to D alpha3/beta4, D alpha3/gamma or D alpha3/delta, these subunit combinations all exhibited high affinity binding of other nicotinic radioligands. Epibatidine showed substantially higher affinity binding to subunit combinations containing neuronal (beta2 or beta4) subunits than it did to combinations containing muscle-type (gamma or delta) subunits. In contrast, alpha-bungarotoxin bound with higher affinity to combinations containing muscle-type subunits. Our results demonstrate that both alpha and non-alpha subunits exert a profound influence upon the affinity of nicotinic ligands for recombinant nAChRs.

Animals↗
PubMed2000

Cloning and heterologous expression of Dalpha4, a Drosophila neuronal nicotinic acetylcholine receptor subunit: identification of an alternative exon influencing the efficiency of subunit assembly.

A neuronal nicotinic acetylcholine receptor (nAChR) subunit, Dalpha4, has been identified and cloned from the fruit fly Drosophila melanogaster, together with several alternatively spliced transcripts. Intron-exon boundaries within the gene encoding Dalpha4 (nAcRalpha-80B) have been identified by comparison of cDNA and genomic sequence data. The influence of amino acids encoded by alternatively spliced exons upon nicotinic radioligand binding and subunit-subunit co-assembly has been examined by heterologous expression in Drosophila S2 cells. The efficiency of subunit assembly has been shown to be influenced by amino acids surrounding the highly conserved 15 amino acid cysteine-loop motif within the N-terminal extracellular domain of the nAChR Dalpha4 subunit. Extensive use has been made of publicly available data determined by the Berkeley Drosophila Genome Project (BDGP). This includes expressed sequence tag (EST) data as well as whole-embryo in situ hybridisation and polytene chromosome in situ hybridisation data. BDGP in situ hybridisation data suggests that the Dalpha4 mRNA is expressed within Drosophila brain and ventral nerve cord and demonstrates that the gene encoding this nAChR subunit is located at position 80B on chromosome 3. The relationship between Dalpha4 and other previously cloned nAChR subunits has been examined and the implications for the nomenclature of insect nAChRs is discussed.

Alternative Splicing↗
PubMed1999

Up-regulation of cell-surface alpha4beta2 neuronal nicotinic receptors by lower temperature and expression of chimeric subunits.

The predominant nicotinic acetylcholine receptor (nAChR) expressed in vertebrate brain is a pentamer containing alpha4 and beta2 subunits. In this study we have examined how temperature and the expression of subunit chimeras can influence the efficiency of cell-surface expression of the rat alpha4beta2 nAChR. Functional recombinant alpha4beta2 nAChRs, showing high affinity binding of nicotinic radioligands (K(d) = 41 +/- 22 pM for [(3)H]epibatidine), are expressed in both stably and transiently transfected mammalian cell lines. Despite this, only very low levels of alpha4beta2 nAChRs can be detected on the cell surface of transfected mammalian cells maintained at 37 degrees C. At 30 degrees C, however, cells expressing alpha4beta2 nAChRs show a 12-fold increase in radioligand binding (with no change in affinity), and a 5-fold up-regulation in cell-surface receptors with no increase in total subunit protein. In contrast to "wild-type" alpha4 and beta2 subunits, chimeric nicotinic/serotonergic subunits ("alpha4chi" and "beta2chi") are expressed very efficiently on the cell surface (at 30 degrees C or 37 degrees C), either as hetero-oligomeric complexes (e.g. alpha4chi+beta2 or alpha4chi+beta2chi) or when expressed alone. Compared with alpha4beta2 nAChRs, expression of complexes containing chimeric subunits typically results in up to 20-fold increase in nicotinic radioligand binding sites (with no change in affinity) and a similar increase in cell-surface receptor, despite a similar level of total chimeric and wild-type protein.

Animals↗
PubMed1999

Molecular characterization and imidacloprid selectivity of nicotinic acetylcholine receptor subunits from the peach-potato aphid Myzus persicae.

The recent introduction of the chloronicotinyl insecticide imidacloprid, targeting insect nicotinic acetylcholine receptors (nAChRs), emphasises the importance of a detailed molecular characterisation of these receptors. We are investigating the molecular diversity of insect nAChR subunit genes in an important agricultural pest, the peach-potato aphid Myzus persicae. Two M. persicae alpha-subunit cDNAs, Mp alpha1 and Mp alpha2, have been cloned previously. Here we report the isolation of three novel alpha-subunit genes (Mp alpha3-5) with overall amino acid sequence identities between 43 and 76% to characterised insect nAChR subunits. Alignment of their amino acid sequences with other invertebrate and vertebrate nAChR subunits suggests that the insect alpha subunits evolved in parallel to the vertebrate neuronal nAChRs and that the insect non-alpha subunits are clearly different from vertebrate neuronal beta and muscle non-alpha subunits. The discovery of novel subtypes in M. persicae is a further indicator of the complexity of the insect nAChR gene family. Heterologous co-expression of M. persicae nAChR alpha-subunit cDNAs with the rat beta2 in Drosophila S2 cells resulted in high-affinity binding of nicotinic radioligands. The affinity of recombinant nAChRs for [3H]imidacloprid was influenced strongly by the alpha subtype. This is the first demonstration that imidacloprid selectively acts on Mp alpha2 and Mp alpha3 subunits, but not Mp alpha1, in M. persicae.

Amino Acid Sequence↗
PubMed1998

A novel octopamine receptor with preferential expression in Drosophila mushroom bodies.

Octopamine is a neuromodulator that mediates diverse physiological processes in invertebrates. In some insects, such as honeybees and fruit flies, octopamine has been shown to be a major stimulator of adenylyl cyclase and to function in associative learning. To identify an octopamine receptor mediating this function in Drosophila, putative biogenic amine receptors were cloned by a novel procedure using PCR and single-strand conformation polymorphism. One new receptor, octopamine receptor in mushroom bodies (OAMB), was identified as an octopamine receptor because human and Drosophila cell lines expressing OAMB showed increased cAMP and intracellular Ca2+ levels after octopamine application. Immunohistochemical analysis using an antibody made to the receptor revealed highly enriched expression in the mushroom body neuropil and the ellipsoid body of central complex, brain areas known to be crucial for olfactory learning and motor control, respectively. The preferential expression of OAMB in mushroom bodies and its capacity to produce cAMP accumulation suggest an important role in synaptic modulation underlying behavioral plasticity.

Adrenergic Agents↗
PubMed1998

Thapsigargin and receptor-mediated activation of Drosophila TRPL channels stably expressed in a Drosophila S2 cell line.

The Drosophila melanogaster genes, transient receptor potential (trp) and transient receptor potential-like (trpl) encode putative plasma membrane cation channels TRP and TRPL, respectively. We have stably co-expressed Drosophila TRPL with a Drosophila muscarinic acetylcholine receptor (DM1) in a Drosophila cell line (S2 cells). Basal Ca2+ levels measured using Fura-2/AM in unstimulated S2-DM1-TRPL cells were low and indistinguishable from untransfected cells, indicating that the TRPL channels were not constitutively active in this expression system. Activation of DM1 receptor in S2-DM1-TRPL cells by 100 microM carbamylcholine induced Ca2+ release from an intracellular Ca2+ pool followed by a Gd(3+)-insensitive Ca2+ influx. Pretreatment of S2-DM1-TRPL cells with 10 microM atropine abolished Gd(3+)-insensitive Ca2+ influx triggered by carbamylcholine, but the response was not blocked by prior incubation with pertussis toxin. TRPL channels could also be reliably activated by bath application of 1 microM thapsigargin for 10 min or 100 nM thapsigargin for 60 min in Ca(2+)-free solution. In some cells, TRPL channels activated by thapsigargin could further be activated by carbamylcholine. The findings suggest that, when stably expressed in the S2 cell line, TRPL may be regulated by two distinct mechanisms: (i) store depletion; and (ii) stimulation of DM1 receptor via pertussis-toxin insensitive G-protein (or the subsequent activation of PLC), but without further requirement for Ca2+ release.

Animals↗
PubMed1998

Host cell-specific folding of the neuronal nicotinic receptor alpha8 subunit.

Heterologous expression of the neuronal nicotinic acetylcholine receptor alpha8 subunit in cultured mammalian cell lines has revealed that the correct folding of this protein is dependent on the host cell type. The alpha8 subunit, which is able to form homo-oligomeric ion channels when expressed in Xenopus oocytes, could be detected in all transfected cell lines by both immunoprecipitation and immunofluorescence microscopy with a monoclonal antibody that recognises a linear epitope. In contrast, the alpha8 subunit could be detected in some but not in all transfected cell lines with a monoclonal antibody that recognises a conformation-sensitive epitope or by nicotinic radioligand binding. It is interesting that although correctly folded alpha8 protein could be detected in transfected rat pituitary (GH4C1) cells, only misfolded alpha8 protein could be detected in a large subpopulation of transfectants (transient or clonal stable isolates). We have also found that the protein encoded by a chimaeric cDNA (constructed from the N-terminal region of alpha8 and the C-terminal domain of the serotonin 5-HT3 receptor subunit) is expressed efficiently, and in a conformation that binds alpha-bungarotoxin, in all cell types examined. These results, together with previous expression studies with the homo-oligomeric alpha7 subunit and hetero-oligomeric nicotinic receptor subunit combinations, suggest that the cell-specific folding described here is a phenomenon that may be characteristic of homo-oligomeric nicotinic receptors.

Animals↗
PubMed1998

Antagonist profile and molecular dynamic simulation of a Drosophila melanogaster muscarinic acetylcholine receptor.

A stably-transfected, Drosophila cell line (S2-DMl-1) expressing the Drosophila DMl muscarinic acetylcholine receptor (mAChR) exhibits high-affinity, saturable, specific binding of the radiolabelled muscarinic antagonist [3H]-N-methyl scopolamine ([3H]-NMS) with an equilibrium dissociation constant (Kd) of 0.67 +/- 0.02 and a Bmax of 1.53 +/- 0.3 pmol/mg protein. Displacement of [3H]-NMS by mAChR antagonists results in the pharmacological profile: 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) > hexahydrosiladifenidol > p-fluorohexahydrosiladifenidol > nitrocaramiphen > pirenzepine > methoctramine > AFDX-116. This antagonist profile most closely resembles that of the vertebrate M3 mAChR subtype. In this study, however, we have demonstrated that the antagonist profile of DM1 is distinct from those of vertebrate mAChR subtypes. Molecular dynamic simulations of the Drosophila muscarinic receptor are presented in the free, carbamylcholine-bound and NMS-bound forms. Theoretical, quantitative structure-activity relationship models have been developed; a good correlation is observed between the interaction energies of the minimized ligand-receptor complexes and the pharmacological affinities of the antagonists tested.

Animals↗
PubMed1997

The ion channel properties of a rat recombinant neuronal nicotinic receptor are dependent on the host cell type.

1. A stable mammalian cell line (L-alpha 3 beta 4) has been established which expresses the cloned rat neuronal nicotinic acetylcholine receptor (nAChR) subunits alpha 3 and beta 4, which are the most abundant in autonomic ganglia. Ion channel properties of nAChRs expressed in L-alpha 3 beta 4 cells were investigated by single-channel and whole-cell recording techniques, and compared with both rat alpha 3 beta 4 nAChRs expressed in Xenopus oocytes, and endogenous nicotinic receptors in rat superior cervical ganglion (SCG) neurones, using identical solutions for all cell types. 2. Acetylcholine (ACh) caused activation of single ion channel currents with a range of amplitudes. Some channels had high conductances (30-40 pS), and relatively brief lifetimes; these resembled the predominant native channel from SCG. Other channels had low conductances (20-26 pS) and long bursts of openings which were quite unlike native channels, but which were similar to channels formed by alpha 3 beta 4 in oocytes. Both types often occurred in the same patch. 3. Cytisine was about 3 times more potent than ACh (low-concentration potency ratio) in L-alpha 3 beta 4 cells, which is not dissimilar to the 5-fold potency ratio found in both SCG and oocytes, whereas 1,1-dimethyl-4-phenylpiperazinium (DMPP) was less potent than ACh in some cells (as in the oocyte), but more potent in others (as in SCG). 4. While the channels expressed in L-alpha 3 beta 4 cells do not mimic exactly those expressed in rat SCG, they differ considerably from the same subunit combination expressed in oocytes. Larger conductance, SCG-like channels were detected frequently in L-alpha 3 beta 4, but were rarely, if ever, seen in oocytes injected with alpha 3 and beta 4 mRNA. Our results indicate that ion channel properties such as single-channel conductance can be influenced by the choice of heterologous expression system.

Acetylcholine↗
PubMed1997

Functional equivalence of native light-sensitive channels in the Drosophila trp301 mutant and TRPL cation channels expressed in a stably transfected Drosophila cell line.

Drosophila photoreceptors express two putative cation channels encoded by the transient receptor potential (trp) and trp-like (trpl) genes, which represent prototypical members of a novel family of phosphoinositide-regulated calcium influx channels. Mutations of both trp and trpl selectively abolish components of the light-sensitive current and, when heterologously expressed, both generate cation permeable conductances; however, a detailed comparison of recombinant and native channel properties is lacking. To more rigorously test the hypothesis that TRPL channels mediate one component of the light-sensitive current we have generated cell lines (Drosophila S2 cells) stably transfected with trpl cDNA and compared the recombinant channel properties with those of the light-sensitive conductance in situ in a Drosophila trp mutant under identical conditions. We found close correspondence in respect of a number of quantifiable biophysical parameters including: current voltage relationships, ionic selectivity, voltage independent block by external Mg2+ ions and effective single channel conductance and gating kinetics derived by noise analysis. Our estimate of 60-70 pS for channel conductance was confirmed directly in patch clamp recordings of single TRPL channels in S2 cells. These findings indicate that channels encoded by the trpl gene can completely account for the component of the light-sensitive conductance remaining in the trp mutant.

Animals↗
PubMed1997

Temperature-sensitive expression of Drosophila neuronal nicotinic acetylcholine receptors.

Heterologous expression of cloned Drosophila nicotinic acetylcholine receptor (nAChR) subunits indicates that these proteins misfold when expressed in mammalian cell lines at 37 degrees C. This misfolding can, however, be overcome either by growing transfected mammalian cells at lower temperatures or by the expression of Drosophila nAChR subunits in a Drosophila cell line. Whereas the Drosophila nAChR beta subunit (SBD) cDNA, reported previously, lacked part of the SBD coding sequence, here we report the construction and expression of a full-length SBD cDNA. We have examined whether problems in expressing functional Drosophila nAChRs in either Xenopus oocytes or mammalian cell lines can be attributed to an inability of these expression systems to assemble correctly Drosophila nAChRs. Despite expression in what might be considered a more native cellular environment, we have been unable to detect functional nAChRs in a Drosophila cell line unless Drosophila nAChR subunit cDNAs are coexpressed with vertebrate nAChR subunits. Our results indicate that the folding of Drosophila nAChR subunits is temperature-sensitive and strongly suggest that the inability of these Drosophila nAChR subunits to generate functional channels in the absence of vertebrate subunits is due to a requirement for coassembly with as yet unidentified Drosophila nAChR subunits.

Amino Acid Sequence↗
PubMed1997

Host cell-specific folding and assembly of the neuronal nicotinic acetylcholine receptor alpha7 subunit.

Expression of the cloned neuronal nicotinic acetylcholine receptor (nAChR) alpha7 subunit in several cultured mammalian cell lines has revealed that the folding, assembly, and subcellular localization of this protein are critically dependent upon the nature of the host cell. In all cell lines that were examined, high levels of alpha7 protein were detected by metabolic labelling and immunoprecipitation after transfection with the cloned alpha7 cDNA. In contrast, elevated levels of alpha-bungarotoxin binding could be detected in only two of the nine cell lines. Both of these "alpha7-permissive" cell lines [rat phaeochromocytoma (PC12) and human neuroblastoma (SH-SY5Y)] express an endogenous alpha7 subunit. However, by expression of an epitope-tagged alpha7 subunit, it has been possible to show that the elevation in surface alpha-bungarotoxin binding in these two cell lines is due to expression of cDNA-encoded alpha7. The cell-specific misfolding of the neuronal nAChR alpha7 subunit is a phenomenon that is not shared by either the hetero-oligomeric muscle nAChR or the homooligomeric serotonin receptor 5-HT3 subunit. Our data also indicate that the cell-specific misfolding cannot be explained by a requirement for the coassembly with other known nAChR subunits and cannot be alleviated by treatments that have been reported to affect the assembly efficiency of other neurotransmitter-gated ion channels.

Animals↗
PubMed1996

Wild-type and insecticide-resistant homo-oligomeric GABA receptors of Drosophila melanogaster stably expressed in a Drosophila cell line.

RDL is an ionotropic GABA receptor subunit, a product of the Rdl gene, originally identified in the Maryland strain of Drosophila melanogaster. Here, we report the generation of a Drosophila melanogaster cell line (S2-RDLA302S) stably expressing a mutated, dieldrin-resistant (A302S) form of RDL. The properties of this dieldrin-resistant, homo-oligomeric receptor have been compared with those of the stably expressed, wild-type form (S2-RDL). Using these stable lines, a striking reduction in sensitivity to both picrotoxinin and dieldrin was observed for responses to GABA of S2-RDLA302S compared to S2-RDL. To determine if these stable insect cell lines generate results similar to those obtained by transient expression in Xenopus laevis oocytes, we have examined the actions of two widely used convulsants, EBOB and TBPS, and a recently developed convulsant BIDN, on RDL-mediated GABA responses in the two expression systems. In both oocytes and S2 cells, the three convulsants suppressed the amplitude of responses to GABA. Thus, in accord with earlier work on agonist and allosteric sites, the S2-RDL cell line is found to yield similar pharmacological results to those obtained in transient expression studies. Stable cell lines are now available expressing susceptible and resistant forms of an ionotropic receptor by GABAergic insecticides.

Animals↗
PubMed1996

DAMB, a novel dopamine receptor expressed specifically in Drosophila mushroom bodies.

The modulatory neurotransmitters that trigger biochemical cascades underlying olfactory learning in Drosophila mushroom bodies have remained unknown. To identify molecules that may perform this role, putative biogenic amine receptors were cloned using the polymerase chain reaction (PCR) and single-strand conformation polymorphism analysis. One new receptor, DAMB, was identified as a dopamine D1 receptor by sequence analysis and pharmacological characterization. In situ hybridization and immunohistochemical analyses revealed highly enriched expression of DAMB in mushroom bodies, in a pattern coincident with the rutabaga-encoded adenylyl cyclase. The spatial coexpression of DAMB and the cyclase, along with DAMB's capacity to mediate dopamine-induced increases in cAMP make this receptor an attractive candidate for initiating biochemical cascades underlying learning.

Amino Acid Sequence↗