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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: Secondly, our preliminary experiments showed that tissue-specific-Gal4/RNAi did not seem to have an effect. Gal4 drivers for fat body, hemocyte, gut and Malpighian tubules were used. In an attempt to maximize Gal4 activity, the experiment was repeated with different pre-assay incubation temperatures (25 or 29°C) and times (2 or 4 days) for the flies, but the observed trends were qualitatively the same (representative figure shown below: Author response image 2). Multiple enhancer trap lines for expression in different segments of the Malpighian tubules were used but none led to any significantly reduced survival, thus for simplicity a representative Gal4 line (that also drives gut expression) is shown in the figure below. The tubulin-Gal4/RNAi results showed consistent susceptibility relative to either tubulin-Gal4 or RNAi only control, indicating that we might be observing a true phenotype (hence we proceeded to generate a genetic knockout). From tissue-specific RNAi experiments, we surmised that either the gene exerts its protective function in a tissue type we did not anticipate, or that it functions in multiple sets of tissues with redundant functions in host defense, thus knocking it down in only one or two sets of tissues was insufficient to recapitulate the whole body phenotype.


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: Secondly, our preliminary experiments showed that tissue-specific-Gal4/RNAi did not seem to have an effect. Gal4 drivers for fat body, hemocyte, gut and Malpighian tubules were used. In an attempt to maximize Gal4 activity, the experiment was repeated with different pre-assay incubation temperatures (25 or 29°C) and times (2 or 4 days) for the flies, but the observed trends were qualitatively the same (representative figure shown below: Author response image 2). Multiple enhancer trap lines for expression in different segments of the Malpighian tubules were used but none led to any significantly reduced survival, thus for simplicity a representative Gal4 line (that also drives gut expression) is shown in the figure below. The tubulin-Gal4/RNAi results showed consistent susceptibility relative to either tubulin-Gal4 or RNAi only control, indicating that we might be observing a true phenotype (hence we proceeded to generate a genetic knockout). From tissue-specific RNAi experiments, we surmised that either the gene exerts its protective function in a tissue type we did not anticipate, or that it functions in multiple sets of tissues with redundant functions in host defense, thus knocking it down in only one or two sets of tissues was insufficient to recapitulate the whole body phenotype.

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