InteractiveFly: GeneBrief

pH-sensitive chloride channel 2: Biological Overview | References

Gene name - pH-sensitive chloride channel 2

Synonyms - hodor

Cytological map position - 100C1-100C1

Function - channel

Keywords - a pH-modulated, zinc-gated chloride channel - regulates fluid secretion in Malpighian tubules - controls growth from a subset of enterocytes (interstitial cells) by promoting food intake and insulin/IGF signalling - reduced food intake of hodor mutants is rescued by activation of Tor signalling in hodor-expressing cells - transport chloride out of interstitial cells-thus maintaining osmolarity and water balance - lysosomal Hodor may transport chloride into the lysosome to sustain V-ATPase function, lysosomal acidification and TOR signalling

Symbol - pHCl-2

FlyBase ID: FBgn0039840

Genetic map position - chr3R:31,300,918-31,305,384

NCBI classification - LGIC_1: ligand-gated ion channel

Cellular location - surface transmembrane

NCBI links: EntrezGene, Nucleotide, Protein

pHCl-2 orthologs: Biolitmine

In cells, organs and whole organisms, nutrient sensing is key to maintaining homeostasis and adapting to a fluctuating environment. In many animals, nutrient sensors are found within the enteroendocrine cells of the digestive system; however, less is known about nutrient sensing in their cellular siblings, the absorptive enterocytes. This study used a genetic screen in Drosophila melanogaster to identify Hodor (pH-sensitive chloride channel 2), an ionotropic receptor in enterocytes that sustains larval development, particularly in nutrient-scarce conditions. Experiments in Xenopus oocytes and flies indicate that Hodor is a pH-sensitive, zinc-gated chloride channel that mediates a previously unrecognized dietary preference for zinc. Hodor controls systemic growth from a subset of enterocytes-interstitial cells-by promoting food intake and insulin/IGF signalling. Although Hodor sustains gut luminal acidity and restrains microbial loads, its effect on systemic growth results from the modulation of Tor signalling and lysosomal homeostasis within interstitial cells. Hodor-like genes are insect-specific, and may represent targets for the control of disease vectors. Indeed, CRISPR-Cas9 genome editing revealed that the single hodor orthologue in Anopheles gambiae is an essential gene. These findings highlight the need to consider the instructive contributions of metals-and, more generally, micronutrients-to energy homeostasis (Redhai, 2020).

To investigate nutrient sensing in enterocytes, 111 putative nutrient sensors in D. melanogaster were selected on the basis of their intestinal expression and their predicted structure or function. Using two enterocyte-specific driver lines, their expression was downregulated in midgut enterocytes throughout development under two dietary conditions, nutrient-rich and nutrient-poor; it was reasoned that dysregulation of nutrient-sensing mechanisms may increase or reduce the normal period of larval growth, and might do so in a diet-dependent manner. Enterocyte-specific knockdown of the gene CG11340, also referred to as pHCl-22, resulted in developmental delay. This delay was exacerbated, and was accompanied by significantly reduced larval viability, under nutrient-poor conditions; these phenotypes were confirmed using a second RNAi transgene and a new CG11340 mutant. In the tradition of naming Drosophila genes according to their loss-of-function phenotype, CG11340 was named 'hodor', an acronym for 'hold on, don't rush', in reference to the developmental delay (Redhai, 2020).

A transcriptional reporter revealed that Hodor was expressed in the intestine. A new antibody revealed that Hodor expression was confined to enterocytes in two midgut portions that are known to store metals: the copper cell region and the iron cell region. Within the copper cell region, Hodor was expressed only in so-called interstitial cells. hodor-Gal4 was also present in the interstitial cells of the copper cell region; however, in the experimental conditions used in this study and in contrast to published results, it was not detected in the iron cell region. Apart from the intestine, Hodor was found only in principal cells of the excretory Malpighian tubules. To identify the cells from which Hodor controls systemic growth, region- or cell-type-specific downregulation and rescue experiments were conducted. Only fly lines in which hodor was downregulated in interstitial cells showed slowed larval development. This developmental delay persisted when hodor knockdown was induced post-embryonically during larval growth, and was rescued only in fly lines in which hodor expression was re-instated in cell types that included interstitial cells. The fat body (analogous to liver and adipose tissue) has long been known to couple nutrient availability with developmental rate; however, recent studies have revealed contributions from the intestine, particularly in nutrient-poor conditions. The current findings confirm a role for the intestine in coupling nutrient availability with larval growth, and further implicate a subpopulation of enterocytes-interstitial cells-as important mediators. Interstitial cells were described decades ago in blowfly, but had remained relatively uncharacterized since; their name refers only to their position, interspersed among the acid-secreting copper cells that control microbiota loads (Redhai, 2020).

This study established that the lethality of hodor mutation or knockdown was apparent only during the larval period. The development of hodor mutants was slower throughout larval life, and surviving mutants attained normal pupal and adult sizes. Consistent with previous findings (Overend, 2016), hodor mutation or knockdown was found to reduce luminal acidity in the copper cell region, suggesting a role specifically for interstitial cells in this process. hodor mutants also had increased gut bacterial titres, which is consistent with the observed functional defects in the copper cell region (Storelli, 2018). Enlarged volumes of both the lumen of the copper cell region and the interstitial cells were also apparent after 1-3 days of (delayed) larval development; ultrastructurally, this was apparent in interstitial cells as a reduction in the complexity of their characteristic basal infoldings. This study was, however, able to rule out all of these defects as reasons for the developmental delay (Redhai, 2020).

During the course of these experiments, it was observed that hodor mutant larvae were more translucent than control larvae. This was suggestive of peripheral lipid depletion, which was confirmed by quantifying and staining for triacylglycerides. Reduced lipid stores did not result from disrupted enterocyte integrity: the intestinal barrier of mutants was intact, both anatomically and functionally. It was observed that hodor mutants had less food in their intestines and accumulated insulin-like peptide Ilp2 in their brains (nutrient-dependent Ilp2 secretion promotes larval development; its accumulation in the brain is commonly interpreted as peptide retention in the absence of transcriptional changes). Consistent with reduced systemic insulin signalling, hodor mutant larval extracts had reduced levels of phospho-Akt and phospho-S6 kinase. As these are all indicators of starvation, food intake was quantified, and it was observed to be reduced in both hodor mutant larvae and in hodor knockdowns targeting interstitial cells. Reduced food intake was apparent soon after hatching and persisted throughout larval development. Ectopic expression of Ilp2-which rescues developmental delay in larvae that lack insulin-like peptides-in hodor mutants partially rescued their developmental delay, but did not increase their food intake. An 'instructive' link between intestinal Hodor and food intake was further suggested by the overexpression of hodor in otherwise wild-type enterocytes; this resulted in larvae that ate more and developed at a normal rate, but had increased lipid stores. Therefore, Hodor controls larval growth from a subset of enterocytes by promoting food intake and systemic insulin signalling. In its absence, larvae fail to eat sufficiently to proceed through development at the normal rate and are leaner. When present in excess, Hodor causes larvae to eat more and accumulate the energy surplus as fat (Redhai, 2020).

In fly adipose tissue, amino acid availability activates Tor signalling to promote systemic growth. This study therefore combined hodor knockout or knockdown with genetic manipulations to alter Tor signalling. In flies with reduced or absent Hodor function, decreasing or increasing Tor signalling in hodor-expressing cells exacerbated or rescued the developmental delay, respectively. The reduced food intake of hodor mutants was also significantly rescued by activation of Tor signalling in hodor-expressing cells. Genetic targeting of Rag GTPases or the Gator1 complex in these cells failed to affect the developmental delay of hodor mutants, which could suggest non-canonical regulation of Tor signalling in Hodor-expressing cells. The systemic effects of Hodor on food intake and larval growth are therefore modulated by Tor signalling within Hodor-expressing interstitial cells (Redhai, 2020).

Hodor belongs to the (typically neuronal) Cys-loop subfamily of ligand-gated ion channels, and is predicted to be a neurotransmitter-gated anion channel (Dent, 2006). It is known to show activity in response to alkaline conditions in Xenopus oocytes, but the acidic pH of the copper cell region prompted a search for additional ligands. Although alkaline pH-induced Hodor activity was confirmed in oocyte expression systems, Hodor did not respond to typical Cys-loop receptor ligands such as neurotransmitters or amino acids. Instead, the screen identified zinc as an unanticipated ligand, which elicited a strong dose-dependent response only in Hodor-expressing oocytes; this response to zinc showed peak current amplitude values much greater than those observed in response to pH or to other metals such as iron or copper. Force-field-based structural stability and binding affinity calculations identified the amino acid pair E255 and E296 as a potential binding site for the divalent zinc ion. Mutating these residues did not abrogate the zinc-elicited currents, but did result in currents with faster rise time and deactivation kinetics, which supports the idea that zinc is a relevant Hodor ligand. On the basis of its sequence and conductance properties, Hodor has been proposed to transport chloride (Feingold, 2016; Remnant, 2016), and the zinc-elicited currents that were observed in oocytes had a reversal potential that is consistent with chloride selectivity. In vivo experiments in flies showed that supplementation of a low-yeast diet with zinc led to a reduction of chloride levels in interstitial cells, whereas hodor mutation increased chloride levels. Thus, Hodor is a pH-modulated, zinc-gated chloride channel (Redhai, 2020).

Attempts were made to establish the relevance of zinc binding in vivo. Zinc enrichment is observed in both the copper and iron cell regions of the larval gut, revealing an unrecognized role for these Hodor-expressing regions in zinc handling. Mutation of hodor failed to affect this zinc accumulation, although dietary yeast levels did, which is consistent with a role for Hodor in sensing rather than transporting zinc. (Notably, the white mutation-which is frequently used in the genetic background of Drosophila experiments-results in a small but significant reduction in both intestinal zinc accumulation and larval growth rate, although the status of the w gene neither exacerbated nor masked the more substantial, hodor-induced developmental delay. Furthermore, larvae that were fed a low-yeast diet ate significantly more when the diet was supplemented with zinc; this effect was abrogated in hodor mutants. In a food choice experiment, control larvae developed a preference for zinc-supplemented food over time, which suggests that the preference develops after ingestion. Consistent with this idea, zinc preference was specifically abrogated in hodor mutants (their general ability to discriminate between other diets was confirmed. Thus, zinc sensing by Hodor is physiologically relevant in vivo. Metals such as zinc are primarily provided by yeasts in nature; Hodor may be one of several sensors used to direct larvae to nutrient-rich food sources (Redhai, 2020).

The subcellular localization of Hodor suggests that it may normally maintain low cytoplasmic chloride concentrations by transporting it out of the interstitial cells and/or into their lysosomes. In accordance with this, and consistent with its putative lysosomal localization signals, Hodor was specifically enriched in apical compartments containing late endosome or lysosomal markers, as well as decorating the brush border of interstitial cells. The presence of Hodor in a subpopulation of lysosomes was of interest, because chloride transport across lysosomal membranes often sustains the activity of the proton-pumping vacuolar-type ATPase (V-ATPase) that maintains lysosomal acidity and Tor activation on the lysosome. To explore a role for Hodor in enabling Tor signalling, whether the absence of hodor induced autophagy-a hallmark of reduced Tor signalling, was tested. First, the induction of common autophagy markers in interstitial cells after genetic interference with the V-ATPase complex, which is known to promote autophagy by reducing lysosomal acidity and Tor signalling, was confirmed. Similar to reduced V-ATPase function, loss of hodor increased autophagy in interstitial cells. Expression of the dual autophagosome and autolysosome reporter UAS-GFP-mCherry-Atg8a in the intestinal cells of hodor mutants confirmed the induction of autophagy, and revealed two additional features. First, the acidification of autophagic compartments was defective in hodor mutants. Second, the increased autophagy and defective acidification observed in hodor mutants were particularly prominent in the two Hodor-expressing intestinal regions (the copper cell region and the iron cell region), consistent with cell-intrinsic roles for Hodor in these processes. Additional support for the roles of lysosomal function and Tor signalling in controlling whole-body growth from interstitial cells was provided by the finding that most V-ATPase subunits were transcriptionally enriched in the copper cell region. Functionally, the downregulation of V-ATPase subunits specifically in Hodor-expressing cells-and not in other subsets of enterocytes, such as those targeted by R2R4-Gal4-led to developmental delay and reduced food intake, phenotypes comparable to those observed as a result of hodor downregulation. Hence, although the directionality of zinc sensing and chloride transport in interstitial cells remains to be established, the data are consistent with roles for brush-border Hodor in transporting chloride out of interstitial cells-thus maintaining osmolarity and water balance. Lysosomal Hodor may transport chloride into the lysosome to sustain V-ATPase function, lysosomal acidification and TOR signalling, pointing to new links between lysosomal homeostasis in specialized intestinal cells, food intake and systemic growth. Nutrients such as amino acids are important regulators of Tor signalling. The genetic data are consistent with novel input from metals and/or micronutrients into Tor signalling. The nutrient-dependent zinc accumulation in lysosomal organelles-recently described in mammalian cells and nematode worms-suggests that links between zinc, lysosomes and Tor may be of broader importance. Two attractive cell types in which to explore such links are the Paneth cells of the mammalian intestine, which accumulate zinc and regulate intestinal immunity and stem cell homeostasis, and the 'lysosome-rich enterocytes' that have recently been described in fish and mice, which have roles in protein absorption (Redhai, 2020).

An extensive reconstruction of the hodor family tree supported the presence of a single member of the family in the ancestor of insects. Because Hodor-like proteins are present only in insects, they may prove to be highly specific targets for the chemical control of disease vectors, particularly given that mosquito genomes contain a single gene rather than the three paralogues that are found in most flies. To test this idea, CRISPR-Cas9 genome editing was used to generate a mutant that lacks the single hodor-like gene (AGAP009616) in the malaria vector Anopheles gambiae. This gene is also expressed in the digestive tract-specifically in the midgut-and in Malphighian tubules. Three independent deletion alleles revealed that AGAP009616 function is essential for the viability of A. gambiae. A target that is expressed in the intestine, such as Hodor, is particularly attractive for vector control as it may circumvent accessibility issues and could be directly targeted using ingestible drugs such as those applied to larval breeding sites (Redhai, 2020).

Metals have received little attention in the contexts of development or whole-body physiology, and are often regarded as passive 'building blocks'. By revealing the roles of a metal sensor in food intake and growth control, these findings highlight the importance of investigating the instructive contributions of metals-and, more generally, micronutrients-to energy homeostasis. These mechanisms could prove to be useful in insect vector control (Redhai, 2020).

The orphan pentameric ligand-gated ion channel pHCl-2 is gated by pH and regulates fluid secretion in Drosophila Malpighian tubules

Pentameric ligand-gated ion channels (pLGICs) constitute a large protein superfamily in metazoa whose role as neurotransmitter receptors mediating rapid, ionotropic synaptic transmission has been extensively studied. Although the vast majority of pLGICs appear to be neurotransmitter receptors, the identification of pLGICs in non-neuronal tissues and homologous pLGIC-like proteins in prokaryotes points to biological functions, possibly ancestral, that are independent of neuronal signalling. This study reports the molecular and physiological characterization of a highly divergent, orphan pLGIC subunit encoded by the pHCl-2 (CG11340/Hodor) gene, in Drosophila melanogaster. pHCl-2 forms a channel that is insensitive to a wide array of neurotransmitters, but is instead gated by changes in extracellular pH. pHCl-2 is expressed in the Malpighian tubules, which are non-innervated renal-type secretory tissues. This study demonstrates that pHCl-2 is localized to the apical membrane of the epithelial principal cells of the tubules and that loss of pHCl-2 reduces urine production during diuresis. The data implicate pHCl-2 as an important source of chloride conductance required for proper urine production, highlighting a novel role for pLGICs in epithelial tissues regulating fluid secretion and osmotic homeostasis (Feingold, 2016).

Pentameric ligand-gated ion channels (pLGICs) constitute a superfamily of ionotropic neurotransmitter receptors that includes vertebrate Cys-loop nicotinic acetylcholine, GABA, glycine and 5HT3 receptors. pLGICs play a central role in mediating rapid ionotropic neurotransmission and are expressed in all characterized bilateria. These channels typically reside on postsynaptic membranes of excitable cells and open in response to the binding of neurotransmitter released from presynaptic axon terminals. Ligand binding induces allosteric changes to protein conformation that result in the opening of a transmembrane, ion-selective pore that initiates the flow of specific ions down their electrochemical gradients, altering the membrane potential of the postsynaptic cell. The subunits of pLGICs have a stereotypical tertiary structure that consists of three general domains: an amino-terminal extracellular ligand-binding domain, four transmembrane domains (M1-M4), which collectively form the ion-permeable channel pore, and an intracellular loop between M3 and M4. Functional channels can exist as homomers, or as heteromers, containing as many as five distinct channel subunits (Feingold, 2016).

Sequencing of invertebrate genomes has led to the recognition that the pLGIC subunit superfamily is much larger and more diverse than was previously realized based on work in vertebrate nervous systems. Vertebrate genomes encode five main classes of pLGICs that have been defined based on ligand specificity: the cation-selective nicotinic acetylcholine receptors, serotonin 5HT3 receptors and zinc-activated receptors and the anion-selective GABA and glycine receptors. Invertebrate genomes, in contrast, encode a greater assortment of channel types with a wider range of ligand specificities and ligand-ion combinations than those found in vertebrates (Dent, 2006). In addition to the nicotinic acetylcholine and GABA receptors found in vertebrates, invertebrate genomes encode anion-selective acetylcholine, glutamate, serotonin, dopamine, tyramine and pH channels, as well as cation-selective GABA and proton channels. Moreover, multiple putative invertebrate pLGICs have been identified that cannot be assigned to any neurotransmitter family based on sequence homology (Feingold, 2016).

The biological functions of pLGICs are also likely to be much more diverse than has generally been appreciated. For instance, the cation-selective, proton-activated PBO-5,-6 channel in Caenorhabditis elegans mediates an intercellular pH signal that stimulates muscle contraction. The proton signal is generated by a proton pump in the intestine rather than by synaptic release from neurons. The function of the Drosophila melanogaster pHCl channel, which is open under alkaline conditions, is not known but its expression in the nervous system and the hindgut suggests non-canonical roles in signalling and/or ion regulation. Finally, the discovery of the proton-gated channel from the cyanobacterium Gloeobacter violaceus suggests that pLGICs originally evolved to regulate ion homeostasis in response to environmental changes (Feingold, 2016).

This study shows that CG11340, a putative pLGIC subunit in D. melanogaster which this study has named pHCl-2, forms a pH-gated chloride channel that is expressed in the Malpighian tubules, which are non-innervated secretory tissues. pHCl-2 channels are localized to the apical (lumen-facing) membrane of Malpighian tubule principal cells, precluding a role in responding to humoral signals originating in the haemolymph. Evidence is presented that, instead, pHCl-2 regulates fluid secretion by the Malpighian tubules in response to the pH of urine by controlling chloride counter-ion availability. Based on these data, a new role is proposed for pLGICs in ion homeostasis and implicate pHCl-2 in a previously unrecognized mechanism regulating urine secretion, a mechanism that will enrich current models of insect secretion (Feingold, 2016).

Previous phylogenetic analysis identified pHCl-2 as a member of an arthropod-specific clade of divergent orphan Cys-loop pLGICs. In Drosophila, pHCl-2 groups with two other orphan pLGIC subunits, Secretory chloride channel and CG6927, which together most closely resemble the Drosophila pH-sensitive chloride channel (pHCl) and the pH-sensitive chloride channel in S. scabiei (SsCl). 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 castaneums and in non-insect arthropods such as the deer tick Ixodes scapularis, 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 half-maximal activity at pH 7.33±0.16 and 7.55±0.06, respectively. In contrast, the other well-characterized pH-gated pLGICs identified to date, the PBO-5,-6 heteromeric cation channel in C. elegans and GLIC from cyanobacterium G. violaceus, are inhibited by alkaline conditions, and instead, are increasingly activated by a rise in proton concentration, displaying half-maximal responses at pH 6.83±0.01 and 5.1±0.1, respectively. While this study has shown that standard neurotransmitters do not gate pHCl-2 channels expressed in oocytes, it cannot be ruled out that pHCl-2 channels are sensitive to other ligands that might be found in gastric fluid or urine (Feingold, 2016).

Fluid secretion in the Malpighian tubules is mediated by active transepithelial ion transport, which establishes the osmotic gradient necessary for the formation of the primary urine. This active transport is powered by an apically localized, electrogenic H+-ATPase that pumps protons into the tubule lumen and generates a net positive apical membrane potential. The proton gradient is used to drive alkali metal cation/H+ antiporters, which replace luminal protons with sodium and potassium. Chloride enters the lumen passively, following its electrochemical gradient, and is a critical regulator of secretion because, in the absence of this negative counter-ion, cation transport into the lumen would result in an increasingly positive apical membrane potential, which would oppose transport by the ATPase before a significant osmotic gradient has formed (Feingold, 2016).

A role for pHCl-2 as an important source of chloride conductance in the Malpighian tubules is supported by the observation that urine production is affected in pHCl-2 mutants. Loss of pHCl-2 did not obviously impair fluid secretion in unstimulated Malpighian tubules, consistent with alternative routes of chloride flow into the lumen, either via known channels in the stellate cells or through a putative paracellular route via septate junctions. However, upon stimulation of the Malpighian tubules with cAMP, a second messenger that enhances the output of the H+ ATPase, pHCl-2 mutant Malpighian tubules showed a significantly reduced diuretic response compared with wild-type. These data suggest that under conditions of enhanced cation transport into the lumen, pHCl-2 provides a necessary source of chloride conductance in the principal cells, without which maximal secretion rates are not achieved (Feingold, 2016).

The expression of pHCl-2 in the apical membrane of principal cells, together with electrophysiology data demonstrating that pHCl-2 forms a pH-sensitive channel, suggests that the pHCl-2-mediated chloride conductance may be regulated by the pH of the luminal environment. Luminal pH is strongly influenced by the relative activities of the proton ATPase and the cation/H+ antiporter, which transport protons into and out of the tubule lumen, respectively, and it is proposed that the gating of pHCl-2 by pH may reveal a homeostatic mechanism that maintains an appropriate balance between these two cation transporters. Under conditions where the activity of the ATPase is high relative to the antiporter, the pH of the lumen would drop because the rate of proton transport into the lumen would exceed proton removal by the antiporter. As secondary active transport of sodium and potassium into the tubule lumen by the antiporter is coupled to active proton transport, an accumulation of excess protons in the tubule lumen would reflect a decrease in the energy efficiency of sodium and potassium secretion. It is proposed that the presence of a pH-sensitive chloride channel like pHCl-2 would counteract such an imbalance. If the pH of the lumen were too acidic, pHCl-2 would be antagonized, chloride permeability would become rate limiting for secretion and the increasingly positive apical membrane potential generated by the electrogenic ATPase would oppose further transport of protons into the lumen. The proton gradient, however, would continue to drive the antiporter, increasing luminal pH, which would, in turn, increase pHCl-2 conductance, and decrease the electrical barrier opposing the ATPase, thus re-establishing homeostasis. pHCl-2 would therefore serve as a 'brake' on the H+-ATPase, providing an upper limit to which the ATPase can operate relative to the antiporter, thus minimizing the expenditure of ATP under conditions where protons are not being put to metabolically efficient use (Feingold, 2016).

If pHCl-2 is in fact inhibited by acidic luminal pH, then one might predict that the stimulatory effects of cAMP on the H+-ATPase would lead to inhibition of pHCl-2-mediated chloride conductance. cAMP signalling in the Malpighian tubules leads to the secretion of a more acidic urine, with pH decreasing from 7.8 to 7.4, consistent with an increase in proton transport by the ATPase. Based on its pH sensitivity in oocytes, the chloride conductance of pHCl-2 channels should decrease ∼80% in response to cAMP, and the normalized chloride conductance-limited secretion rate increase should be significantly smaller in the presence of a pH-sensitive channel (i.e. wild-type) than in its absence (i.e. pHCl-2 knockout). Yet, the opposite was observed: the normalized response of secretion to cAMP in the pHCl-2 knockout was smaller than in wild-type, indicating that a simple physiological model does not account for the pHCl-2 phenotype. Instead, the pHCl-2 knockout may have indirect feedback effects on lumen pH or membrane potential, for example by directly affecting the ability of the H+-ATPase to respond to cAMP (Feingold, 2016).

Another effect of down-regulating pHCl-2 may be to increase the relative chloride current through stellate cell chloride channels. Decreased luminal pH that inhibits pHCl-2, resulting in a rise in apical membrane potential, should increase the driving force for chloride through apically localized chloride channels in stellate cells, thus increasing the relative contribution of stellate cells to apical chloride current. The increased activity of stellate cell chloride channels could in turn drive anion exchange via the basal membrane-localized Cl-/HCO3- transporter in stellate cells, thereby alkalinizing the haemolymph. Interestingly, pharmacological block of the Cl-/HCO3- transporter in A. aegypti has little effect on resting secretion rate but inhibits stimulated secretion, similar to the pHCl-2 mutant phenotype (Piermarini, 2010). As bicarbonate (HCO3-) is thought to be produced by carbonic anhydrase in the principal cells and enter the stellate cells through intracellular junctions, the effect of the pHCl-2 mutant on stimulated transport may be an indirect effect on the Cl-/HCO3- balance in principal cells feeding back and affecting chloride transport through stellate cells (Feingold, 2016).

Recent work by Remnant (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. While a clear relationship between H+-ATPase output and copper uptake has yet to be elucidated, it has been shown that copper uptake is impaired in flies whose copper cells are deficient in acid secretion, and that the copper cell midgut region is less acidic following copper feeding. Similar to the proposed model in the Malpighian tubules, pHCl-2 may influence ATPase output in copper cells by regulating chloride counter-ion availability, which, in turn, could affect the rate of copper uptake. Curiously, the role of pHCl-2 both in the Malpighian tubules and in the copper cells points to a somewhat counter-intuitive physiological model in which the activity of an alkaline-gated chloride channel provides the necessary counter-current for an acid-secreting transporter (Feingold, 2016).

Demonstration that pHCl-2 regulates secretion in the Malpighian tubules, a polarized epithelial tissue that is not directly associated with the nervous system, underscores the ability of pLGICs to function in a wide array of biological contexts beyond their canonical function in the nervous system. Previous work, for instance the discovery of pLGIC-like proteins in bacteria, has also hinted at possible roles for this ion channel superfamily that are entirely independent of neuronal signalling. There are also examples of pLGICs with well-characterized roles in the nervous system that appear to function in non-neuronal tissues. For example, the mammalian immune system is rich in pLGICs, including nicotinic acetylcholine (nACh), GABAA and glycine receptors. While such non-neuronal roles of the GABA and glycine receptors are poorly understood, the α7 nicotinic acetylcholine receptor (nAChR) is expressed in macrophages where it regulates tumour necrosis factor-α in response to acetylcholine released from spleen lymphocytes. nAChR function has also been reported in bronchial epithelia, where nAChRs expressed on the apical membrane respond to non-neuronal autocrine/paracrine ACh release to regulate chloride permeability through the cystic fibrosis transmembrane conductance regulator (CFTR) channel. Non-neural roles for nAChRs have also been identified in vascular endothelia and in keratinocytes (Grando, 1995) (Feingold, 2016).

pHCl-2 represents an extreme in the evolution of pLGIC functions. Like epithelial nAChRs, it is expressed in non-innervated tissues, but pHCl-2 is unique in that it is not obviously responding to an autocrine/paracrine signal. Whether pHCl-2 has an additional function in the nervous system remains unclear; microarray data suggest that it is not expressed in the head, brain and eyes of adult flies, or in the larval central nervous system, but this broad survey would not necessarily detect expression in a small subset of neurons. Additionally, althoug pHCl-2 localizes to the apical membrane of principal cells, the possibility cannot be ruled out that it functions in apically enriched endosomal vesicles to regulate secretion, similar to CUP-4 in C. elegans, which is localized to endosomes and is necessary for endosomal trafficking, although its activating ligand, if any, is unknown. Nevertheless, whether it acts in endosomes or the apical membrane, characterization of pHCl-2 illustrates the remarkable ability of the pLGICs to evolve diverse physiological functions (Feingold, 2016).

Evolution, expression, and function of nonneuronal ligand-gated chloride channels in Drosophila melanogaster

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 (Secretory chloride channel), CG6927, and CG11340 (pHCl-2), 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. This study shows that the Dipteran lineage has undergone two gene duplications followed by expression-based functional differentiation. Promoter-GFP expression analysis, RNA-sequencing, and in situ hybridization were used 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 this study has highlighted 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 (Remnant, 2016).


Search PubMed for articles about Drosophila pHCl-2

Dent, J. A. (2006). Evidence for a diverse Cys-loop ligand-gated ion channel superfamily in early bilateria. J Mol Evol 62(5): 523-535. PubMed ID: 16586016

Feingold, D., Starc, T., O'Donnell, M.J., Nilson, L., Dent, J.A. (2016). The orphan pentameric ligand-gated ion channel pHCl-2 is gated by pH and regulates fluid secretion in Drosophila Malpighian tubules. J. Exp. Biol. 219(17): 2629--2638. PubMed ID: 27358471

Grando, S. A., Zelickson, B. D., Kist, D. A., Weinshenker, D., Bigliardi, P. L., Wendelschafer-Crabb, G., Kennedy, W. R. and Dahl, M. V. (1995). Keratinocyte muscarinic acetylcholine receptors: immunolocalization and partial characterization. J Invest Dermatol 104(1): 95-100. PubMed ID: 7528248

Overend, G., Luo, Y., Henderson, L., Douglas, A. E., Davies, S. A. and Dow, J. A. (2016). Molecular mechanism and functional significance of acid generation in the Drosophila midgut. Sci Rep 6: 27242. PubMed ID: 27250760

Piermarini, P. M., Grogan, L. F., Lau, K., Wang, L. and Beyenbach, K. W. (2010). A SLC4-like anion exchanger from renal tubules of the mosquito (Aedes aegypti): evidence for a novel role of stellate cells in diuretic fluid secretion. Am J Physiol Regul Integr Comp Physiol 298(3): R642-660. PubMed ID: 20042685

Redhai, S., Pilgrim, C., Gaspar, P., Giesen, L. V., Lopes, T., Riabinina, O., Grenier, T., Milona, A., Chanana, B., Swadling, J. B., Wang, Y. F., Dahalan, F., Yuan, M., Wilsch-Brauninger, M., Lin, W. H., Dennison, N., Capriotti, P., Lawniczak, M. K. N., Baines, R. A., Warnecke, T., Windbichler, N., Leulier, F., Bellono, N. W. and Miguel-Aliaga, I. (2020). An intestinal zinc sensor regulates food intake and developmental growth. Nature 580(7802): 263-268. PubMed ID: 32269334

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Biological Overview

date revised: 15 July 2021

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