Yoshihisa Ozoe

B96Bom encodes a Bombyx mori tyramine receptor negatively coupled to adenylate cyclase

A cDNA encoding a biogenic amine receptor (B96Bom) was isolated from silkworm (Bombyx mori) larvae, and the ligand response of the receptor stably expressed in HEK‐293 cells was examined. Tyramine (TA) at 0.1–100 µm reduced forskolin (10 µm)‐stimulated intracellular cAMP levels by approximately 40%. The inhibitory effect of TA at 1 µm was abolished by yohimbine and chlorpromazine (each 10 µm). Although octopamine (OA) also reduced the cAMP levels, the potency was at least two orders of magnitude lower than that of TA. Furthermore, unlabelled TA (IC50 = 5.2 nm) inhibited specific [3H]TA binding to the membranes of B96Bom‐transfected HEK‐293 cells more potently than did OA (IC50 = 1.4 µm) and dopamine (IC50 = 1.7 µm). Taken together with the result of phylogenetic analysis, these findings indicate that the B96Bom receptor is a B. mori TA receptor, which is negatively coupled to adenylate cyclase. The use of this expression system should facilitate physiological studies of TA receptors as well as structure–activity studies of TA receptor ligands.

Functional characterization of Musca glutamate‐ and GABA‐gated chloride channels expressed independently and coexpressed in Xenopus oocytes

Ligand‐gated chloride channels (LGICs) are important targets for insecticides and parasiticides. Genes encoding subunits of two LGICs, a glutamate‐gated chloride channel (MdGluCl‐α) and a γ‐aminobutyric acid (GABA)‐gated chloride channel (MdRdl), were cloned from house‐flies (Musca domestica L.). These genes were first expressed independently in Xenopus laevis oocytes by cRNA injection in order to investigate the pharmacology of these ligand‐gated channels using two‐electrode voltage‐clamp electrophysiology. It was found that l‐glutamate and GABA activated the MdGluCl‐α homo‐oligomers with an EC50 value of 30 µm and the MdRdl homo‐oligomers with an EC50 value of 101 µm, respectively. Both channels were chloride ion‐permeable, and the MdRdl channel was more sensitive to chloride channel blockers, such as γ‐hexachlorocyclohexane (γ‐HCH), fipronil and picrotoxinin, than the MdGluCl‐α channel. MdGluCl‐α required only 1–2 days of incubation after cRNA injection to be expressed in oocytes, whereas 4–7 days of incubation was necessary to achieve MdRdl expression. However, when the cRNA of MdGluCl‐α was injected at a dose of 1% (w/w) 1 day after the injection of the cRNA of MdRdl, a significant increase in the current amplitude of responses to GABA was observed, and the incubation period necessary for MdRdl expression became shorter. These results suggest that MdGluCl‐α assists in the expression of MdRdl when the two are coexpressed.

γ-Aminobutyrate- and Glutamate-gated Chloride Channels as Targets of Insecticides

γ-Aminobutyrate (GABA)- and glutamate-gated chloride channels (GABACls and GluCls) are members of the Cys-loop receptor channel family. Both channels are widely distributed in the nervous system of invertebrates and function as neurotransmitter receptors to mediate inhibitory synaptic transmission. These receptor channels are important targets for widely used insecticides and parasiticides. Diverse noncompetitive antagonists, including the phenylpyrazole insecticide fipronil, inhibit inhibitory neurotransmission by binding to a site deep within the pentameric channel. Macrocyclic lactones, such as the insecticide/parasiticide avermectin, activate GluCls by irreversibly binding to the subunit interfaces. Recent genome sequencing and molecular biology studies have revealed a diversity of subunit isoforms arising from the duplication, alternative splicing, and RNA editing of the genes encoding these channels in various insect species, which may lead to physiological and pharmacological diversification of the receptor channels. Fipronil resistance due to target-site insensitivity in GABACls is a growing concern in several insect pest species. Nevertheless, identification of new chemistry and multiple potential binding sites for agonists, antagonists, and modulators in these channels suggests potential for developing novel pest control chemicals.

Molecular cloning and pharmacological characterization of a Bombyx mori tyramine receptor selectively coupled to intracellular calcium mobilization.

Tyramine (TA) is a biogenic amine in invertebrates. cDNA encoding the TA receptor (TAR) BmTAR2 was cloned from the nerve tissue of the silkworm Bombyx mori. The receptors functional and pharmacological properties were examined in BmTAR2-transfected HEK-293 cells. In [(3)H]TA binding assays, BmTAR2 showed considerably higher affinity for TA than for other biogenic amines, with an IC(50) value of 57.5 nM. Moreover, TA induced a dose-dependent increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in cells, with an EC(50) value of 11.6 nM, whereas octopamine and dopamine increased [Ca(2+)](i) only at concentrations above 100 microM. A few antagonists were found to inhibit the TA-induced increases in [Ca(2+)](i); the rank order of potency was yohimbine > chlorpromazine > mianserin. TA showed no effect on intracellular cAMP concentration. The data indicate that BmTAR2 belongs to the second class of TARs, which are selectively coupled to intracellular Ca(2+) mobilization. RT-PCR analysis revealed that BmTAR2 was expressed predominantly in the nervous tissue of B. mori larvae, suggesting that TA has neurotransmitter and neuromodulatory roles that are mediated by BmTAR2.

Molecular cloning and heterologous expression of an α-adrenergic-like octopamine receptor from the silkworm Bombyx mori

A cDNA encoding an octopamine (OA) receptor (BmOAR1) was isolated from the nerve tissue of silkworm (Bombyx mori) larvae. Comparison of amino acid sequences showed that BmOAR1 is highly identical to OA receptors isolated from Periplaneta americana (Pa oa1), Apis mellifera (AmOA1), and Drosophila melanogaster (OAMB or DmOA1A). BmOAR1 was stably expressed in HEK‐293 cells. OA above 1 µm led to an increase in intracellular cyclic AMP concentration ([cAMP]i). The synthetic OA‐receptor agonist demethylchlordimeform also elevated [cAMP]i to the same maximal level (≈ 5‐fold over the basal level) as that induced by OA. However, other biogenic amines, tyramine and dopamine, and chlordimeform were without effects. The [cAMP]i level raised by OA was lowered by antagonists; the rank order of antagonist activity was chlorpromazine > mianserin = yohimbine. Cyproheptadine and metoclopramide had little effect. OA above 100 nm induced a transient or sustained increase in intracellular Ca2+ concentration ([Ca2+]i), depending on the concentration of OA. Sequence homology and functional analysis data indicate that BmOAR1 is an α‐adrenergic‐like OA receptor of B. mori.

Picrodendrin and related terpenoid antagonists reveal structural differences between ionotropic GABA receptors of mammals and insects.

Twenty-eight picrotoxane terpenoids, including picrodendrins isolated from the Euphorbiaceae plant, Picrodendron baccatum (L.) Krug and Urban, have been evaluated for their ability to inhibit the specific binding of [3H]EBOB, the noncompetitive antagonist of ionotropic GABA receptors, to rat-brain and housefly (Musca domestica L.)-head membranes. Picrodendrin Q was the most potent competitive inhibitor of [3H]EBOB binding, with IC50 values of 16 nM (rat) and 22 nM (Musca). We find that the spiro gamma-butyrolactone moiety at the 13-position, which contains a carbonyl group conjugated with an unsaturated bond, and the substituents at the 4-position play important roles in the interaction of picrodendrins with their binding site in rat receptors. In contrast, such structural features are not strictly required in the case of the interaction with Musca receptors; the spiro saturated gamma-butyrolactone moiety at the 13-position, which bears the 16-sp3 carbon atom, and the hydroxyl groups at various positions are somewhat tolerated. Quantitative structure-activity studies have clearly shown that the electronegativity of the 16-carbon atom and the presence or absence of the 4- and 8-hydroxyl groups are important determinants of the potency of nor-diterpenes in Musca receptors, while the negative charge on the 17-carbonyl oxygen atom is likely important in the case of rat receptors. These findings indicate that there are significant differences between the structures of the complementary binding sites in rat GABA receptors and Musca GABA receptors. We also infer differences between native Musca GABA receptors and the Drosophila Rdl subunit-containing homo-oligomeric GABA receptors in the structures of their binding sites.

Comparison of a coq7 deletion mutant with other respiration‐defective mutants in fission yeast

Among the steps in ubiquinone biosynthesis, that catalyzed by the product of the clk‐1/coq7 gene has received considerable attention because of its relevance to life span in Caenorhabditis elegans. We analyzed the coq7 ortholog (denoted coq7) in Schizosaccharomyces pombe, to determine whether coq7 has specific roles that differ from those of other coq genes. We first confirmed that coq7 is necessary for the penultimate step in ubiquinone biosynthesis, from the observation that the deletion mutant accumulated the ubiquinone precursor demethoxyubiquinone‐10 instead of ubiquinone‐10. The coq7 mutant displayed phenotypes characteristic of other ubiquinone‐deficient Sc. pombe mutants, namely, hypersensitivity to hydrogen peroxide, a requirement for antioxidants for growth on minimal medium, and an elevated production of sulfide. To compare these phenotypes with those of other respiration‐deficient mutants, we constructed cytochrome c (cyc1) and coq3 deletion mutants. We also assessed accumulation of oxidative stress in various ubiquinone‐deficient strains and in the cyc1 mutant by measuring mRNA levels of stress‐inducible genes and the phosphorylation level of the Spc1 MAP kinase. Induction of ctt1, encoding catalase, and apt1, encoding a 25 kDa protein, but not that of gpx1, encoding glutathione peroxidase, was indistinguishable in four ubiquinone‐deficient mutants, indicating that the oxidative stress response operates at similar levels in the tested strains. One new phenotype was observed, namely, loss of viability in stationary phase (chronological life span) in both the ubiquinone‐deficient mutant and in the cyc1 mutant. Finally, Coq7 was found to localize in mitochondria, consistent with the possibility that ubiquinone biosynthesis occurs in mitochondria in yeasts. In summary, our results indicate that coq7 is required for ubiquinone biosynthesis and the coq7 mutant is not distinguishable from other ubiquinone‐deficient mutants, except that its phenotypes are more pronounced than those of the cyc1 mutant.

Molecular cloning of a GABA receptor subunit from Laodelphax striatella (Fallén) and patch clamp analysis of the homo‐oligomeric receptors expressed in a Drosophila cell line

A cDNA encoding a γ‐aminobutyric acid (GABA) receptor subunit was cloned from the small brown planthopper Laodelphax striatella. The L. striatella GABA receptor subunit was found to have high amino acid sequence similarity to the bd‐type splice variant of the Drosophila GABA receptor Rdl subunit and several other GABA receptor subunits, with identities of over 70%. The cDNA was inserted into the expression vector pAc5.1‐lac‐Hygro. Clonal cell lines stably expressing homo‐oligomeric L. striatella GABA receptors were generated by transfecting the vector into D.mel‐2 cells. Expression of functional GABA receptors in the cell lines was demonstrated by whole‐cell patch clamp recordings. GABA induced inward currents with an EC50 value of 29 µM and a Hill coefficient of 1.7. The GABA‐evoked responses reversed close to the Nernst equilibrium potential for chloride ions. The amplitudes of agonist‐induced currents were found to be in the order muscimol (100 µM) ≥ GABA (100 µM) > isoguvacine (100 µM) > cis‐4‐aminocrotonic acid (CACA) (100 µM) > 5‐(4‐piperidyl)‐3‐isoxazolol (4‐PIOL) (1 mM). Antagonists such as fipronil (100 nM), 4′‐ethynyl‐4‐n‐propylbicycloorthobenzoate (EBOB) (100 nM), dieldrin (100 nM) and SR95531 (gabazine) (1 µM) suppressed GABA‐induced currents. The functional expression of a GABA receptor from an agricultural pest presents a unique opportunity to discover new molecules active at this important target site.

Functional and pharmacological characterization of a β-adrenergic-like octopamine receptor from the silkworm Bombyx mori

A cDNA encoding a seven-transmembrane receptor was cloned from the nervous tissues of silkworm (Bombyx mori) larvae. Sequence analysis indicated that the gene is an ortholog of CG6989, which encodes a Drosophila beta-adrenergic-like octopamine (OA) receptor (DmOct beta 2R). As very little information is available regarding this class of receptors, we generated a cell line that stably expressed the gene in HEK-293 cells and we then performed functional and pharmacological studies of this receptor. [(3)H]OA-binding assays using membrane preparations of this cell line showed that the receptor possesses a higher affinity for OA than for tyramine (TA) or dopamine (DA). The cell line elicited a bell-shaped, OA concentration-dependent increase in intracellular cAMP levels, with a maximum at 100 nM. (R)-OA was more potent than (S)-OA. TA and DA had weak or marginal effects on cAMP production. The OA receptor agonist demethylchlordimeform elicited a similar biphasic response, although the maximum response was attained at a concentration as low as 1 nM. The rank order of potency of other agonists was as follows: naphazoline > tolazoline, clonidine. Among the antagonists tested, only chlorpromazine significantly attenuated the OA-induced increase in cAMP levels. No increase in intracellular Ca(2+) levels was observed with OA at concentrations up to 100 microM. These findings indicate that the cloned receptor is a beta-adrenergic-like OA receptor with unique functional and pharmacological properties.

The channel-lining 6′ amino acid in the second membrane-spanning region of ionotropic GABA receptors has more profound effects on 4′-ethynyl-4-n-propylbicycloorthobenzoate binding than the 2′ amino acid

The noncompetitive antagonist of ionotropic γ-aminobutyric acid (GABA) receptors 4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) is a useful tool to probe the antagonist-binding site. In the present study, four mutants of the human GABAA receptor β3 subunit were stably expressed in S2 cells and examined for their abilities to bind [3H]EBOB to identify the binding site of EBOB. The homo-oligomeric β3 GABA receptor was used as a housefly GABA receptor model, as the β3 subunit has a high sequence similarity with the housefly Rdl subunit in the second membrane-spanning (M2) region. The A274S mutation at the -1′ position in the M2 region had no effect on [3H]EBOB binding. The A277S mutation at the 2′ position led to a decrease in the affinity of EBOB for the GABA receptor. The T281V mutant at the 6′ position and the A277S/T281V double mutant completely abolished the binding ability. A β3 GABA receptor homology model predicts these interactions between the receptor and EBOB. These results suggest that EBOB interacts with threonine 281 and alanine 277, and that threonine 281 plays a more critical role in interacting with EBOB than alanine 277.