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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.

Ivermectin (IVM) is a macrocyclic lactone that exerts antifilarial, antiparasitic, and insecticidal effects on nematodes and insects by acting on l-glutamic acid-gated chloride channels (GluCls). IVM also acts as an allosteric modulator of various other ion channels. Although the IVM binding site in the Caenorhabditis elegans GluCl was identified by X-ray crystallographic analysis, the mechanism of action of IVM in insects is not well defined. We therefore examined the action of IVM on the housefly (Musca domestica) GluCl and γ-aminobutyric acid (GABA)-gated ion channel (GABACl). For both channels, IVM induced currents by itself, potentiated currents induced by low concentrations of agonists, and inhibited currents induced by high concentrations of agonists. Despite exerting common actions on both types of channels, GluCls were more susceptible to IVM actions than GABACls, indicating that GluCls are the primary target of IVM. Substitution of an amino acid residue in the third transmembrane segment (G312M in GluCls, and G333A and G333M in GABACls) resulted in significantly reduced levels or loss of activation, potentiation, and antagonism of the channels, indicating that these three actions result from the interaction of IVM with amino acid residues in the transmembrane intersubunit crevice.

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 receptor's 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 yohimbinechlorpromazinemianserin. 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.