A Novel Chloride Channel in Drosophila melanogaster Is Inhibited by Protons

Abstract

A systematic analysis of the Drosophila genome data reveals the existence of pHCl, a novel member of ligand-gated ion channel subunits. pHCl shows nearly identical similarity to glutamate-, glycine-, and histamine-gated ion channels, does however not belong to any of these ion channel types. We identified three different sites, where splicing generates multiple transcripts of the pHCl mRNA. The pHCl is expressed in Drosophila embryo, larvae, pupae, and the adult fly. In embryos, in situ hybridization detected pHCl in the neural cord and the hindgut. Functional expression of the three different splice variants of pHCl in oocytes of Xenopus laevis and Sf9 cells induces a chloride current with a linear current-voltage relationship that is inhibited by extracellular protons and activated by avermectins in a pH-dependent manner. Further, currents through pHCl channels were induced by a raise in temperature. Our data give genetic and electrophysiological evidence that pHCl is a member of a new branch of ligand-gated ion channels in invertebrates with, however, a hitherto unique combination of pharmacological and biophysical properties.Ligand-gated ion channels (LGICs) 1 mediate the fast inhibitory and excitatory responses of neuronal and muscle cells to neurotransmitters. A universal feature of the type of "Cys-loop" class of LGIC is a common topology of four membrane-spanning segments (M1-M4) and a huge N-terminal extracellular domain with a hyperconservated cysteinebridge motive (1). In vertebrates this "Cys-bridge" family of phylogenetically related genes codes for cation channels activated by acetylcholine and serotonin or for anion channels activated by GABA and glycine (1). In addition, glutamateand serotonin-gated anion channel genes are known in invertebrates (2, 3). Recently, genes for histamine-gated chloride channels and GABA-gated cation channels were identified in invertebrates (4 -7). The molecular basis of further channel types like acetylcholine-gated chloride channels in invertebrates is, however, still unknown (8). Information from the Drosophila melanogaster genome sequencing project allows identifying all members of the superfamily of ligand-gated ion channels occurring in this species by bioinformatic analysis of new homologous genes. The summarized data obtained from several published bioinformatic analyses (5,6,9,10) show that the group of ligand-gated "chloride" channels consists of 12 genes that are coding for GABA, histamine, and glutamate receptors or new, homologous ion channel types. Four members of this group cannot be directly assigned to the GABA, glutamate, or histamine branches and thus code for putative new types of ligand-gated chloride channels with yet unknown function. In a systematic expression approach of these predicted novel types of ion channels in Xenopus oocytes, it was found that none of the typical neurotransmitters activated these novel types of channels (6). Therefore, we extended the molecular biological analysis of the mRNA and found that the gene CG6112 encodes fo...

An Internally Modulated, Thermostable, pH-sensitive Cys Loop Receptor from the Hydrothermal Vent Worm Alvinella pompejana

“…Upon raising the pH to above pH 7.3, Schnizler et al (26) observed the onset of a chloride current with a pH response (Hill number 1.07) that did not desensitize as long as the pH was in the alkaline range. Temperature (in the range of 15-30°C) is a positive modulator for the activation of these channels.…”

Characterization of the target of ivermectin, the glutamate-gated chloride channel, fromAnopheles gambiae

“…The primary target of IVM is the invertebrate glutamate-gated chloride channel (GluCl) (Cully et al, 1994(Cully et al, , 1996Janssen et al, 2007Janssen et al, , 2010McCavera et al, 2009;Moreno et al, 2010), though it also has efficacy against other members of the invertebrate Cys-loop family of neurotransmitter receptors including the γ-aminobutyric acid- (Brown et al, 2012), histamine- (Zheng et al, 2002) and pHsensitive chloride channels (Schnizler et al, 2005). Because IVM is used to control and treat parasitic nematode diseases (Õmura, 2008), the majority of research on IVM targets has occurred in nematodes or model organisms, but the function of GluCl in mosquito disease vectors is unknown.…”

ClC-3 is a candidate of the channel proteins mediating acid-activated chloride currents in nasopharyngeal carcinoma cells

“…It was shown that an inward-rectified Cl Ϫ current was induced by extracellular acidification in rat cortical astrocytes (16). Furthermore, Schnizler and colleagues (26) found that there was a novel member of ligand-gated ion channel subunits, pHCl, expressed in Drosophila; pHCl mediated a chloride current that was inhibited by extracellular protons and activated by avermectins in a pH-dependent manner, with a linear current-voltage relationship.…”

“…Chloride channels were activated by extracellular acidification in human skeletal muscle cells (13), rat carotid body cells (21), and rat Sertoli cells (2) but were inhibited in Drosophila melanogaster (26) and Xenopus oocytes (8). Nobles and colleagues (20) showed that though some differences existed, many properties of acid-activated Cl Ϫ current are similar to those of the swelling-activated Cl Ϫ current, implying that the two currents are related and that extracellular acidification reflects a novel stimulus for volume-activated Cl Ϫ current.…”