Limits...
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.

Show MeSH

Related in: MedlinePlus

Subdued is a calcium and voltage-dependent channel.(A) Subdued-transfected HEK 293T cells were used for recording in whole cell patch clamp experiments. No current was observed without calcium in the pipette (left), but large time dependent currents were observed (right) when calcium was added to the pipette (200 µM free calcium). Unless otherwise mentioned, all recordings in this study were done with 200 µM free calcium in the pipette and symmetrical NaCl in the pipette and bath solution. (B) Representative traces showing outward rectification of Subdued current in voltage ramps from −100 to +100 mV. Traces 1–4 were taken sequentially and show an increase in current and decrease in rectification over time (20 µM free calcium in pipette). The ramps were done at a rate (dV/dT) of 0.067 V/s.DOI:http://dx.doi.org/10.7554/eLife.00862.004
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3814000&req=5

fig2: Subdued is a calcium and voltage-dependent channel.(A) Subdued-transfected HEK 293T cells were used for recording in whole cell patch clamp experiments. No current was observed without calcium in the pipette (left), but large time dependent currents were observed (right) when calcium was added to the pipette (200 µM free calcium). Unless otherwise mentioned, all recordings in this study were done with 200 µM free calcium in the pipette and symmetrical NaCl in the pipette and bath solution. (B) Representative traces showing outward rectification of Subdued current in voltage ramps from −100 to +100 mV. Traces 1–4 were taken sequentially and show an increase in current and decrease in rectification over time (20 µM free calcium in pipette). The ramps were done at a rate (dV/dT) of 0.067 V/s.DOI:http://dx.doi.org/10.7554/eLife.00862.004

Mentions: 48 hr post-transfection, HEK 293T cells expressing Subdued were recorded from using whole-cell patch clamp. Upon activation with 20–200 µM free intracellular calcium and symmetric NaCl solutions, the cells showed large time-dependent currents. In zero-calcium pipette solutions, no currents were observed in the transfected cells (Figure 2A). For this study, all recordings were done with 200 µM calcium unless otherwise mentioned. Mock-transfected cells showed little to no CaCC (data not shown). Voltage ramps showed that Subdued was outwardly rectifying, giving rise to more current at depolarized potentials (Figure 2B). In these experiments with calcium infused from the whole-cell patch clamp pipette into the cytosol, rectification decreased over time as current amplitude increased. Run-up of current likely results from the process of calcium diffusing from the pipette solution into the cell, suggesting that rectification is also calcium-dependent, as has been shown for CaCCs (Yang et al., 2008).10.7554/eLife.00862.004Figure 2.Subdued is a calcium and voltage-dependent channel.


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)

Subdued is a calcium and voltage-dependent channel.(A) Subdued-transfected HEK 293T cells were used for recording in whole cell patch clamp experiments. No current was observed without calcium in the pipette (left), but large time dependent currents were observed (right) when calcium was added to the pipette (200 µM free calcium). Unless otherwise mentioned, all recordings in this study were done with 200 µM free calcium in the pipette and symmetrical NaCl in the pipette and bath solution. (B) Representative traces showing outward rectification of Subdued current in voltage ramps from −100 to +100 mV. Traces 1–4 were taken sequentially and show an increase in current and decrease in rectification over time (20 µM free calcium in pipette). The ramps were done at a rate (dV/dT) of 0.067 V/s.DOI:http://dx.doi.org/10.7554/eLife.00862.004
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Subdued is a calcium and voltage-dependent channel.(A) Subdued-transfected HEK 293T cells were used for recording in whole cell patch clamp experiments. No current was observed without calcium in the pipette (left), but large time dependent currents were observed (right) when calcium was added to the pipette (200 µM free calcium). Unless otherwise mentioned, all recordings in this study were done with 200 µM free calcium in the pipette and symmetrical NaCl in the pipette and bath solution. (B) Representative traces showing outward rectification of Subdued current in voltage ramps from −100 to +100 mV. Traces 1–4 were taken sequentially and show an increase in current and decrease in rectification over time (20 µM free calcium in pipette). The ramps were done at a rate (dV/dT) of 0.067 V/s.DOI:http://dx.doi.org/10.7554/eLife.00862.004
Mentions: 48 hr post-transfection, HEK 293T cells expressing Subdued were recorded from using whole-cell patch clamp. Upon activation with 20–200 µM free intracellular calcium and symmetric NaCl solutions, the cells showed large time-dependent currents. In zero-calcium pipette solutions, no currents were observed in the transfected cells (Figure 2A). For this study, all recordings were done with 200 µM calcium unless otherwise mentioned. Mock-transfected cells showed little to no CaCC (data not shown). Voltage ramps showed that Subdued was outwardly rectifying, giving rise to more current at depolarized potentials (Figure 2B). In these experiments with calcium infused from the whole-cell patch clamp pipette into the cytosol, rectification decreased over time as current amplitude increased. Run-up of current likely results from the process of calcium diffusing from the pipette solution into the cell, suggesting that rectification is also calcium-dependent, as has been shown for CaCCs (Yang et al., 2008).10.7554/eLife.00862.004Figure 2.Subdued is a calcium and voltage-dependent channel.

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