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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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Subdued displays hallmark ionic selectivity of classic CaCCs and is blocked by a known CaCC inhibitor.(A) NaCl gradients were introduced across the membrane by varying NaCl concentrations of external solutions. The reversal potential (Erev) at each concentration was obtained and fitted to the Goldman-Hodgkin-Katz (GHK) equation from which the PNa/PCl was determined to be 0.16. (B) Representative I/V plots obtained by varying external NaCl (in mM). A diagram of the voltage protocol used to measure Erev is shown below. After a 750 ms activating pre-pulse to +100 mV, instantaneous tail currents were measured from test voltages −100 to +100 mV in 20 mV steps. After an initial estimate of Erev using this protocol, test potentials and voltage increments were refined, while pre-pulse conditions and the length of voltage steps remained constant. For each NaCl concentration, n = 4 to n = 9. (C) Subdued preferentially permeates larger anions with the selectivity sequence: SCN > I > Br > Cl. Bi-ionic conditions were introduced by varying the external solution. Erev and permeability ratios were obtained by the same methodology as described for (B). Representative I/V plots obtained by varying the anion in external solutions using the same voltage protocols as described in (B) are shown. (D) Subdued is significantly and reversibly blocked by 20 µM benzbromarone (n = 4, p<0.05, Student’s t-test).DOI:http://dx.doi.org/10.7554/eLife.00862.005
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fig3: Subdued displays hallmark ionic selectivity of classic CaCCs and is blocked by a known CaCC inhibitor.(A) NaCl gradients were introduced across the membrane by varying NaCl concentrations of external solutions. The reversal potential (Erev) at each concentration was obtained and fitted to the Goldman-Hodgkin-Katz (GHK) equation from which the PNa/PCl was determined to be 0.16. (B) Representative I/V plots obtained by varying external NaCl (in mM). A diagram of the voltage protocol used to measure Erev is shown below. After a 750 ms activating pre-pulse to +100 mV, instantaneous tail currents were measured from test voltages −100 to +100 mV in 20 mV steps. After an initial estimate of Erev using this protocol, test potentials and voltage increments were refined, while pre-pulse conditions and the length of voltage steps remained constant. For each NaCl concentration, n = 4 to n = 9. (C) Subdued preferentially permeates larger anions with the selectivity sequence: SCN > I > Br > Cl. Bi-ionic conditions were introduced by varying the external solution. Erev and permeability ratios were obtained by the same methodology as described for (B). Representative I/V plots obtained by varying the anion in external solutions using the same voltage protocols as described in (B) are shown. (D) Subdued is significantly and reversibly blocked by 20 µM benzbromarone (n = 4, p<0.05, Student’s t-test).DOI:http://dx.doi.org/10.7554/eLife.00862.005

Mentions: Given that the mammalian TMEM16 family contains both anion and cation channels (Yang et al., 2012), we wanted to determine the ionic selectivity of the channel. This was done by varying concentrations of NaCl externally while keeping the intracellular NaCl concentration constant at 140 mM. A series of positive reversal potentials (Erev) was obtained upon decreasing the external NaCl concentration, indicating anionic selectivity (Figure 3A,B). Using the Goldman-Hodgkin-Katz equation (Figure 3A inset) , which describes experimentally obtained reversal potentials as a function of ion concentrations and their respective permeabilities (Px, where X is any ion in the system), PNa/PCl was calculated to be 0.16, indicating a small permeability for cations, as has been found for mammalian TMEM16A and B (Pifferi et al., 2009; Yang et al., 2012). Substitution of chloride with larger halide anions in the external solution revealed that Subdued preferentially permeates the larger anions SCN−, I− and Br− relative to Cl− (Figure 3C). This hallmark feature of CaCCs implicates hydration energy as a factor in anionic selectivity, a feature shared with cystic fibrosis transmembrane conductance regulator (CFTR) channels (Qu and Hartzell, 2000). However, classic CaCC blockers niflumic acid (NFA), flufenamic acid (FFA) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) as well as a more recently developed TMEM16A inhibitor T16Ainh-A01 (Namkung et al., 2011) did not block the channel (data not shown). Benzbromarone, a TMEM16A blocker identified from a high throughput screen (Huang et al., 2012a), blocked Subdued current significantly and reversibly (Figure 3D).10.7554/eLife.00862.005Figure 3.Subdued displays hallmark ionic selectivity of classic CaCCs and is blocked by a known CaCC inhibitor.


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 displays hallmark ionic selectivity of classic CaCCs and is blocked by a known CaCC inhibitor.(A) NaCl gradients were introduced across the membrane by varying NaCl concentrations of external solutions. The reversal potential (Erev) at each concentration was obtained and fitted to the Goldman-Hodgkin-Katz (GHK) equation from which the PNa/PCl was determined to be 0.16. (B) Representative I/V plots obtained by varying external NaCl (in mM). A diagram of the voltage protocol used to measure Erev is shown below. After a 750 ms activating pre-pulse to +100 mV, instantaneous tail currents were measured from test voltages −100 to +100 mV in 20 mV steps. After an initial estimate of Erev using this protocol, test potentials and voltage increments were refined, while pre-pulse conditions and the length of voltage steps remained constant. For each NaCl concentration, n = 4 to n = 9. (C) Subdued preferentially permeates larger anions with the selectivity sequence: SCN > I > Br > Cl. Bi-ionic conditions were introduced by varying the external solution. Erev and permeability ratios were obtained by the same methodology as described for (B). Representative I/V plots obtained by varying the anion in external solutions using the same voltage protocols as described in (B) are shown. (D) Subdued is significantly and reversibly blocked by 20 µM benzbromarone (n = 4, p<0.05, Student’s t-test).DOI:http://dx.doi.org/10.7554/eLife.00862.005
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fig3: Subdued displays hallmark ionic selectivity of classic CaCCs and is blocked by a known CaCC inhibitor.(A) NaCl gradients were introduced across the membrane by varying NaCl concentrations of external solutions. The reversal potential (Erev) at each concentration was obtained and fitted to the Goldman-Hodgkin-Katz (GHK) equation from which the PNa/PCl was determined to be 0.16. (B) Representative I/V plots obtained by varying external NaCl (in mM). A diagram of the voltage protocol used to measure Erev is shown below. After a 750 ms activating pre-pulse to +100 mV, instantaneous tail currents were measured from test voltages −100 to +100 mV in 20 mV steps. After an initial estimate of Erev using this protocol, test potentials and voltage increments were refined, while pre-pulse conditions and the length of voltage steps remained constant. For each NaCl concentration, n = 4 to n = 9. (C) Subdued preferentially permeates larger anions with the selectivity sequence: SCN > I > Br > Cl. Bi-ionic conditions were introduced by varying the external solution. Erev and permeability ratios were obtained by the same methodology as described for (B). Representative I/V plots obtained by varying the anion in external solutions using the same voltage protocols as described in (B) are shown. (D) Subdued is significantly and reversibly blocked by 20 µM benzbromarone (n = 4, p<0.05, Student’s t-test).DOI:http://dx.doi.org/10.7554/eLife.00862.005
Mentions: Given that the mammalian TMEM16 family contains both anion and cation channels (Yang et al., 2012), we wanted to determine the ionic selectivity of the channel. This was done by varying concentrations of NaCl externally while keeping the intracellular NaCl concentration constant at 140 mM. A series of positive reversal potentials (Erev) was obtained upon decreasing the external NaCl concentration, indicating anionic selectivity (Figure 3A,B). Using the Goldman-Hodgkin-Katz equation (Figure 3A inset) , which describes experimentally obtained reversal potentials as a function of ion concentrations and their respective permeabilities (Px, where X is any ion in the system), PNa/PCl was calculated to be 0.16, indicating a small permeability for cations, as has been found for mammalian TMEM16A and B (Pifferi et al., 2009; Yang et al., 2012). Substitution of chloride with larger halide anions in the external solution revealed that Subdued preferentially permeates the larger anions SCN−, I− and Br− relative to Cl− (Figure 3C). This hallmark feature of CaCCs implicates hydration energy as a factor in anionic selectivity, a feature shared with cystic fibrosis transmembrane conductance regulator (CFTR) channels (Qu and Hartzell, 2000). However, classic CaCC blockers niflumic acid (NFA), flufenamic acid (FFA) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) as well as a more recently developed TMEM16A inhibitor T16Ainh-A01 (Namkung et al., 2011) did not block the channel (data not shown). Benzbromarone, a TMEM16A blocker identified from a high throughput screen (Huang et al., 2012a), blocked Subdued current significantly and reversibly (Figure 3D).10.7554/eLife.00862.005Figure 3.Subdued displays hallmark ionic selectivity of classic CaCCs and is blocked by a known CaCC inhibitor.

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