Evolution, Expression, and Function of Nonneuronal Ligand-Gated Chloride Channels inDrosophila melanogaster

Abstract

Ligand-gated chloride channels have established roles in inhibitory neurotransmission in the nervous systems of vertebrates and invertebrates. Paradoxically, expression databases in Drosophila melanogaster have revealed that three uncharacterized ligand-gated chloride channel subunits, CG7589, CG6927, and CG11340, are highly expressed in nonneuronal tissues. Furthermore, subunit copy number varies between insects, with some orders containing one ortholog, whereas other lineages exhibit copy number increases. Here, we show that the Dipteran lineage has undergone two gene duplications followed by expression-based functional differentiation. We used promoter-GFP expression analysis, RNA-sequencing, and in situ hybridization to examine cell type and tissue-specific localization of the three D. melanogaster subunits. CG6927 is expressed in the nurse cells of the ovaries. CG7589 is expressed in multiple tissues including the salivary gland, ejaculatory duct, malpighian tubules, and early midgut. CG11340 is found in malpighian tubules and the copper cell region of the midgut. Overexpression of CG11340 increased sensitivity to dietary copper, and RNAi and ends-out knockout of CG11340 resulted in copper tolerance, providing evidence for a specific nonneuronal role for this subunit in D. melanogaster. Ligand-gated chloride channels are important insecticide targets and here we highlight copy number and functional divergence in insect lineages, raising the potential that order-specific receptors could be isolated within an effective class of insecticide targets.

The orphan pentameric ligand-gated ion channelpHCl-2is gated by pH and regulates fluid secretion inDrosophilaMalpighian tubules

“…Although pLGICs are typically expressed throughout the nervous system and in muscle cells, where they mediate fast, ionotropic synaptic transmission (Sine and Engel, 2006), previously generated gene expression assays have not detected pHCl-2 expression in the nervous system, but instead have identified expression enriched in the midgut and Malpighian tubules (Chintapalli et al, 2007;Graveley et al, 2011;Remnant et al, 2016). Using polyclonal antibodies raised against the poorly conserved M3-M4 intracellular loop of pHCl-2, we confirmed that the pHCl-2 protein is expressed in the Malpighian tubules.…”

“…In Drosophila, pHCl-2 groups with two other orphan pLGIC subunits, CG7589 and CG6927 (Dent, 2006;Jones and Sattelle, 2006;Remnant et al, 2016), which together most closely resemble the Drosophila pH-sensitive chloride channel ( pHCl) and the pHsensitive chloride channel in S. scabiei (SsCl) (Mounsey et al, 2007;Schnizler et al, 2005). Clades of channel subunits orthologous to the clade of subunits defined by pHCl-2, CG7589 and CG6927 have been reported in other insects such as Apis mellifera, A. aegypti, Nasonia vitripennis and Tribolium cataneums Sattelle, 2006, 2007;Jones et al, 2010;Remnant et al, 2016; www.flybase.org) and in non-insect arthropods such as the deer tick Ixodes scapularis (www.flybase.org), but not in nematodes, molluscs, annelids or chordates (Dent, 2006). The pH response of pHCl-2 closely resembles that of the two other characterized pH-sensitive arthropod pLGICs, Drosophila pHCl and Sarcoptes SsCl; both pHCl and SsCl are inhibited by protons and are increasingly activated by a rise in alkalinity, exhibiting halfmaximal activity at pH 7.33±0.16 and 7.55±0.06, respectively (Mounsey et al, 2007;Schnizler et al, 2005).…”

“…Recent work by Remnant et al (2016) demonstrated that pHCl-2 is expressed in the copper cells of the midgut and influences sensitivity to dietary copper: flies deficient for pHCl-2 display increased copper tolerance, whereas the opposite is observed when pHCl-2 is over-expressed. Copper cells are the principal site of acid secretion in the midgut, and, like the Malpighian tubules, are thought to transport protons via apically localized V-type H + -ATPases (Dubreuil, 2004;Shanbhag and Tripathi, 2005).…”

Anatomy and Physiology of the Digestive Tract of Drosophila melanogaster

“…RNA interference (RNAi) knockdown experiments have also explored the contribution of ion transporters enriched in the acidic region to pH maintenance, and have identified five ion transporters that sustain low pH ( Overend et al 2016 ). These include: the potassium/chloride symporter Kazachoc (Kcc), a member the Slc12 family of electroneutral cation-chloride transporters previously shown to be expressed in several organs including the intestine ( Filippov et al 2003 ; Sun et al 2010 ); the Slowpoke pore-forming subunit of a calcium-activated K + channel, expressed in neurons, muscles, tracheal cells, and two types of midgut ECs in the copper and iron cell regions ( Brenner and Atkinson 1997 ); the ligand-gated chloride channel pHCL-2 which, in addition to regulating fluid secretion in Malpighian tubules, is expressed in the copper cell, iron, and large flat cell regions of the midgut ( Feingold et al 2016 ; Remnant et al 2016 ); the carbonic anhydrase CAH1; and the bicarbonate/chloride exchanger CG8177, belonging to the Slc4a1-3 subfamily of anion exchangers expressed in a specific midgut pattern similar to that of pHCl-2 ( Dubreuil et al 2010 ). Of note, a CG8177 mutant generated in the latter study failed to revel a contribution of this transporter to gut acidity, in contrast to the RNAi knockdown in Overend et al (2016) .…”

An intestinal zinc sensor regulates food intake and developmental growth

“…A transcriptional reporter revealed that Hodor was expressed in the intestine 3 . A new antibody (Extended Data Fig.…”