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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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Knockout (KO) flies do not display significant lethality relative to wild-type (WT) flies upon ingestion of UV-killed Db11.Db11/sucrose solution was irradiated with UV for 15 min prior to administration to the flies. While non-irradiated Db11 caused high mortality in KO strains 96 hr post-infection, UV-irradiated Db11 did not bring about noticeable lethality in all three strains at this time point. Increased lethality at later time points could arise from general reduced fitness of the KO strains. UV-irradiated bacterial feeding was done in a separate Db11-free incubator (n = 3).DOI:http://dx.doi.org/10.7554/eLife.00862.008
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fig5s1: Knockout (KO) flies do not display significant lethality relative to wild-type (WT) flies upon ingestion of UV-killed Db11.Db11/sucrose solution was irradiated with UV for 15 min prior to administration to the flies. While non-irradiated Db11 caused high mortality in KO strains 96 hr post-infection, UV-irradiated Db11 did not bring about noticeable lethality in all three strains at this time point. Increased lethality at later time points could arise from general reduced fitness of the KO strains. UV-irradiated bacterial feeding was done in a separate Db11-free incubator (n = 3).DOI:http://dx.doi.org/10.7554/eLife.00862.008

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)

Knockout (KO) flies do not display significant lethality relative to wild-type (WT) flies upon ingestion of UV-killed Db11.Db11/sucrose solution was irradiated with UV for 15 min prior to administration to the flies. While non-irradiated Db11 caused high mortality in KO strains 96 hr post-infection, UV-irradiated Db11 did not bring about noticeable lethality in all three strains at this time point. Increased lethality at later time points could arise from general reduced fitness of the KO strains. UV-irradiated bacterial feeding was done in a separate Db11-free incubator (n = 3).DOI:http://dx.doi.org/10.7554/eLife.00862.008
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5s1: Knockout (KO) flies do not display significant lethality relative to wild-type (WT) flies upon ingestion of UV-killed Db11.Db11/sucrose solution was irradiated with UV for 15 min prior to administration to the flies. While non-irradiated Db11 caused high mortality in KO strains 96 hr post-infection, UV-irradiated Db11 did not bring about noticeable lethality in all three strains at this time point. Increased lethality at later time points could arise from general reduced fitness of the KO strains. UV-irradiated bacterial feeding was done in a separate Db11-free incubator (n = 3).DOI:http://dx.doi.org/10.7554/eLife.00862.008
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