br Introduction The cys loop ligand gated ion channels LGICs
Introduction The cys-loop ligand gated ion channels (LGICs) are membrane-bound proteins mediating fast transduction from chemical to electrical signals at neuronal synapses and neuromuscular junctions , . Each subunit that makes up the pentameric LGIC architecture possesses a dicysteine bridge in the large extracellular N terminal regions, and four transmembrane (TM) domains with TM2 forming an ion channel lining. The cys-loop LGICs bind a neurotransmitter at the orthosteric site to open the ion channel. For example, nicotinic prostaglandin endoperoxide synthase receptors open a cation selective channel upon acetylcholine binding to induce membrane depolarization, while γ-aminobutyric acid receptors open a chloride channel upon the ligand binding to counteract the nicotinic receptor mediated depolarization. Insecticides are required to control pests as fast as possible and some of them target LGICs such as nicotinic acetylcholine receptors, γ-aminobutyric acid receptors and glutamate-gated chloride channels. These major insect LGICs have been studied intensively in terms of structural features underlying pesticide action and selectivity , , , , , ,  as well as characteristics of stage-dependent expression profile and subunit partners , , . However, the pH-sensitive chloride channels (pHCls), although expressed broadly in the nervous system of Drosophila , are poorly understood in terms of their contribution to insect homeostasis because no endogenous ligand has been identified. Bombyx mori is a beneficial insect species used for silk production and its genome has been sequenced , . Since many agriculturally important pests belong to Lepidoptera, Bombyx may also be viewed as a model organism useful for studying pesticide screens and modes of action. Hence we have isolated cDNAs encoding two B. mori pHCl variants (BmpHCl variants) “A” and “B” from the head of the silkworm larvae and characterized their receptor functions and pesticide sensitivity. We report here that only the A variant formed a functional pHCl in Xenopus laevis oocytes and was modulated significantly by ivermectin at a sub-micromolar concentration.
Materials and methods
Discussion In this study, we have for the first time isolated full-length cDNA encoding pHCl from the silkworm larval head and characterized the channel function and sensitivity to some pesticides. We found that two pHCl variants A and B were expressed in the larval head of B. mori (Fig. 1). Our result, however, does not preclude the possibility that more variants may be found in body parts other than the head or in other developmental stages as in Drosophila; this issue needs to be studied in future. Of the two pHCl variants, only A variant was capable of forming a pH-sensitive ion channel when expressed alone in Xenopus oocytes (Fig. 2A). The inability of the B variant to form pHCl in oocytes is attributable to its structure lacking TM1 and a part of TM2 (Fig. 1A). A question arises as to the physiological role of expressing the B variant. It could conceivably influence the properties of the A variant if the two variants co-assemble. The reversal potential for the peak current amplitude of the pH-shift induced response of the A variant was close to the chloride equilibrium potential and reducing the extracellular chloride concentration shifted the potential (Fig. 2B), suggesting chloride permeability. In Drosophila, ivermectin activates the pHCl not only at acidic pHs but also at basic pHs . Also, the pHCl was shown to open the channel at pH7.0 in Sarcoptes scabiei . However in Bombyx, ivermectin only slightly activated the pHCl at pH7.0 and had no apparent activating action at pH7.6 or higher pHs (Fig. 3), indicating a peculiarity of the silkworm channel. Such pH sensitivity itself is not limited to pHCl. For example, it was observed that extracellular pH strikingly affected the agonist action of synthetic agonists THIP and ZAPA on the Drosophila GABACl RDL expressed in oocytes . Perhaps pHCl binds an unidentified native ligand to induce chloride currents for maintaining homeostasis and such a pH sensing nature is not its primary function as the response is not robust (Fig. 2A).