Fumiyo Ozoe

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.

The 14-3-3 Proteins Rad24 and Rad25 Negatively Regulate Byr2 by Affecting Its Localization in Schizosaccharomyces pombe

ABSTRACT In Schizosaccharomyces pombe, rad24 and rad25 have been identified to be homologous to mammalian 14-3-3 genes and found to be involved in many cellular events, including checkpoint and meiosis. In the present study, we present evidences that Rad24 and Rad25 act as negative regulators of Byr2 (mitogen-activated protein kinase [MAPK] kinase kinase). Overexpression of rad24 or rad25 reduced mating and sporulation in homothallic wild-type cells. In contrast, the mating and sporulation efficiency of rad24- or rad25-null cells was higher than that of wild-type cells. Deletion of rad24 or rad25 increased sporulation efficiency in ras1-null diploid cells but not in byr2-, ste4-, byr1-, and spk1-null cells. Rad24 and Rad25 had no effect on the activity of constitutively active Byr1S214DT218D. Rad24 and Rad25 bound to both the N-terminal and the C-terminal domains of Byr2 when these bacterially expressed proteins were examined. The formation of complexes in vivo between Byr2 and either Rad24 or Rad25 was also confirmed by immunocoprecipitation. Furthermore, we showed negative regulation of Byr2 by Rad25, by monitoring the mRNA level of mam2, which is regulated by both the Ras1/MAPK pathway and ste11, in various combinations of mutants. In addition, the cellular localization of Byr2 in living cells was observed by using fusion to green fluorescent protein. Byr2 was mainly localized in the cytoplasm during vegetative growth and then concentrated at the plasma membrane in response to nitrogen starvation. Deletion of rad24 or rad25 fastened the timing of Byr2 translocation. Our results are consistent with the hypothesis that one of the roles of 14-3-3 is to keep Byr2 in the cytoplasm and to affect the timing of Byr2 translocation in response to sexual developmental signal.

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.

Activation of the pheromone-responsive MAP kinase drives haploid cells to undergo ectopic meiosis with normal telomere clustering and sister chromatid segregation in fission yeast.

Meiosis is a process of importance for sexually reproducing eukaryotic organisms. In the fission yeast Schizosaccharomyces pombe, meiosis normally proceeds in a diploid zygote which is produced by conjugation of haploid cells of opposite mating types. We demonstrate that activation of the pheromone-responsive MAPK, Spk1, by the ectopic expression of a constitutively active form of Byr1 (MAPKK for Spk1) induced the cells to undergo meiosis while in the haploid state. Moreover, the induction of meiosis required Mei2 (a key positive regulator of meiosis), but did not require Mei3; Mei3 is normally required to inactivate the Pat1 kinase (a negative regulator of Mei2) thereby allowing Mei2 to drive meiosis. Therefore, expression of a constitutively active form of Byr1 activates Mei2 without the need of Mei3. In cells induced to undergo meiosis by activating the Spk1 MAPK signaling pathway, telomeres clustered at the spindle pole body (SPB) and centromeres detached normally from the SPB during meiotic prophase, and the cells showed the correct segregation of sister chromatids during meiotic divisions. In contrast, in meiosis induced by inactivation of Pat1, sister chromatids segregate precociously during the first meiotic division. Thus, these results suggest that activation of Spk1 drives meiosis in S. pombe.

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.

A Novel Gene, msa1, Inhibits Sexual Differentiation in Schizosaccharomyces pombe

Sexual differentiation in the fission yeast Schizosaccharomyces pombe is triggered by nutrient starvation or by the presence of mating pheromones. We identified a novel gene, msa1, which encodes a 533-aa putative RNA-binding protein that inhibits sexual differentiation. Disruption of the msa1 gene caused cells to hypersporulate. Intracellular levels of msa1 RNA and Msa1 protein diminished after several hours of nitrogen starvation. Genetic analysis suggested that the function of msa1 is independent of the cAMP pathway and stress-responsive pathway. Deletion of the ras1 gene in diploid cells inhibited sporulation and in haploid cells decreased expression of mating-pheromone-induced genes such as mei2, mam2, ste11, and rep1; simultaneous deletion of msa1 reversed both phenotypes. Overexpression of msa1 decreased activated Ras1Val17-induced expression of mam2. Phenotypic hypersporulation was similar between cells with deletion of only rad24 and both msa1 and rad24, but simultaneous deletion of msa1 and msa2/nrd1 additively increased hypersporulation. Therefore, we suggest that the primary function of Msa1 is to negatively regulate sexual differentiation by controlling the expression of Ste11-regulated genes, possibly through the pheromone-signaling pathway.

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.

zds1, a novel gene encoding an ortholog of Zds1 and Zds2, controls sexual differentiation, cell wall integrity, and cell morphology in fission yeast

While screening for genes that reverse the sporulation-deficient phenotype of the ras1Δ diploid Schizosaccharomyces pombe strain, we identified zds1. This gene shares sequence homology with the ZDS1 and ZDS2 genes from Saccharomyces cerevisiae, which appear to be involved in multiple cellular events. Expression of Zds1 in ras1Δ diploid cells elevated their sporulation rate from 0.3 to 11.2%. Expression of the Zds1 C-terminal region increased the sporulation rate further (to 21.9%) while introduction of the Zds1 N-terminal region had no effect. zds1 expression did not induce sporulation in strains with mutations in genes participating in the downstream MAP kinase cascade. The zds1-disrupted strain is sensitive to CaCl2, and this effect is suppressed by the C-terminal region of Zds1. The growth of the zds1Δ strain is markedly inhibited by cold temperatures, while its viability decreased in the stationary phase. Moreover, the zds1Δ strain is round in shape and very sensitive to zymolyase, and its cell wall becomes thicker than that of wild type. Thus, zds1 must be required to maintain cell wall integrity. The Zds1–GFP fusion protein localized to the cytosol, the septum, and the cell cortex. Its localization in the septum was dependent on its C-terminal region. Overexpression of the C-terminal region of Zds1 induced multi-septa and abnormal zygotes. We propose that the C-terminal region is the functional domain of Zds1 while the N-terminal region is a negative regulatory region. Thus, Zds1 is involved in multiple cellular events in fission yeast, including sexual differentiation, Ca2+ tolerance, cell wall integrity, viability in the stationary phase, and cell morphology.

Expression pattern and function of alternative splice variants of glutamate-gated chloride channel in the housefly Musca domestica

Glutamate-gated chloride channels (GluCls) mediate fast inhibitory neurotransmission in invertebrate nervous systems. cDNAs encoding two alternative splice variants (MdGluClB and C) of the GluCl subunit were cloned from the housefly Musca domestica. The expression patterns of three variants, including the previously reported MdGluClA, differed among the body parts (head, thorax, abdomen, and leg) of the adult housefly and among developmental stages (embryo, larva, pupa, and adult). The MdGluClA and B transcripts were abundant in the central nervous system of the adult, whereas the MdGluClC transcript was expressed in the central nervous system and as the predominant variant in the peripheral tissues. The sensitivities to the agonist glutamate and the allosteric activator ivermectin B1a did not differ between channels containing MdGluCl variants when they were singly or co-expressed in Xenopus oocytes. By contrast, MdGluClA and B channels were more sensitive to the channel blockers fipronil and picrotoxinin than was MdGluClC channels. Heteromeric channels containing different subunit variants were more sensitive to picrotoxinin than were homomeric channels. Heteromeric channels were more sensitive to fipronil than were homomeric MdGluClC channels but not than homomeric MdGluClA and B channels. These results suggest that functionally indistinguishable but pharmacologically distinct GluCls are expressed in a spatially and temporally distinct manner in the housefly.