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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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Mentions: The suggested DUOX induction experiment was performed as described in the above mentioned paper. Dissecting about 20 guts per experiment (n=3), we observed a statistically non-significant trend (p=0.07, unpaired t-test) of reduced DUOX induction in the knockout flies. A feeding assay showed a slight trend towards higher feeding for the knockout flies (5 guts, n=3) (see Author response image 1 below). Thus, even though the knockout flies may have consumed a greater amount of uracil, they appear to display less DUOX induction. However in our hands, high background fluorescence was observed from flies fed with only uracil and not the R19S dye. This occurred despite feeding the flies sucrose solution for a few days after eclosion and pre-starving them for 18 hr on water to reduce auto-fluorescence from normal food. Normalizing to this background, we were only able to observe less than half the number of ROS-positive guts as compared to what has been previously reported for the wildtype control (70%). This technical problem has rendered it difficult to ascertain a statistically significant reduction of DUOX induction by uracil in knockout flies. By retaining a discussion of the hypothesis of a requirement for Subdued in DUOX function, we hope to stimulate future studies that may yield more definitive assessments of this possibility.


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)

© Copyright Policy - open-access
Related In: Results  -  Collection

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

Mentions: The suggested DUOX induction experiment was performed as described in the above mentioned paper. Dissecting about 20 guts per experiment (n=3), we observed a statistically non-significant trend (p=0.07, unpaired t-test) of reduced DUOX induction in the knockout flies. A feeding assay showed a slight trend towards higher feeding for the knockout flies (5 guts, n=3) (see Author response image 1 below). Thus, even though the knockout flies may have consumed a greater amount of uracil, they appear to display less DUOX induction. However in our hands, high background fluorescence was observed from flies fed with only uracil and not the R19S dye. This occurred despite feeding the flies sucrose solution for a few days after eclosion and pre-starving them for 18 hr on water to reduce auto-fluorescence from normal food. Normalizing to this background, we were only able to observe less than half the number of ROS-positive guts as compared to what has been previously reported for the wildtype control (70%). This technical problem has rendered it difficult to ascertain a statistically significant reduction of DUOX induction by uracil in knockout flies. By retaining a discussion of the hypothesis of a requirement for Subdued in DUOX function, we hope to stimulate future studies that may yield more definitive assessments of this possibility.

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