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

Abstract

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. Here, we report the molecular and physiological characterization of a highly divergent, orphan pLGIC subunit encoded by the pHCl-2 (CG11340) gene, in Drosophila melanogaster. We show that 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. We demonstrate 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. Our 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.

secCl is a cys-loop ion channel necessary for the chloride conductance that mediates hormone-induced fluid secretion in Drosophila

“…Application of 1 mM acetylcholine, GABA, glutamate, glycine, histamine, serotonin, dopamine, nicotine, tyramine and octopamine to oocytes expressing secCl did not induce changes in holding current, indicating that none of these putative neurotransmitters gate secCl (data not shown). Even though secCl displays greatest homology to a class of pH-sensitive Cys-loop LGICs 14,15,17 , secCl -expressing oocytes were not sensitive to changes in pH (pH 6–pH 9.5). However, the holding currents of oocytes injected with secCl cRNA were significantly higher than water injected controls (302 nA ± 59.6 vs. 38.9 nA ± 10.7; p < 0.002, two tailed t-test) (Fig.…”

“…secCl contains a motif that is highly predictive of chloride selectivity among Cys-loop LGICs 17–19 (Supplemental Figure S1). In oocytes injected with secCl cRNA we observed a significant 8.5 ± 2.5 mV average positive shift in reversal potential in low-chloride buffer (13.6 mM Cl) relative to normal saline (103.6 mM Cl) compared to an insignificant 0.5 mV shift in low-sodium buffer (6 mM Na) relative to normal saline (96 mM Na) (Fig.…”

“…However, it is becoming increasingly clear that this family of ion channels has functions beyond ligand-gated neurotransmission. We previously showed that pHCl-2 , which forms a proton gated anion channel, functions in the principal cells of MTs to modulate urine secretion 17 . Here we present evidence that a related channel subunit, secCl , forms a channel that mediates chloride flux in Malpighian tubules in response to diuretic hormones.…”

“…Oocytes were harvested from mature Xenopus laevis and injected with cRNA according to standard procedures 39 and Two Electrode Voltage Clamp (TEVC) recordings were performed in ND96 buffer (‘Normal Saline’) as described in Feingold et al . 17 . For ion substitutions experiments, we substituted sodium acetate for sodium chloride (“Low Cl”) and cesium chloride for sodium chloride (“Low Na”).…”

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) .…”