Download Developmental and functional studies of the SLC12 gene family

Articles in PresS. Am J Physiol Cell Physiol (October 14, 2009). doi:10.1152/ajpcell.00376.2009
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Developmental and functional studies of the SLC12 gene family
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members from Drosophila melanogaster
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Qifei Sun1, E Tian2, R. James Turner1 and Kelly G. Ten Hagen2
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Membrane Biology Section, Molecular Physiology and Therapeutics Branch, and
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Developmental Glycobiology Unit, Laboratory of Cell and Developmental Biology, National
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Institute of Dental and Craniofacial Research, National Institutes of Health, DHHS, Bethesda,
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Maryland 20892-1190, USA
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Running title: SLC12 gene family of Drosophila melanogaster
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Corresponding authors: Dr R. James Turner, Bldg. 10, Rm. 1A01, 10 Center Drive MSC 1190,
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National Institutes of Health, Bethesda MD 20892-1190. Tel. (301) 402-1060, FAX (301) 402-
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1228, e-mail [email protected] and Dr. Kelly G. Ten Hagen, Bldg. 30, Rm. 426, 30 Convent Dr
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MSC 4370, National Institutes of Health, Bethesda MD 20892-4370. Tel. (301) 301 451-6318,
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FAX (301) 402-0897, e-mail [email protected]
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Copyright © 2009 by the American Physiological Society.
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ABSTRACT
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The electroneutral cation-chloride cotransporter gene family, SLC12, contains nine members in
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vertebrates. These include seven sodium and/or potassium-coupled chloride transporters and two
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membrane proteins of unknown function. Although SLC12 family members have been identified
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in a number of lower species, the functional properties of these proteins are unknown. There are
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five SLC12 homologues in Drosophila melanogaster including at least one member on each of
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the four main branches of the vertebrate phylogenetic tree. We have employed in situ
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hybridization to study the expression patterns of the Drosophila SLC12 proteins during
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embryonic development. Our studies indicate that all five members of this family are expressed
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during early embryogenesis (stages 1-6) but that spatial and temporal expression patterns become
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more refined as development proceeds. Expression during late embryogenesis was seen
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predominantly in the ventral nerve cord, salivary gland, gut and anal pad. In parallel studies we
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have carried out transport assays on each of the five Drosophila homologues expressed as
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recombinant proteins in the cultured insect cell line High Five. Under our experimental conditions
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we found that only one of these proteins, CG4357, transported the potassium congener 86Rb.
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Additional experiments established that rubidium transport via CG4357 was saturable (Km =
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0.29+0.05 mM), sodium-dependent (K1/2 = 53+11 mM), chloride-dependent (K1/2 = 48+5 mM)
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and potently inhibited by bumetanide (KI = 1.17+0.08 μM), a specific inhibitor of Na+-K+-2Cl-
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cotransporters. Taken together our results provide strong evidence that CG4357 is an insect
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orthologue of the vertebrate Na+-K+-2Cl- cotransporters.
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Key words: Drosophila embryogenesis, in situ hybridization, Na+-K+-2Cl- cotransport, cation-
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chloride cotransport, chloride transport
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INTRODUCTION
SLC12 is a small gene family of integral membrane proteins. To date all of the SLC12
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family members that have been successfully functionally characterized have proven to be
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electroneutral cation-chloride cotransporters (2; 4). The existence and physiological significance
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of a number of these transporters was established in functional studies that began to appear in the
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early 1980s (13), long before it was realized that they were genetically related proteins. The first
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of the SLC12 proteins to be identified at the molecular level was the Na+-K+-2Cl- cotransporter
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(NKCC1) of the shark rectal gland which was cloned in 1994 (18). It is now known that there are
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nine SLC12 family members in vertebrates (2; 4); two Na+-K+-2Cl- cotransporters (NKCC1 and
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NKCC2), a Na+-Cl- cotransporter (NCC), four K+-Cl- cotransporters (KCC1, KCC2, KCC3 and
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KCC4), and two additional proteins, commonly referred to as CIP and CCC9, whose function
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remains uncertain. In addition, SLC12 sequences have been identified in a number of lower
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species including crustaceans, insects, worms, plants, fungi and some bacteria.
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In vertebrates SLC12 family members are known to play important roles in numerous
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physiological processes including exocrine fluid secretion, renal salt and water absorption,
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hearing, olfaction, spermatogenesis, pain perception, visual processing and other neuronal
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functions (2; 4; 5; 8). However, little is known about the properties or significance of the SLC12
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proteins in lower species. There are five SLC12 homologues in the model organism Drosophila
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melanogaster including at least one member on each of the four main branches of the vertebrate
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phylogenetic tree. Our purpose here is to begin to characterize these Drosophila SLC12 proteins
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in order to understand their significance in Drosophila as well as to eventually better understand
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the properties of their vertebrate orthologues. We have used in situ hybridization to study the
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expression patterns of the Drosophila SLC12 family members during embryonic development. In
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addition we have explored their functional properties when they are expressed in the cultured
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insect cell line High Five. Interestingly one of these proteins, CG4357, was found to have the
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functional properties of a Na+-K+-2Cl- cotransporter with kinetic properties very similar to
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vertebrate NKCCs.
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MATERIALS AND METHODS
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Molecular Biology
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Molecular biological procedures employed standard methods. Ligations of PCR products
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were carried out using restriction sites incorporated into the PCR primers. The correctness of all
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clones used in the paper was confirmed by direct sequencing.
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Preparation of Drosophila SLC12 RNA probes
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Full length cDNA clones of the 5 Drosophila SLC12 proteins CG4357 (clone GH27027),
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CG31547-PB (clone GH09711), CG12773 (clone LD15480), CG10413 (clone GH08340) and
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CG5594-PB (clone GH09271) were purchased from the Drosophila Genomics Resource Center
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(DGRC). The complete coding sequences of CG4357, CG12773 and CG10413 were amplified by
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PCR and ligated into pBlueScript SK+ (Stratagene) between EcoRI and NotI, EcoRI and NotI,
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and XhoI and NotI, respectively. The nucleotide sequences corresponding to amino acids 93-1068
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of CG31547-PB and amino acids 31-898 of CG5594-PB were amplified by PCR and ligated into
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pBlueScript SK+ between EcoRI and NotI, and XhoI and NotI, respectively; these regions of
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CG31547 and CG5594 were common to all alternatively spliced versions of their respective
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genes. Riboprobes were synthesized using the DIG RNA labeling kit (Roche). To generate sense
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probes, plasmids were linearized with NotI and transcribed with T7 RNA polymerase, and to
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generate antisense probes plasmids were linearized with EcoRI or XhoI, as appropriate, and
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transcribed with T3 RNA polymerase.
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Whole mount in situ hybridization
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Embryos were collected at 6 and 18 hours after egg lay on grape juice agar plates then
dechorionated and fixed according to Tautz and Pfeifle (14). Briefly, embryos were
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dechorionated in 100% bleach for 1-3 min and then fixed in 2.5 ml 4% formaldehyde, 160 mM
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KCl, 40 mM NaCl, 4 mM EGTA and 30 mM Pipes. To this mixture, 2.5 ml heptane was added
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and the embryos were vortexed vigorously. The lower aqueous phase was removed and methanol
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was added. Fixed embryos were stored in methanol at -20oC or used immediately for in situ
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hybridization. For in situ hybridization embryos were treated with formaldehyde/PBT (PBS with
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0.1% Tween 20), followed by 3 µg/mL Proteinase K for 3-5 min and then fixed again with 4%
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formaldehyde/PBT. Hybridization was performed at 58oC overnight using 0.1 µg/ml DIG-labeled
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antisense or sense probes as described previously (16). Washing was performed using serial
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dilutions of the hybridization buffer in PBT. Embryos were then incubated with anti-DIG-AP
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antibody (Roche) (1:2000 in PBT) overnight at 4oC. Wash and color development were
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performed using PBT and BM Purple AP substrate (Roche). Embryos were equilibrated in 70%
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glycerol/PBT and stored at 4oC. Embryo staging was performed according to Hartenstein (6).
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Drosophila strains and crosses
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Crosses were performed using the following Drosophila melanogaster stocks obtained
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from the Bloomington Stock Center (Indiana University): #20377 (y1, w67c23;
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P{wHy}Ncc69DG04311) which contains a P-element transposon insertion in the 5’UTR of the
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CG4357 gene; and #5492 (w*; Df(3L)eygC1/TM3, P{ftz/lacC}SC1, ryRK Sb1 Ser1), which deletes
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the 69A4-69D6 region of chromosome 3L. The wild type strain used was Oregon R. All
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Drosophila crosses were performed at 25oC on MM media (KD Medical, Inc.).
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Quantitative PCR
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Parents and progeny from the Drosophila crosses were collected to quantitate CG4357
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gene expression using quantitative PCR (QPCR). DNase-free RNA and cDNA were prepared
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from five adult females of each genotype using the FastRNA Pro Green kit (Q-BIOgene) with
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DNase removal treatment (Ambion) and reverse transcription (Bio-Rad), according to the
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manufacturers’ instructions. Primers for quantitative PCR were designed using Beacon Designer
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software (Bio-Rad). Primer sequences used to detect CG4357 gene expression are:
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TACTCCCGCAGCCGCAAATACC (antisense), ACCAGCAGCAGGGCAACTCTC (sense).
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Quantitative PCR was performed for 40 cycles (95 °C for 10 seconds and 62 °C for 30 seconds)
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using SYBR-GREEN PCR Master Mix, 1 ng of each cDNA sample and a My IQ real time PCR
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thermocycler (Bio-Rad). Gene expression was normalized to 18S rRNA. Reactions were run in
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triplicate, and each experiment was repeated three times.
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Insect expression vectors for Drosophila SLC12 proteins
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The complete coding sequences, including their native stop codons, of CG4357,
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CG31547-PB, CG12773, CG10413, and CG5594-PB were amplified by PCR and ligated into the
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insect expression vector pIB/V5-His (Invitrogen) between HindIII and NotI (CG4357, CG31547-
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PB, CG10413 and CG5594-PB) or SacI and NotI (CG12773). A CG4357 clone without its native
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stop codon and in frame with the V5 C-terminal tag of the pIB/V5-His vector was also
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constructed. All clones included an upstream Kozak consensus sequence. pIB/V5-His-CAT, a
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control vector expressing V5-tagged chloramphenicol acetyltransferase (CAT) was used as a
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control in some experiments.
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Anti-CG4357 antibody production
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The nucleotide sequence corresponding to amino acids 899-1171 of CG4357 was
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amplified by PCR and ligated into the bacterial expression vector pQE30 (Qiagen) between
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BamHI and HindIII in frame with a 6xHis N-terminal tag. The resulting 6xHis fusion protein was
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expressed in E. coli and purified on Ni-NTA Agarose (Qiagen) following the manufacturer’s
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instructions. Anti-CG4357 antibody was raised in rabbits against this recombinant protein by
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Sigma-Genosys. The ability of the antibody to detect the CG4357 protein expressed in the insect
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cell line High Five was confirmed by Western blotting (see below).
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High Five cell culture, transfection and preparation of crude membranes
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The High Five insect cell line was grown in Express Five SFM medium supplemented
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with 25 µg/mL Gentamycin Sulfate and 2mM glutamine at 27oC (the High Five cells and all
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media were from Invitrogen). Cells were grown and maintained in 6 cm culture dishes and
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vigorously pipetted into suspension for splitting. High Five cells growing in 6 cm culture dishes
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were transfected with the plasmids indicated using Cellfection (Invitrogen) according to the
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manufacturer's instructions. Crude membranes were prepared from High Five cells transiently
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transfected with the plasmids indicated as previously described (9).
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Western Blotting and Confocal Microscopy
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SDS-polyacrylamide gel electrophoresis and Western blotting were carried out as
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previously described (12). The primary antibodies used were anti-CG4357 (dilution 1:5000) or an
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anti-V5 monoclonal (Invitrogen; dilution 1:1000). Horseradish peroxidase-conjugated secondary
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antibodies (Pierce) were used at a dilution of 1:10000 and detection was carried out using the
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ECL kit from Amersham Biosciences.
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For the confocal studies High Five cells transiently transfected with the plasmids
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indicated were re-plated onto glass coverslips. Immunostaining was carried out as previously
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described (10) using anti-CG4357 (dilution 1:600) or the anti-V5 monoclonal (dilution 1:100) as
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primary antibodies. The secondary antibodies (diluted 1:200) were Alexa Flour® 594 donkey
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anti-rabbit IgG (H+L) and Alexa Flour® 488 goat anti-mouse IgG (H+L), respectively (both from
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Invitrogen).
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Rb Flux Assay
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The following media were used in the flux experiments. "Preincubation medium"
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contained 50 mM NaCl, 4 mM CaCl2, 11 mM MgCl2, 15 mM Na-HEPES (pH 7.3 with NaOH), 5
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mM glucose and 170 mM sucrose. Unless indicated otherwise "uptake medium" was
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preincubation medium containing in addition 20 µM RbCl and ~0.3 µCi/mL 86Rb (Perkin Elmer).
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"Termination medium" was preincubation medium containing in addition 5 mM RbCl, and 250
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µM bumetanide. All media are ~340 mOs.
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The procedure for the 86Rb flux assays was similar to the one previously described from
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our laboratory for HEK-293 cells (1) with some modifications. High Five cells growing in 6 cm
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culture dishes were transfected with the plasmids indicated. The next day the plasmid was
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removed and the cells were re-plated into 24 well dishes in fresh culture medium. The flux assay
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was then carried out the following day as described below (variations in the flux protocol, if any,
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are noted in the figure legends). A row of four wells was used for each experimental point, three
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wells were used to measure 86Rb flux (i.e. fluxes were carried out in triplicate) and the fourth well
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was used to determine the protein/well using the BCA protein assay kit (Pierce). All of the wells
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were treated identically with the exception that 86Rb was omitted from the uptake medium in the
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fourth well. For each row of wells, the culture medium was removed, the wells were washed twice
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in preincubation medium (in every case, additions to the wells were 0.5 ml), and then incubated in
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preincubation medium for 40 min at room temperature (20 °C, all steps hereafter were carried out
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at room temperature). 86Rb uptake was initiated by replacing the preincubation medium with
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uptake medium. After 7 min of incubation, the uptake medium was removed and the well was
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washed 3 times with termination medium. Finally, a 0.5-ml aliquot of 1% Triton X-100 was
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added to each well, and samples were taken for liquid scintillation counting and protein
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determination. In control experiments (data not shown) we have verified that 86Rb uptake is linear
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with time for at least 7 min under the experimental conditions described here.
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Data Presentation
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All quantitative results are expressed as means ± S.E. for three or more independent
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experiments. Theoretical fits to the data were carried out by non-linear least squares regression
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using the program SigmaPlot.
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RESULTS
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Phylogenetic Analysis of the SLC12 Gene Family
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Blast searches of the Drosophila melanogaster genome reveal five members of the
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SLC12 gene family: CG4357 (also called Ncc69), CG31547 (occurring in two alternatively
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spliced versions, CG31547-PA and CG31547-PB), CG12773, CG10413, and CG5594 (occurring
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in four alternatively spliced versions, CG5594-PA, CG5594-PB, CG5594-PC, and CG5594-PD).
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Fig. 1 shows a phylogenetic tree of SLC12 family members from human (shaded in gray),
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Drosophila melanogaster (in bold), C. elegans (C el 1 through 6) and several other representative
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species (see Figure caption for further details). It can be seen that the vertebrate (human) family
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members lie on 4 main branches: one containing the sodium-dependent transporters NKCC1,
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NKCC2 and NCC; one containing the potassium-dependent transporters KCC1, KCC2, KCC3
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and KCC4; and two other branches, one containing CIP and the other CCC9, both of whose
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function is presently unknown. There is at least one Drosophila orthologue and one C. elegans
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orthologue on each of these four branches.
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Expression patterns of the SLC12 family during Drosophila embryogenesis
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To determine the temporal and spatial expression pattern of members of the SLC12 gene
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family during Drosophila development, we performed whole mount in situ hybridization using
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non-radioactive DIG-labeled RNA probes. In general, members of the SLC12 gene family
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exhibited unique expression patterns during Drosophila embryonic development, suggesting
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distinct biological roles for the membrane proteins they encode.
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Expression of CG4357 was first detectable during early embryonic stages 1-3 (Fig. 2).
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Expression at these stages usually represents maternal RNA contributed to the oocyte by the
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mother. CG4357 expression was then detected at the cellular blastoderm stages (stages 4-6),
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when individual cells surround the central yolk sac and zygotic expression is first observed (Fig.
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2). As development proceeds, three germ layers are generated during gastrulation (stages 6-8),
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followed by germ band elongation at stages 9-11 and germ band retraction at stages 12-13.
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CG4357 expression was seen in the developing gut and nervous system during germ band
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elongation and retraction stages (stages 9-13; Fig. 2). During stages 14-15, which represents the
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completion of a number of different morphogenic movements, CG4357 expression is detected
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predominantly in the foregut and anterior region of the developing embryo. The expression
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pattern of CG4357 persists through stages 16-17, during which organ system differentiation is
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completed just prior to the onset of larval development.
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CG12773 showed patterns of expression similar to those of CG4357 during
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embryogenesis (Fig. 3). CG12773 was expressed broadly during stages 1-6, followed by
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expression in the developing gut and nervous system throughout most of the later stages of
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embryonic development (stages 9-17).
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CG5594 displayed maternal expression at stages 1-3, followed by expression during
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cellular blastoderm (stages 4-6) (Fig. 4).
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hindgut, anal pads and the ventral nerve cord were seen during stages 12-13. Expression in the
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hindgut, anal pads and nervous system continued throughout stages 14-17.
Expression in the anterior and posterior midgut,
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CG31547 was expressed during stages 1-6 (Fig. 5). At stages 9-11, expression was seen
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in the developing anterior and posterior midgut and the hindgut. At stages 12-13, expression of
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CG31547 was seen in the nervous system and gut, with very intense expression in the anal pads
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(Fig. 4). At later stages (14-17), expression was most intense in the salivary glands and the anal
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pads.
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Finally, expression of CG10413 was distinct from that seen for other family members.
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Maternally contributed CG10413 transcripts were seen during stages 1-3, followed by expression
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during cellular blastoderm (stages 4-6) (Fig. 6). Thereafter, specific expression of CG10413
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during later stages of embryogenesis was not detected.
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Functional activity
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We tested for functional activity of the five Drosophila SLC12 family members by
measuring the uptake of 86Rb into High Five cells transiently transfected with CG4357,
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CG31547-PB, CG12773, CG10413, and CG5594-PB, each cloned into the insect expression
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vector pIB/V5-His (see Methods). Rubidium is a potassium congener that is known to substitute
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for potassium on a number of biological membrane transporters, including the vertebrate KCCs
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and NKCCs. For this reason we felt that 86Rb transport was an appropriate first functional test of
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the Drosophila SLC12 family. As shown in Fig. 7, under our experimental conditions only the
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protein CG4357 was found to exhibit 86Rb transport levels significantly above those seen with the
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empty vector pIB/V5-His.
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Characterization of CG4357 expression in High Five cells
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In order to further characterize the CG4357 protein we first examined its expression and
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localization in the High Five cells. In Western blots anti-CG4357, an antibody raised against the
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C-terminal 273 residues of CG4357 (see Methods), showed no reaction with membranes prepared
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from High Five cells transfected with the empty vector pIB/V5-His, but recognized a single
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protein of molecular weight ~130 kD in membranes prepared from High Five cells transfected
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with either CG4357 or CG4357 tagged with the V5 epitope (Fig. 8A; the predicted molecular
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weight of CG4357 is 129 kDa). A protein of the same molecular weight was recognized by an
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anti-V5 antibody in cells transfected with V5-tagged CG4357 (Fig. 8A), confirming the
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specificity of the anti-CG4357 antibody.
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Fig. 8B shows confocal micrographs of High Five cells transfected with the empty vector
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pIB/V5-His (panel a), pIB/V5-His-CAT, a control vector expressing the cytosolic protein V5-
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tagged chloramphenicol acetyltransferase (panel b), CG4357 (panel c), and V5-tagged CG4357
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(panel d). All cells were simultaneously probed with both anti-V5 (green) and anti-CG4357 (red)
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antibodies. It is clear from panels c and d that both CG4357 and V5-tagged CG4357 are localized
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at or very near the plasma membrane of the High Five cells (the yellow staining in panel d is
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presumably due to the labeling of V5-tagged CG4357 by both anti-CG4357 and anti-V5
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antibodies). In Fig. 8C we examined the transport of 86Rb in High Five cells transiently
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transfected as in Fig. 8B. Interestingly, although both CG4357 and V5-tagged CG4357 appear to
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be trafficked to the plasma membrane of the High Five cells (Fig. 8B), only cells expressing the
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untagged protein exhibit CG4357-dependent 86Rb transport activity.
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Characterization of transport via CG4357 in High Five cells
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We next examined the properties of 86Rb flux via CG4357. Because the High Five cells
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exhibit significant endogenous rubidium transport we always measured 86Rb uptake in both cells
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transfected with empty vector (pIB/V5-His) and cells transfected with CG4357 on the same day
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and under the same experimental conditions. Specific fluxes via CG4357 could then be
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determined by subtracting the fluxes observed from cells transfected with empty vector. In Fig.
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9A we illustrate the rubidium concentration dependence of 86Rb uptake into High Five cells
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transfected with empty vector. This endogenous flux is clearly saturable and analysis of these
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data via non-linear least squares regression reveals an excellent fit to the Michaelis-Menten
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equation indicating the presence of a single endogenous rubidium transport system in the High
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Five cells with Km = 2.56+0.15 mM (see Fig. 9 caption). The flux induced by expression of
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CG4357 is likewise saturable and also conforms well to Michaelis-Menten kinetics with Km =
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0.29+0.05 mM (Fig. 9 caption).
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In Fig. 10 we show the sodium concentration dependence of 86Rb uptake into High Five
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cells transfected with empty vector (panel A) and CG4357 (panel B). No significant sodium
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dependence is seen for the endogenous High Five rubidium transport system (panel A) but the
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flux via CG4357 is clearly sodium-dependent (panel B) and conforms well to the Michaelis-
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Menten equation with K1/2 = 53+11 mM (Fig. 10 caption).
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The effect of chloride concentration is illustrated in Fig. 11. The endogenous High Five
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transport system shows a saturable dependence on chloride (panel A) which can be modeled by
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the Michaelis-Menten equation with K1/2 = 80+13 mM (see figure caption). In contrast the
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dependence of rubidium transport via CG4357 on chloride concentration is clearly sigmoidal
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(panel B) indicating the involvement of more than one chloride ion in the rubidium transport
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event. These latter data conform well to the Hill equation with K1/2 = 48+5 mM and n = 2.6+0.3
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(see Fig. 11 caption), where n is a measure of the Cl:Rb stoichiometry (17).
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Effects of SLC12 inhibitors
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In Fig. 12 we examine the effects of the SLC12 inhibitors bumetanide (white bars), a
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specific inhibitor of NKCCs, furosemide (dark gray bars), an inhibitor of NKCCs and KCCs, and
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metolazone (light gray bars), a specific inhibitor of NCCs; in each case fluxes have been
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normalized to that observed in the absence of inhibitors (black bars). All three of the compounds
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tested are employed clinically as diuretics in humans. These data indicate that metolazone has no
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significant inhibitory effect on either endogenous High Five 86Rb transport (panel A) or on 86Rb
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transport via GC4357 (panel B). On the other hand, furosemide and bumetanide inhibit both
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transporters with bumetanide appearing to be somewhat more effective on the endogenous system
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and dramatically more effective on CG4357. The bumetanide dose response for inhibition of
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CG4357 is shown in Fig. 13. These data indicate that the KI for bumetanide inhibition of CG4357
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is 1.17+0.08 μM (see figure caption), a value very similar to that found for vertebrate NKCC1s
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(1; 7).
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Loss of CG4357 gene expression does not affect viability
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To further investigate the role of the SLC12 family member CG4357 during Drosophila
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development, we generated flies deficient in CG4357 expression by crossing a Drosophila strain
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that contains a P-element transposon insertion mutation in the 5’UTR of CG4357
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(P{wHy}Ncc69DG04311) and a strain that contains a chromosomal deletion that encompasses
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CG4357 (Df(3L)). Progeny from this cross that were heterozygous for either the transposon
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(P{wHy}Ncc69DG04311/+) or the deletion (Df(3L)/+) were viable and had an approximately 50%
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reduction in the level of CG4357 expression relative to wild type as determined by QPCR (Fig.
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14). This indicates that both the transposon and the deletion decrease CG4357 expression.
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Progeny from this cross that contained both the transposon insertion and the chromosomal
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deletion together (P{wHy}Ncc69DG04311/Df(3L)) had greatly reduced CG4357 expression relative
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to wild type (Fig. 14). However, no significant decrease in viability relative to their heterozygous
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siblings was observed (data not shown).
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DISCUSSION
The long-term goal of our studies is to understand the developmental and functional roles
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of the cation-chloride-coupled cotransporter gene family SLC12 in the model organism
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Drosophila melanogaster. We anticipate that this information will not only lead to a better
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appreciation of the significance of these proteins in Drosophila but also to increased
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understanding of their vertebrate orthologues. Here we employed in situ hybridization to study
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the expression patterns of the five Drosophila SLC12 family members CG4357, CG12773,
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CG5594, CG31547, and CG10413 during embryonic development (Figs. 2-6). We found that all
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five members of this family are expressed during early embryogenesis (stages 1-6). Specific
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expression of CG10413 during later stages of embryogenesis was not detected. Expression of the
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remaining family members during late embryogenesis varied but was seen predominantly in the
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ventral nerve cord, salivary gland, gut and anal pad. In parallel studies we carried out functional
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assays on each of the five Drosophila homologues by measuring the uptake of 86Rb into
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transiently transfected High Five cells. Rubidium is known to be able to substitute for potassium
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on a number of vertebrate SLC12 transporters, but under our experimental conditions we found
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that only one of the Drosophila SLC12 proteins, CG4357, exhibited significant 86Rb transport
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activity (Fig. 7).
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In confocal micrographs we found that CG4357 was expressed primarily in, or very close
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to, the plasma membrane of the High Five cells (Fig. 8B). In additional experiments we found
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that 86Rb transport via CG4357 was saturable with Km = 0.29+0.05 mM (Fig. 9B). 86Rb flux via
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CG4357 was also sodium- and chloride-dependent. A plot of 86Rb uptake vs. sodium
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concentration conformed well to the Michaelis-Menten equation with K1/2 = 53+11 mM (Fig.
352
10B) indicating a Na:Rb stoichiometry of 1:1 (17). In contrast, a plot of 86Rb flux via CG4357 vs.
353
chloride concentration was sigmoidal indicating the involvement of more than one chloride ion in
354
the Rb transport event (Fig. 11B). A fit of these data to the Hill equation yielded K1/2 = 48+5 mM
355
and n = 2.6+0.3 suggesting a Cl:Rb stoichiometry of > 2:1. 86Rb flux via CG4357 was also
17
356
inhibited by bumetanide (KI = 1.17+0.08 μM; Figs. 12 and 13), a potent and specific inhibitor of
357
vertebrate Na+-K+-2Cl- cotransporters. In previous studies from our laboratory carried out on rat
358
NKCC1 expressed in the human embryonic kidney cell line HEK-293 (1) we found Km = 1.85
359
mM+0.26 mM for rubidium, K1/2 = 60.8+4.0 mM for sodium, K1/2 = 48.1+4.7 mM with n =
360
2.47+0.45 for chloride, and K1/2 = 2.4+0.7 μM for bumetanide. Thus the kinetic parameters for
361
CG4357 and rat NKCC1 are quite similar, the most notable difference being the higher affinity of
362
CG4357 for rubidium. Taken together these results provide strong evidence that CG4357 is an
363
insect orthologue of the vertebrate Na+-K+-2Cl- cotransporters.
364
The functional properties of the remaining four Drosophila SLC12 proteins have yet to
365
be determined. We note, however, that the endogenous 86Rb transport system that we observe in
366
the High Five cells is saturable (Fig. 9A), sodium-independent (Fig. 10A), chloride-dependent
367
(Fig. 11A) and furosemide-dependent (Fig. 12A), and thus appears to be a K+-Cl- cotransporter. It
368
is possible that corresponding Drosophila protein (possibly CG5594 which lies closest to the
369
vertebrate KCCs phylogenetically – see Fig. 1) is inactive in the High Five cells under our
370
experimental conditions or that an SLC12 homologue is found in the High Five cells (derived
371
from the cabbage looper, Trichoplusia ni) that is not found in Drosophila. Further studies to
372
explore these questions were beyond the scope of the present paper.
373
Expression of CG4357, the one SLC12 family member for which transporter activity was
374
detected, was seen to change throughout Drosophila development, indicating dynamic temporal
375
regulation of this gene (Fig. 2). Upon organ formation, CG4357 expression was found
376
predominantly in the gut and nervous system, suggesting a role for this transporter in the ionic
377
homeostasis of these developing organ systems. Indeed, the gut, anal pads and malpighian
378
tubules are intimately involved in maintaining the proper ionic balance in insects, suggesting that
379
expression of transporters in these tissues may be crucial for both development and homeostasis.
380
Support for this is seen when examining the expression patterns of other members of the SLC12
381
family. Three additional family members (CG31547, CG12773 and CG5594) are also expressed
18
382
in the gut and two of these (CG31547 and CG5594) have very intense expression in the anal pad,
383
a structure present at the posterior end of the hindgut that is involved in regulating salt and water
384
balance. Multiple family members are also expressed in the developing nervous system. Taken
385
together, our data suggest that members of this transporter family are likely to be involved in the
386
ion transport and osmoregulation that occurs in the digestive system of the insect as well as in
387
nervous system function. Finally, the expression of multiple family members in the gut and
388
nervous system also suggests that there may be functional redundancy built into these organ
389
systems. The potential for functional redundancy within this family is supported by our genetic
390
experiments examining mutations in CG4357. Flies that are homozygous mutants for CG4357
391
were viable and fertile. While these data suggest that CG4357 is not required for viability, it
392
remains possible that subtle defects in ionic concentrations, digestion or nervous system function
393
may still be present. Interestingly, Filippov et al. (3) also found that disruption of another family
394
member, CG10413, did not produce an obvious phenotype or affect viability. Future studies will
395
be required to examine the effects of mutations in multiple co-expressed family members to
396
address the issue of redundancy.
397
19
398
REFERENCES
399
400
401
Reference List
1. Dehaye JP, Nagy A, Premkumar A and Turner RJ. Identification of a
402
functionally important conformation-sensitive region of the secretory Na+-K+-2Cl-
403
cotransporter (NKCC1). J Biol Chem 278: 11811-11817, 2003.
404
405
406
2. Delpire E and Mount DB. Human and murine phenotypes associated with defects
in cation-chloride cotransport. Annu Rev Physiol 64: 803-843, 2002.
3. Filippov V, Aimanova K and Gill SS. Expression of an Aedes aegypti cation-
407
chloride cotransporter and its Drosophila homologues. Insect Mol Biol 12: 319-331,
408
2003.
409
410
4. Gamba G. Molecular physiology and pathophysiology of electroneutral cationchloride cotransporters. Physiol Rev 85: 423-493, 2005.
411
5. Gavrikov KE, Nilson JE, Dmitriev AV, Zucker CL and Mangel SC. Dendritic
412
compartmentalization of chloride cotransporters underlies directional responses of
413
starburst amacrine cells in retina. Proc Natl Acad Sci U S A 103: 18793-18798,
414
2006.
415
416
6. Hartenstein V.In: Atlas of Drosophila Development, New York: Cold Spring
Harbour Laboratory Press, 1993, p. 2-52.
20
417
7. Isenring P, Jacoby SC, Payne JA and Forbush B, III. Comparison of Na-K-Cl
418
cotransporters. NKCC1, NKCC2, and the HEK cell Na- L-Cl cotransporter. J Biol
419
Chem 273: 11295-11301, 1998.
420
421
8. Kaneko H, Putzier I, Frings S, Kaupp UB and Gensch T. Chloride accumulation
in mammalian olfactory sensory neurons. J Neurosci 24: 7931-7938, 2004.
422
9. Moore-Hoon ML and Turner RJ. The structural unit of the secretory Na+-K+-
423
2Cl- cotransporter (NKCC1) is a homodimer. Biochemistry 39: 3718-3724, 2000.
424
10. Nezu A, Parvin MN and Turner RJ. A conserved hydrophobic tetrad near the C
425
terminus of the secretory Na+-K+-2Cl- cotransporter (NKCC1) is required for its
426
correct intracellular processing. J Biol Chem 284: 6869-6876, 2009.
427
428
429
11. Page RD. TreeView: an application to display phylogenetic trees on personal
computers. Comput Appl Biosci 12: 357-358, 1996.
12. Parvin MN, Gerelsaikhan T and Turner RJ. Regions in the cytosolic C-terminus
430
of the secretory Na(+)-K(+)-2Cl(-) cotransporter NKCC1 are required for its
431
homodimerization. Biochemistry 46: 9630-9637, 2007.
432
433
13. Russell JM. Sodium-potassium-chloride cotransport. Physiol Rev 80: 211-276,
2000.
21
434
14. Tautz D and Pfeifle C. A non-radioactive in situ hybridization method for the
435
localization of specific RNAs in Drosophila embryos reveals translational control of
436
the segmentation gene hunchback. Chromosoma 98: 81-85, 1989.
437
15. Thompson JD, Higgins DG and Gibson TJ. CLUSTAL W: improving the
438
sensitivity of progressive multiple sequence alignment through sequence weighting,
439
position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:
440
4673-4680, 1994.
441
16. Tian E and Ten Hagen KG. Expression of the UDP-GalNAc: polypeptide N-
442
acetylgalactosaminyltransferase family is spatially and temporally regulated during
443
Drosophila development. Glycobiology 16: 83-95, 2006.
444
445
446
17. Turner RJ. Stoichiometry of coupled transport systems in vesicles. Methods
Enzymol 191: 479-494, 1990.
18. Xu JC, Lytle C, Zhu TT, Payne JA, Benz E Jr and Forbush B, III. Molecular
447
cloning and functional expression of the bumetanide-sensitive Na-K-Cl
448
cotransporter. Proc Natl Acad Sci U S A 91: 2201-2205, 1994.
449
450
22
451
FIGURE LEGENDS
452
Fig. 1. Phylogenetic tree of representative members of the SLC12 family. Amino acid sequences
453
were aligned using the program AlignX (a component of the Vector NTI Advance package from
454
Invitrogen) which uses the Clustal W algorithm (15). The tree was displayed using the program
455
Treeview (11). Human sequences are shaded in gray and indicate the 4 main branches of the
456
vertebrate phylogenetic tree. Drosophila SLC12 family members are shown in bold. In addition
457
to the Drosophila proteins, the SLC12 amino acid sequences included are Crab (Callinectes
458
sapidus; AF190129), C el 1 (Caenorhabditis elegans; T22488), C el 2 (Caenorhabditis elegans;
459
NP_501141), C el 3 (Caenorhabditis elegans; H16O14), C el 4 (Caenorhabditis elegans;
460
NP_495469), C el 5 (Caenorhabditis elegans; P34261), C el 6 (Caenorhabditis elegans;
461
NP_495555), C el 7 (Caenorhabditis elegans; NP_493773), Cyanobacterium (Trichodesmium
462
erythraeum; ZP_00074455), CCC9 (human; AAO49174), CIP (human; AAK21008), KCC1
463
(human; NP_005063), KCC2 (human; NP_065759), KCC3 (human; AAF24986), KCC4 (human;
464
AAD39741), NCC (human; NP_000330), NKCC1 (human; NP_001037), NKCC2 (human;
465
AAB07364), Archaea (Methanosarcina acetivorans; NP_619366), Rice (Oryza sativa;
466
BAB20646), and Yeast (Saccharomyces cerevisiae; NP_009794).
467
Fig. 2. CG4357 gene expression during Drosophila embryogenesis by whole mount in situ
468
hybridization. Expression patterns using antisense riboprobes and sense riboprobes are shown.
469
Orientation of the embryo is anterior to the left, posterior to the right. Dorsal is up in lateral
470
images. amg, anterior midgut rudiment; fg, foregut; pmg, posterior midgut rudiment; vne, ventral
471
neurogenic region; vnc, ventral nerve cord.
472
Fig. 3. CG12773 gene expression during Drosophila embryogenesis by whole mount in situ
473
hybridization. Expression patterns using antisense riboprobes and sense riboprobes are shown.
474
Orientation of the embryo is anterior to the left, posterior to the right. Dorsal is up in lateral
475
images. amg, anterior midgut rudiment; pmg, posterior midgut rudiment; hg, hindgut; vne, ventral
476
neurogenic region; vnc, ventral nerve cord.
23
477
Fig. 4. CG5594 gene expression during Drosophila embryogenesis by whole mount in situ
478
hybridization. Expression patterns using antisense riboprobes and sense riboprobes are shown.
479
Orientation of the embryo is anterior to the left, posterior to the right. Dorsal is up in lateral
480
images. amg, anterior midgut rudiment; pmg, posterior midgut rudiment; hg, hindgut; ap, anal
481
pads; vnc, ventral nerve cord.
482
Fig. 5. CG31547 gene expression during Drosophila embryogenesis by whole mount in situ
483
hybridization. Expression patterns using antisense riboprobes and sense riboprobes are shown.
484
Orientation of the embryo is anterior to the left, posterior to the right. Dorsal is up in lateral
485
images. amg, anterior midgut rudiment; pmg, posterior midgut rudiment; hg, hindgut; ap, anal
486
pads; sg, salivary glands; vnc, ventral nerve cord.
487
Fig. 6. CG10413 gene expression during Drosophila embryogenesis by whole mount in situ
488
hybridization. Expression patterns using antisense riboprobes and sense riboprobes are shown.
489
Orientation of the embryo is anterior to the left, posterior to the right. Dorsal is up in lateral
490
images.
491
Fig. 7. Uptake of 86Rb into High Five cells transiently transfected with the Drosophila SLC12
492
proteins indicated. The Drosophila SLC12 proteins were cloned into the insect expression vector
493
pIB/V5-His and transfected into the High Five cells as described in Methods. All clones included
494
their native stop codons so that the C-terminal V5-His tag of the pIB/V5-His vector was not
495
appended to the proteins. 86Rb uptake was measured as described in Methods except that 20 µM
496
unlabeled RbCl was omitted from the uptake medium so that only tracer 86Rb was present. The
497
results of 3 independent experiments were averaged to produce the figure. In each experiment
498
fluxes were normalized to that observed in cells transfected with the empty vector pIB/V5-His
499
(empty).
500
Fig. 8. Expression, location and function of CG4357 and V5-tagged CG4357 in the cultured
501
insect cell line High Five. A. Western blots of membranes prepared from High Five cells
502
transiently transfected with the empty vector pIB/V5-His (empty), CG4357, or V5-tagged
24
503
CG4357, as indicated. The left-hand-panel was probed with the anti-CG4357 antibody and the
504
right-hand-panel with anti-V5 antibody (see Methods for details). B. Confocal images of High
505
Five cells transiently transfected with the empty vector pIB/V5-His (panel a), pIB/V5-His-CAT
506
(panel b), CG4357 (panel c), and V5-tagged CG4357 (panel d). All cells were prepared as
507
described in Methods and simultaneously probed with both anti-V5 (green) and anti-CG4357
508
(red) antibodies. C. 86Rb fluxes measured in High Five cells transiently transfected with the same
509
plasmids used in panel B. Fluxes, measured as described in Methods, have been normalized to
510
that observed in cells transfected with the empty vector pIB/V5-His. The results of 3 independent
511
experiments were averaged to produce the figure.
512
Fig. 9. Rubidium concentration dependence of 86Rb fluxes via the endogenous High Five cell
513
transport system and via CG4357. 86Rb fluxes were measured as a function of rubidium
514
concentration in High Five cells transiently transfected with the empty vector pIB/V5-His (panel
515
A) or with CG4357 (panel B) as indicated. Other experimental details are as described in
516
Methods. Each data point represents the average + S.E. for 3 or more independent determinations.
517
Fluxes in panel B have been corrected for 86Rb transport via the endogenous High Five system
518
(panel A) by subtracting uptakes measured in cells transfected with empty vector from those
519
measured in cells transfected with CG4357 for each experimental condition (measurements were
520
carried out on the same cell passage on the same day). Fluxes were fit to the Michaelis-Menton
521
equation via non-linear least squares regression. The fit to the data in panel A yielded Km =
522
2.56+0.15 mM with r = 0.999 while that to the data in panel B yielded Km = 0.29+0.05 mM with r
523
= 0.990. The lines drawn through the data points correspond to these theoretical fits.
524
Fig. 10. Sodium concentration dependence of 86Rb fluxes via the endogenous High Five cell
525
transport system and via CG4357. 86Rb fluxes were measured as a function of sodium
526
concentration in High Five cells transiently transfected with the empty vector pIB/V5-His (panel
527
A) or with CG4357 (panel B) as indicated. In these experiments Na-HEPES (pH 7.3) was
528
replaced with N-methyl-D-glucamine-HEPES (pH 7.3) in all media. To obtain an uptake medium
25
529
containing 100 mM NaCl, 100 mM sucrose was replaced with an additional 50 mM NaCl (see
530
Methods for composition of uptake medium). The ionic strength of the uptake medium was then
531
held constant by replacing sodium with N-methyl-D-glucamine to obtain the various sodium
532
concentrations indicated. Other experimental details are as described in Methods. The fluxes in
533
panel B have been corrected for 86Rb transport via the endogenous High Five system (panel A) as
534
described in the caption to Fig. 9. The results of 3 independent experiments were averaged to
535
produce the figures. In each experiment fluxes were normalized to those observed at 100 mM
536
NaCl. The dashed line in panel A indicates a relative flux of 1.0 (i.e., no dependence on sodium
537
concentration). The data in panel B were fit to the Michaelis-Menton equation via non-linear least
538
squares regression yielding K1/2 = 53+11 mM with r = 0.998. The line drawn through the data in
539
panel B corresponds to this theoretical fit.
540
Fig. 11. Chloride concentration dependence of 86Rb fluxes via the endogenous High Five cell
541
transport system and via CG4357. 86Rb fluxes were measured as a function of chloride
542
concentration in High Five cells transiently transfected with the empty vector pIB/V5-His (panel
543
A) or with CG4357 (panel B) as indicated. To obtain the chloride concentrations indicated while
544
keeping the ionic strength of the uptake medium (normally 80 mM chloride; see Methods)
545
constant, chloride was replaced with gluconate. Other experimental details are as described in
546
Methods. The fluxes in panel B have been corrected for 86Rb transport via the endogenous High
547
Five system (panel A) as described in the caption to Fig. 9. In each data set fluxes were
548
normalized to those observed at 80 mM chloride. The results of 3 independent experiments were
549
averaged to produce the figures. The data in panel A were fit to the Michaelis-Menton equation
550
via non-linear least squares regression yielding K1/2 = 80+13 mM with r = 0.998. The data in
551
panel B were fit to the Hill equation yielding K1/2 = 48+5 mM and n = 2.6+0.3 with r = 0.998.
552
The lines drawn through the data points correspond to these theoretical fits.
553
Fig. 12. Effects of SLC12 inhibitors on 86Rb fluxes via the endogenous High Five cell transport
554
system and via CG4357. 86Rb fluxes were measured in the presence of bumetanide (white bars),
26
555
furosemide (dark gray bars) or metolazone (light gray bars), at the concentrations indicated, in
556
High Five cells transiently transfected with the empty vector pIB/V5-His (panel A) or with
557
CG4357 (panel B). Fluxes have been normalized to that observed in the absence of inhibitors
558
(black bars). Cells were incubated with the inhibitors in preincubation medium for 10 min before
559
the flux measurements; other experimental details are as described in Methods. The fluxes in
560
panel B have been corrected for 86Rb transport via the endogenous High Five system (panel A) as
561
described in the caption to Fig. 9. Each data point represents the average + S.E. for 3 independent
562
determinations.
563
Fig. 13. CG4357 bumetanide dose response. 86Rb fluxes were measured in High Five cells
564
transiently transfected with CG4357 in the presence of bumetanide at the concentrations indicated.
565
Experimental details are as in the caption of Fig. 12. Fluxes were corrected for 86Rb transport via
566
the endogenous High Five system as described in the caption to Fig. 9 and then normalized to that
567
observed in the absence of bumetanide. The data were fit to a model that assumes a single
568
inhibitory site for bumetanide. This fit yields KI = 1.17+0.08 μM for bumetanide with r = 0.995.
569
The line drawn through the data points corresponds to this theoretical fit.
570
Fig. 14. CG4357 gene expression in Drosophila lines containing mutations in CG4357.
571
Quantitative PCR analysis of CG4357 gene expression was performed using the primer pairs
572
described in Materials and Methods. CG4357 gene expression levels were determined for wild
573
type flies (WT), flies heterozygous for the transposon insertion mutation
574
(P{wHy}Ncc69DG04311/+), flies heterozygous for the chromosomal deletion encompassing
575
CG4357 (Df(3L)/+) and flies that were homozygous mutants for CG4357, containing both the
576
transposon insertion and the chromosomal deletion (P{wHy}Ncc69DG04311/Df(3L)). RNA levels
577
were normalized to 18S rRNA. Values shown are the average of one experiment performed in
578
triplicate + standard deviation.
579
27
580
ACKNOWLEDGEMENTS
581
We thank Drs. William D. Swaim, Marilyn Moore-Hoon and Most. Nahid Parvin for advice and
582
help with the confocal and flux studies. This research was supported by the Intramural Research
583
Program of the National Institute of Dental and Craniofacial Research, National Institutes of
584
Health, Bethesda MD.