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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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Mutations of subdued change properties of observed currents.(A) Different kinetic properties in the wild-type (WT) and Q672K mutant channel revealed by a voltage step protocol (750 ms in 15 mV increments). (B) Semi-log plots of mean activation time constants (τ) as a function of voltage. τ was derived from the single exponential fitting of the current traces obtained from 750 ms (WT, n = 6) and 5 s (Q672K, n = 4) voltage steps. Time constants at 60 and 75 mV were significantly different for the two channels (p<0.001, Student’s t-test). (C) A Y489H mutation decreases chloride selectivity compared to the WT channel. A representative I/V plot showing the shift in Erev in a 20 mM external NaCl solution. WT Erev was determined to be (25 ± 4) mV, n=5; the Y489H mutant Erev was significantly different at (11 ± 2) mV, (n = 4, p<0.05, Student’s t-test). Data were obtained using methodology described in Figure 2B.DOI:http://dx.doi.org/10.7554/eLife.00862.006
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fig4: Mutations of subdued change properties of observed currents.(A) Different kinetic properties in the wild-type (WT) and Q672K mutant channel revealed by a voltage step protocol (750 ms in 15 mV increments). (B) Semi-log plots of mean activation time constants (τ) as a function of voltage. τ was derived from the single exponential fitting of the current traces obtained from 750 ms (WT, n = 6) and 5 s (Q672K, n = 4) voltage steps. Time constants at 60 and 75 mV were significantly different for the two channels (p<0.001, Student’s t-test). (C) A Y489H mutation decreases chloride selectivity compared to the WT channel. A representative I/V plot showing the shift in Erev in a 20 mM external NaCl solution. WT Erev was determined to be (25 ± 4) mV, n=5; the Y489H mutant Erev was significantly different at (11 ± 2) mV, (n = 4, p<0.05, Student’s t-test). Data were obtained using methodology described in Figure 2B.DOI:http://dx.doi.org/10.7554/eLife.00862.006

Mentions: (A) Multiple sequence alignment of protein sequences was done with ClustalW2 and phylogenetic tree construction was done in PHYLIP 3.67 (Drawgram). (B) Putative transmembrane segments are highlighted with boxes, and mutated residues (Figure 4) featured in this report are marked in color on the primary sequence alignment Y489 is shown in green and Q672 is shown in red. Bracketed values at the end of the alignment indicate the number of residues in the whole 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)

Mutations of subdued change properties of observed currents.(A) Different kinetic properties in the wild-type (WT) and Q672K mutant channel revealed by a voltage step protocol (750 ms in 15 mV increments). (B) Semi-log plots of mean activation time constants (τ) as a function of voltage. τ was derived from the single exponential fitting of the current traces obtained from 750 ms (WT, n = 6) and 5 s (Q672K, n = 4) voltage steps. Time constants at 60 and 75 mV were significantly different for the two channels (p<0.001, Student’s t-test). (C) A Y489H mutation decreases chloride selectivity compared to the WT channel. A representative I/V plot showing the shift in Erev in a 20 mM external NaCl solution. WT Erev was determined to be (25 ± 4) mV, n=5; the Y489H mutant Erev was significantly different at (11 ± 2) mV, (n = 4, p<0.05, Student’s t-test). Data were obtained using methodology described in Figure 2B.DOI:http://dx.doi.org/10.7554/eLife.00862.006
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Mutations of subdued change properties of observed currents.(A) Different kinetic properties in the wild-type (WT) and Q672K mutant channel revealed by a voltage step protocol (750 ms in 15 mV increments). (B) Semi-log plots of mean activation time constants (τ) as a function of voltage. τ was derived from the single exponential fitting of the current traces obtained from 750 ms (WT, n = 6) and 5 s (Q672K, n = 4) voltage steps. Time constants at 60 and 75 mV were significantly different for the two channels (p<0.001, Student’s t-test). (C) A Y489H mutation decreases chloride selectivity compared to the WT channel. A representative I/V plot showing the shift in Erev in a 20 mM external NaCl solution. WT Erev was determined to be (25 ± 4) mV, n=5; the Y489H mutant Erev was significantly different at (11 ± 2) mV, (n = 4, p<0.05, Student’s t-test). Data were obtained using methodology described in Figure 2B.DOI:http://dx.doi.org/10.7554/eLife.00862.006
Mentions: (A) Multiple sequence alignment of protein sequences was done with ClustalW2 and phylogenetic tree construction was done in PHYLIP 3.67 (Drawgram). (B) Putative transmembrane segments are highlighted with boxes, and mutated residues (Figure 4) featured in this report are marked in color on the primary sequence alignment Y489 is shown in green and Q672 is shown in red. Bracketed values at the end of the alignment indicate the number of residues in the whole 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