Feingold D , Knogler L , Starc T , Drapeau P , O'Donnell MJ , Nilson LA , Dent JA .

P40 OD018537 NIH HHS

	Figure 1. secCl forms a constitutively open homomeric chloride channel. (a) Bars indicate baseline currents of oocytes clamped at −80 mV that were injected with either water (n = 5) or secCl cRNA (n = 10). Error bars represent standard error of the mean. *P < 0.002 (two-tailed t-test). (b) Sample trace from an oocyte clamped at −80mV expressing secCl. The upward deflection corresponds to channels closing or being blocked upon treatment with 5 mM tyramine. (c) Sample current-voltage (IV) curves from an oocyte injected with secCl cRNA (solid traces) and a control oocyte injected with water (dashed traces). “Normal Saline” (ND96: 96 mM Na+ and 103.6 Cl−) “low Na” (6 mM Na+ and 103.6 mM Cl−) and”Low Cl” (96 mM Na+ and 13.6 mM Cl−). (d) Reversal potential shifts of secCl-expressing and control oocytes in low-chloride and low-sodium buffers relative to normal saline. ***P < 0.0001. (e–g) Coexpression of secCl with CG6927 results in a heteromeric pH-sensitive chloride channel. (e) Representative traces from an oocyte clamped at −80 mV expressing secCl and CG6927, showing responses to changes in pH. Control buffer was ND96 at pH 6.0. (f) The response profile of the channel to changes in pH. The responses were normalized to the maximum current response of each oocyte. The curve represents the fit to the Hill equation (n = 5). (g) Representative traces of the current voltage relationship in “normal” (n = 6), “low sodium” (n = 4), and “low chloride” (n = 4) buffer. Error bars represent standard error of the mean.
	Figure 2. secCl gene expression patterns. Expression patterns of a secCl promoter-GFP fusion transgene. (a–d) Tissues of third instar larvae with (a,c; GFP) and without (b,d; WT) transgene. (a–b) Salivary glands. (c,d) Gastric cecae. (e,f) Main segments of MTs of third instar larvae (e) and adult (f) showing expression in stellate cells. Scale bars represent 100 μm.
	Figure 3. secCl protein is expressed in the apical membrane of stellate cells. Immunostaining of adult MTs using anti-secCl (green). Nuclei are labelled with DAPI (blue). (a) secCl is expressed in the stellate cells but not the principal cells of MTs. (b) Immunostaining is not observed in homozygous mutant secClG689/G68933 MTs. (c) Immunostaining is restored to stellate cells in homozygous mutant secClG6893/G6893 bearing a single copy of a wild-type secCl transgene, p[secCl]. (d) An optical cross-section of MTs stained with anti-secCl showing localization to apical membrane (luminal side of the nucleus). (e) Expression of the transgenic mCherry-tagged CD8 (red), which is a marker of the apical membrane in polarized epithelia. (f) Merge of (d) and (e) showing co-localization of secCl and CD8 (yellow). (g–i) A z-stack rendered in 3D and rotated showing single stellate cell. Colors as in (d–f). Scale bars in (a–c) represent 50 μm; (d–f) 10 μm; (g–i) 5 μm.
	Figure 4. secCl is required for the tyramine-mediated diuretic response. Fluid secretion assays conducted on tubules with the genotypes: w (secCl(+)), w; secClG6893/G6893 (secCl(−)) and w; p[secCl], secClG6893/G6893 (secCl(−) rescue). (a) Basal fluid secretion rate (FSR) in Drosophila saline42 over 40 minutes. (b) Change in FSR in response to 2.9 μM tyramine or Drosophila saline (mock treatment) normalized to the first 40 minute interval. Red horizontal bar indicates the mean and error bars represent standard error of the mean. *** indicates p < 10−5. Significance was estimated by ANOVA and pairwise comparisons by Tukey’s HSD.
	Figure 5. secCl is necessary for the decrease in transepithelial potential in response to tyramine and leucokinin. Responses of the transepithelial potential (TEP) to tyramine. (a–d) Recordings of TEP in isolated Malpighian tubules. ‘*’ indicates penetration of the electrode into the lumen and ‘**’ indicates exit of electrode. The shaded area indicates the period of perfusion with saline or 2.9 μM tyramine as indicated. The beginning of perfusion was marked by a recording artifact. (a) The TEP of control w (secCl(+)) tubules did not respond to perfusion with saline. (b) In response to tyramine, the TEP of control tubules dropped to near zero mV and began to oscillate. (c) Tubules from w; secClG6893/G6893 (secCl(−)) flies did not respond to tyramine with a voltage drop. (d) Rescuing the secClG689/G68933 with a wild-type transgene (w; p[secCl]/+; secClG6893/G6893) restored the voltage drop and oscillations in response to tyramine. (e) P-values indicate the significance of the median change in absolute TEP pre- vs post-treatment (15 seconds after perfusion with saline control, 2.9 μM tyramine or 5 μM leucokinin) by Mann-Whitney U test.
	Figure 6. Genetic knockdown of secCl in stellate cells does not alter viability on a high-salt diet but does increase resistance to desiccation. (a–c) Immunostaining of adult MTs using anti-secCl (green). Nuclei are labelled with DAPI (blue). secCl expression is observed in MTs from undriven RNAi (a) and c724-Gal4 (b) control lines. secCl protein is not detected in MTs where the RNAi is driven in stellate cells (c). (d) Survival rates of w; RNAi/+, w; c724/+ and w; c724/+; RNAi/+ maintained on a NaCl rich diet over six days. N = 11–12 vials of 20 flies (see methods) for each genotype. Survival rates of w; RNAi/+, w; c724/+ and w; c724/+; RNAi/+ under conditions of desiccation stress over the course of 60 hours. N = 11–14 vials of 10 flies (see methods) per genotype. Scale bars represent 100 μm. For (d) and (e), error bars represent standard error of the mean. For (e) survival rates for w; c724/+; RNAi/+ are significantly different from w; RNAi/+ and w; c724/+ at all points between18–45 hours (p < 0.002, two tailed t-test).
	Figure 7. Model: The role of secCl within the tyramine/leucokinin diuretic pathway. A model describing the role of secCl within the signaling pathways of the diuretic hormones tyramine (Tyr) and Drosophila leucokinin (DK). Diuretic hormones bind to their respective GPCRs on the basolateral membrane of stellate cells, which triggers the release of calcium from intracellular stores via the PLC/IP3 pathway. Through an unknown mechanism, this rise in intracellular [Ca++] results in the activation of ClC-a (orange), and secCl (red). ClC-a, localized to the basolateral membrane, provides necessary Cl− entry into the cell from the hemolymph and secCl, localized to the apical membrane, provides a route for Cl− exit into the lumen. FSRs increase as a consequence of increased chloride secretion.

Beyenbach, Transcellular and paracellular pathways of transepithelial fluid secretion in Malpighian (renal) tubules of the yellow fever mosquito Aedes aegypti. 2011, Pubmed

Beyenbach, Transcellular and paracellular pathways of transepithelial fluid secretion in Malpighian (renal) tubules of the yellow fever mosquito Aedes aegypti. 2011, Pubmed
Beyenbach, The developmental, molecular, and transport biology of Malpighian tubules. 2010, Pubmed
Beyenbach, Signaling to the apical membrane and to the paracellular pathway: changes in the cytosolic proteome of Aedes Malpighian tubules. 2009, Pubmed
Blumenthal, Characterization of transepithelial potential oscillations in the Drosophila Malpighian tubule. 2001, Pubmed
Blumenthal, Regulation of chloride permeability by endogenously produced tyramine in the Drosophila Malpighian tubule. 2003, Pubmed
Cabrero, Chloride channels in stellate cells are essential for uniquely high secretion rates in neuropeptide-stimulated Drosophila diuresis. 2014, Pubmed
Cabrero, A biogenic amine and a neuropeptide act identically: tyramine signals through calcium in Drosophila tubule stellate cells. 2013, Pubmed
Chahine, Effects of genetic knock-down of organic anion transporter genes on secretion of fluorescent organic ions by Malpighian tubules of Drosophila melanogaster. 2012, Pubmed
Chintapalli, Using FlyAtlas to identify better Drosophila melanogaster models of human disease. 2007, Pubmed
Dent, Evidence for a diverse Cys-loop ligand-gated ion channel superfamily in early bilateria. 2006, Pubmed
Dow, The malpighian tubules of Drosophila melanogaster: a novel phenotype for studies of fluid secretion and its control. 1994, Pubmed
Dow, Extremely high pH in biological systems: a model for carbonate transport. 1984, Pubmed
Feingold, The orphan pentameric ligand-gated ion channel pHCl-2 is gated by pH and regulates fluid secretion in Drosophila Malpighian tubules. 2016, Pubmed
Galzi, Mutations in the channel domain of a neuronal nicotinic receptor convert ion selectivity from cationic to anionic. 1992, Pubmed , Xenbase
Goldin, Maintenance of Xenopus laevis and oocyte injection. 1992, Pubmed , Xenbase
Graveley, The developmental transcriptome of Drosophila melanogaster. 2011, Pubmed
Gunthorpe, Conversion of the ion selectivity of the 5-HT(3a) receptor from cationic to anionic reveals a conserved feature of the ligand-gated ion channel superfamily. 2001, Pubmed
Gunthorpe, Conversion of the ion selectivity of the 5-HT(3a) receptor from cationic to anionic reveals a conserved feature of the ligand-gated ion channel superfamily. 2001, Pubmed
Huang, The Drosophila inebriated-encoded neurotransmitter/osmolyte transporter: dual roles in the control of neuronal excitability and the osmotic stress response. 2002, Pubmed
Jaiteh, Evolution of Pentameric Ligand-Gated Ion Channels: Pro-Loop Receptors. 2016, Pubmed
Jones, The cys-loop ligand-gated ion channel superfamily of the honeybee, Apis mellifera. 2006, Pubmed
Keyser, The Drosophila NFAT homolog is involved in salt stress tolerance. 2007, Pubmed
Mounsey, Molecular characterisation of a pH-gated chloride channel from Sarcoptes scabiei. 2007, Pubmed , Xenbase
O'Connor, Chloride channels in apical membrane patches of stellate cells of Malpighian tubules of Aedes aegypti. 2001, Pubmed
O'Donnell, Separate control of anion and cation transport in malpighian tubules of Drosophila Melanogaster. 1996, Pubmed
O'Donnell, Hormonally controlled chloride movement across Drosophila tubules is via ion channels in stellate cells. 1998, Pubmed
Pannabecker, Regulation of epithelial shunt conductance by the peptide leucokinin. 1993, Pubmed
Patton, Endocytosis function of a ligand-gated ion channel homolog in Caenorhabditis elegans. 2005, Pubmed
Phillips, Comparative physiology of insect renal function. 1981, Pubmed
Piwon, ClC-5 Cl- -channel disruption impairs endocytosis in a mouse model for Dent's disease. 2000, Pubmed
Pollock, NorpA and itpr mutants reveal roles for phospholipase C and inositol (1,4,5)- trisphosphate receptor in Drosophila melanogaster renal function. 2003, Pubmed
Remnant, Evolution, Expression, and Function of Nonneuronal Ligand-Gated Chloride Channels in Drosophila melanogaster. 2016, Pubmed
Rosay, Cell-type specific calcium signalling in a Drosophila epithelium. 1997, Pubmed
Rousso, Apical targeting of the formin Diaphanous in Drosophila tubular epithelia. 2013, Pubmed
Rubin, Genetic transformation of Drosophila with transposable element vectors. 1982, Pubmed
Semenov, Diversification of Drosophila chloride channel gene by multiple posttranscriptional mRNA modifications. 1999, Pubmed
Shanbhag, Epithelial ultrastructure and cellular mechanisms of acid and base transport in the Drosophila midgut. 2009, Pubmed
Sine, Recent advances in Cys-loop receptor structure and function. 2006, Pubmed
Sözen, Functional domains are specified to single-cell resolution in a Drosophila epithelium. 1997, Pubmed
Stergiopoulos, Salty dog, an SLC5 symporter, modulates Drosophila response to salt stress. 2009, Pubmed
Talwar, Phosphorylation mediated structural and functional changes in pentameric ligand-gated ion channels: implications for drug discovery. 2014, Pubmed
Tasneem, Identification of the prokaryotic ligand-gated ion channels and their implications for the mechanisms and origins of animal Cys-loop ion channels. 2005, Pubmed
Terhzaz, Isolation and characterization of a leucokinin-like peptide of Drosophila melanogaster. 1999, Pubmed