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Subdued, a TMEM16 family Ca²⁺-activated Cl⁻channel in Drosophila melanogaster with an unexpected role in host defense.

Wong XM, Younger S, Peters CJ, Jan YN, Jan LY - Elife (2013)

Bottom Line: Whether distant relatives of the vertebrate TMEM16 families also form CaCCs is an intriguing open question.Amino acid substitutions of Subdued alter the ion selectivity and kinetic properties of the CaCC channels heterologously expressed in HEK 293T cells.This Drosophila channel displays characteristics of classic CaCCs, thereby providing evidence for evolutionarily conserved biophysical properties in the TMEM16 family.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco, San Francisco, United States.

ABSTRACT
TMEM16A and TMEM16B are calcium-activated chloride channels (CaCCs) with important functions in mammalian physiology. Whether distant relatives of the vertebrate TMEM16 families also form CaCCs is an intriguing open question. Here we report that a TMEM16 family member from Drosophila melanogaster, Subdued (CG16718), is a CaCC. Amino acid substitutions of Subdued alter the ion selectivity and kinetic properties of the CaCC channels heterologously expressed in HEK 293T cells. This Drosophila channel displays characteristics of classic CaCCs, thereby providing evidence for evolutionarily conserved biophysical properties in the TMEM16 family. Additionally, we show that knockout flies lacking subdued gene activity more readily succumb to death caused by ingesting the pathogenic bacteria Serratia marcescens, suggesting that subdued has novel functions in Drosophila host defense. DOI: http://dx.doi.org/10.7554/eLife.00862.001.

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Drosophila guts express the subdued gene.RNA extraction was performed on four whole dissected guts and subdued expression was determined by performing reverse transcription and quantitative PCR (A, n = 3, p<0.001), using KO guts as a control. For further verification, a different primer set was used to perform regular PCR using the same cDNA as a template (B).DOI:http://dx.doi.org/10.7554/eLife.00862.010
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fig5s3: Drosophila guts express the subdued gene.RNA extraction was performed on four whole dissected guts and subdued expression was determined by performing reverse transcription and quantitative PCR (A, n = 3, p<0.001), using KO guts as a control. For further verification, a different primer set was used to perform regular PCR using the same cDNA as a template (B).DOI:http://dx.doi.org/10.7554/eLife.00862.010

Mentions: Wild-type flies and two knockout strains generated from independent crosses, KO2 and KO11, were fed Db11 mixed with sucrose solution, and their survival monitored for 8 days, as in previous studies of fly immunity (Cronin et al., 2009). Confirming the susceptibility phenotype previously reported, the knockout strains had significantly higher lethality upon being fed Db11 (Figure 5A). Over the same timescale as the infection experiment, UV-killed Db11 did not bring about early lethality in any of the strains (Figure 5—figure supplement 1). We also determined that subdued mRNA was indeed expressed in the gut of wild-type flies but not the genetic knockout strains (Figure 5—figure supplement 3).10.7554/eLife.00862.007Figure 5.Subdued plays a role in host defense in Drosophila melanogaster.


Subdued, a TMEM16 family Ca²⁺-activated Cl⁻channel in Drosophila melanogaster with an unexpected role in host defense.

Wong XM, Younger S, Peters CJ, Jan YN, Jan LY - Elife (2013)

Drosophila guts express the subdued gene.RNA extraction was performed on four whole dissected guts and subdued expression was determined by performing reverse transcription and quantitative PCR (A, n = 3, p<0.001), using KO guts as a control. For further verification, a different primer set was used to perform regular PCR using the same cDNA as a template (B).DOI:http://dx.doi.org/10.7554/eLife.00862.010
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814000&req=5

fig5s3: Drosophila guts express the subdued gene.RNA extraction was performed on four whole dissected guts and subdued expression was determined by performing reverse transcription and quantitative PCR (A, n = 3, p<0.001), using KO guts as a control. For further verification, a different primer set was used to perform regular PCR using the same cDNA as a template (B).DOI:http://dx.doi.org/10.7554/eLife.00862.010
Mentions: Wild-type flies and two knockout strains generated from independent crosses, KO2 and KO11, were fed Db11 mixed with sucrose solution, and their survival monitored for 8 days, as in previous studies of fly immunity (Cronin et al., 2009). Confirming the susceptibility phenotype previously reported, the knockout strains had significantly higher lethality upon being fed Db11 (Figure 5A). Over the same timescale as the infection experiment, UV-killed Db11 did not bring about early lethality in any of the strains (Figure 5—figure supplement 1). We also determined that subdued mRNA was indeed expressed in the gut of wild-type flies but not the genetic knockout strains (Figure 5—figure supplement 3).10.7554/eLife.00862.007Figure 5.Subdued plays a role in host defense in Drosophila melanogaster.

Bottom Line: Whether distant relatives of the vertebrate TMEM16 families also form CaCCs is an intriguing open question.Amino acid substitutions of Subdued alter the ion selectivity and kinetic properties of the CaCC channels heterologously expressed in HEK 293T cells.This Drosophila channel displays characteristics of classic CaCCs, thereby providing evidence for evolutionarily conserved biophysical properties in the TMEM16 family.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco, San Francisco, United States.

ABSTRACT
TMEM16A and TMEM16B are calcium-activated chloride channels (CaCCs) with important functions in mammalian physiology. Whether distant relatives of the vertebrate TMEM16 families also form CaCCs is an intriguing open question. Here we report that a TMEM16 family member from Drosophila melanogaster, Subdued (CG16718), is a CaCC. Amino acid substitutions of Subdued alter the ion selectivity and kinetic properties of the CaCC channels heterologously expressed in HEK 293T cells. This Drosophila channel displays characteristics of classic CaCCs, thereby providing evidence for evolutionarily conserved biophysical properties in the TMEM16 family. Additionally, we show that knockout flies lacking subdued gene activity more readily succumb to death caused by ingesting the pathogenic bacteria Serratia marcescens, suggesting that subdued has novel functions in Drosophila host defense. DOI: http://dx.doi.org/10.7554/eLife.00862.001.

Show MeSH
Related in: MedlinePlus