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Rescue of volume-regulated anion current by bestrophin mutants with altered charge selectivity.

Chien LT, Hartzell HC - J. Gen. Physiol. (2008)

Bottom Line: The F81E mutant was 1.3 times more permeable to Cs(+) than Cl(-).The finding that VRAC was rescued by F81C and F81E mutants with different biophysical properties shows that bestrophin-1 is a VRAC in S2 cells and not simply a regulator or an auxiliary subunit.F81C overexpressed in HEK293 cells also exhibits a shift of ionic selectivity after MTSES(-) treatment, although the effect is quantitatively smaller than in S2 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, GA 30322, USA.

ABSTRACT
Mutations in human bestrophin-1 are linked to various kinds of retinal degeneration. Although it has been proposed that bestrophins are Ca(2+)-activated Cl(-) channels, definitive proof is lacking partly because mice with the bestrophin-1 gene deleted have normal Ca(2+)-activated Cl(-) currents. Here, we provide compelling evidence to support the idea that bestrophin-1 is the pore-forming subunit of a cell volume-regulated anion channel (VRAC) in Drosophila S2 cells. VRAC was abolished by treatment with RNAi to Drosophila bestrophin-1. VRAC was rescued by overexpressing bestrophin-1 mutants with altered biophysical properties and responsiveness to sulfhydryl reagents. In particular, the ionic selectivity of the F81C mutant changed from anionic to cationic when the channel was treated with the sulfhydryl reagent, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) (P(Cs)/P(Cl) = 0.25 for native and 2.38 for F81C). The F81E mutant was 1.3 times more permeable to Cs(+) than Cl(-). The finding that VRAC was rescued by F81C and F81E mutants with different biophysical properties shows that bestrophin-1 is a VRAC in S2 cells and not simply a regulator or an auxiliary subunit. F81C overexpressed in HEK293 cells also exhibits a shift of ionic selectivity after MTSES(-) treatment, although the effect is quantitatively smaller than in S2 cells. To test whether bestrophins are VRACs in mammalian cells, we compared VRACs in peritoneal macrophages from wild-type mice and mice with both bestrophin-1 and bestrophin-2 disrupted (best1(-/-)/best2(-/-)). VRACs were identical in wild-type and best1(-/-)/best2(-/-) mice, showing that bestrophins are unlikely to be the classical VRAC in mammalian cells.

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Quantification of relative cation/chloride permeability. Current–voltage relationships of MTSES−-modified native (A) and F81C (B) currents and unmodified F81E currents (C) in response to different external [CsCl]. Whole cell currents were activated under isosmotic condition (304 mosmol kg−1) with high Ca2+ in the pipet and symmetrical CsCl in the bath and pipet (150 mM). The extracellular solution was replaced by solutions containing different [CsCl] as indicated. (D) Changes in Erev (ΔErev) as a function of extracellular salt concentration. ΔErev is Erev at the indicated salt concentration minus the Erev with 150 mM extracellular salt. Salt is either CsCl or NaCl as indicated. Each data point represents the mean Erev ± SEM of two to nine cells. Dashed lines were calculated from the GHK equation (ΔErev = 25.7 · ln [([X+]o + [Cl−]i · PCl/PX) / ([X+]i + [Cl−]o · PCl/PX)]), assuming that the channel is exclusively permeable to Cl− (PX/PCl = 0) or to the cation X+ (PCl/PX = 0). Filled symbols: CsCl solutions; •, MTSES−-treated native dBest1 (n = 2–7); ▴, MTSES−-modified F81C (n = 4–9); ▪, F81E (n = 2–5). Open symbols: NaCl solutions; ○, MTSES−-treated native dBest1 (n = 3–5); ▵, F81C (n = 3–8).
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fig5: Quantification of relative cation/chloride permeability. Current–voltage relationships of MTSES−-modified native (A) and F81C (B) currents and unmodified F81E currents (C) in response to different external [CsCl]. Whole cell currents were activated under isosmotic condition (304 mosmol kg−1) with high Ca2+ in the pipet and symmetrical CsCl in the bath and pipet (150 mM). The extracellular solution was replaced by solutions containing different [CsCl] as indicated. (D) Changes in Erev (ΔErev) as a function of extracellular salt concentration. ΔErev is Erev at the indicated salt concentration minus the Erev with 150 mM extracellular salt. Salt is either CsCl or NaCl as indicated. Each data point represents the mean Erev ± SEM of two to nine cells. Dashed lines were calculated from the GHK equation (ΔErev = 25.7 · ln [([X+]o + [Cl−]i · PCl/PX) / ([X+]i + [Cl−]o · PCl/PX)]), assuming that the channel is exclusively permeable to Cl− (PX/PCl = 0) or to the cation X+ (PCl/PX = 0). Filled symbols: CsCl solutions; •, MTSES−-treated native dBest1 (n = 2–7); ▴, MTSES−-modified F81C (n = 4–9); ▪, F81E (n = 2–5). Open symbols: NaCl solutions; ○, MTSES−-treated native dBest1 (n = 3–5); ▵, F81C (n = 3–8).

Mentions: To quantify relative cation+/Cl− permeability, we performed dilution potential experiments (Franciolini and Nonner, 1987) for native dBest1, F81C, and F81E currents. For these experiments, we chose to activate the current by high intracellular Ca2+ because the dBest1 current activated by cell swelling often ran down after the current was fully activated, making several Erev determinations in the same cell problematic. In contrast, the Ca2+-activated dBest1 current does not run down significantly for up to 10 min. The nature of this rundown and why it is only seen when dBest1 current is activated by hyposmotic cell swelling but not by Ca2+ are not clear. Nevertheless, we chose to activate dBest1 current by intracellular application of Ca2+ to obtain data to avoid the possible interference of the rundown. Cells were treated with MTSES−, and Erev was measured with 150 mM CsCl inside and various concentrations of CsCl outside. I-V curves from typical cells recorded with 10, 50, and 150 mM external CsCl are superimposed in Fig. 5 (A–C). For native dBest1 currents (Fig. 5 A), Erev moved toward positive values as external [CsCl] was decreased, as would be predicted if dBest1 is selectively permeable to Cl−. In addition, the conductance in the outward direction at +100 mV increased significantly with increasing external [CsCl], consistent with Cl− carrying the majority of the outward current. In contrast, the Erev of F81C shifted toward negative potentials with decreasing external [CsCl] (Fig. 5 B). This negative shift in Erev showed that F81C had become more selective to Cs+ than to Cl− after MTSES− modification. The augmented inward rectification with increasing external [CsCl] was also consistent with a higher Cs+ conductance relative to Cl− after MTSES− treatment.


Rescue of volume-regulated anion current by bestrophin mutants with altered charge selectivity.

Chien LT, Hartzell HC - J. Gen. Physiol. (2008)

Quantification of relative cation/chloride permeability. Current–voltage relationships of MTSES−-modified native (A) and F81C (B) currents and unmodified F81E currents (C) in response to different external [CsCl]. Whole cell currents were activated under isosmotic condition (304 mosmol kg−1) with high Ca2+ in the pipet and symmetrical CsCl in the bath and pipet (150 mM). The extracellular solution was replaced by solutions containing different [CsCl] as indicated. (D) Changes in Erev (ΔErev) as a function of extracellular salt concentration. ΔErev is Erev at the indicated salt concentration minus the Erev with 150 mM extracellular salt. Salt is either CsCl or NaCl as indicated. Each data point represents the mean Erev ± SEM of two to nine cells. Dashed lines were calculated from the GHK equation (ΔErev = 25.7 · ln [([X+]o + [Cl−]i · PCl/PX) / ([X+]i + [Cl−]o · PCl/PX)]), assuming that the channel is exclusively permeable to Cl− (PX/PCl = 0) or to the cation X+ (PCl/PX = 0). Filled symbols: CsCl solutions; •, MTSES−-treated native dBest1 (n = 2–7); ▴, MTSES−-modified F81C (n = 4–9); ▪, F81E (n = 2–5). Open symbols: NaCl solutions; ○, MTSES−-treated native dBest1 (n = 3–5); ▵, F81C (n = 3–8).
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2571971&req=5

fig5: Quantification of relative cation/chloride permeability. Current–voltage relationships of MTSES−-modified native (A) and F81C (B) currents and unmodified F81E currents (C) in response to different external [CsCl]. Whole cell currents were activated under isosmotic condition (304 mosmol kg−1) with high Ca2+ in the pipet and symmetrical CsCl in the bath and pipet (150 mM). The extracellular solution was replaced by solutions containing different [CsCl] as indicated. (D) Changes in Erev (ΔErev) as a function of extracellular salt concentration. ΔErev is Erev at the indicated salt concentration minus the Erev with 150 mM extracellular salt. Salt is either CsCl or NaCl as indicated. Each data point represents the mean Erev ± SEM of two to nine cells. Dashed lines were calculated from the GHK equation (ΔErev = 25.7 · ln [([X+]o + [Cl−]i · PCl/PX) / ([X+]i + [Cl−]o · PCl/PX)]), assuming that the channel is exclusively permeable to Cl− (PX/PCl = 0) or to the cation X+ (PCl/PX = 0). Filled symbols: CsCl solutions; •, MTSES−-treated native dBest1 (n = 2–7); ▴, MTSES−-modified F81C (n = 4–9); ▪, F81E (n = 2–5). Open symbols: NaCl solutions; ○, MTSES−-treated native dBest1 (n = 3–5); ▵, F81C (n = 3–8).
Mentions: To quantify relative cation+/Cl− permeability, we performed dilution potential experiments (Franciolini and Nonner, 1987) for native dBest1, F81C, and F81E currents. For these experiments, we chose to activate the current by high intracellular Ca2+ because the dBest1 current activated by cell swelling often ran down after the current was fully activated, making several Erev determinations in the same cell problematic. In contrast, the Ca2+-activated dBest1 current does not run down significantly for up to 10 min. The nature of this rundown and why it is only seen when dBest1 current is activated by hyposmotic cell swelling but not by Ca2+ are not clear. Nevertheless, we chose to activate dBest1 current by intracellular application of Ca2+ to obtain data to avoid the possible interference of the rundown. Cells were treated with MTSES−, and Erev was measured with 150 mM CsCl inside and various concentrations of CsCl outside. I-V curves from typical cells recorded with 10, 50, and 150 mM external CsCl are superimposed in Fig. 5 (A–C). For native dBest1 currents (Fig. 5 A), Erev moved toward positive values as external [CsCl] was decreased, as would be predicted if dBest1 is selectively permeable to Cl−. In addition, the conductance in the outward direction at +100 mV increased significantly with increasing external [CsCl], consistent with Cl− carrying the majority of the outward current. In contrast, the Erev of F81C shifted toward negative potentials with decreasing external [CsCl] (Fig. 5 B). This negative shift in Erev showed that F81C had become more selective to Cs+ than to Cl− after MTSES− modification. The augmented inward rectification with increasing external [CsCl] was also consistent with a higher Cs+ conductance relative to Cl− after MTSES− treatment.

Bottom Line: The F81E mutant was 1.3 times more permeable to Cs(+) than Cl(-).The finding that VRAC was rescued by F81C and F81E mutants with different biophysical properties shows that bestrophin-1 is a VRAC in S2 cells and not simply a regulator or an auxiliary subunit.F81C overexpressed in HEK293 cells also exhibits a shift of ionic selectivity after MTSES(-) treatment, although the effect is quantitatively smaller than in S2 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, GA 30322, USA.

ABSTRACT
Mutations in human bestrophin-1 are linked to various kinds of retinal degeneration. Although it has been proposed that bestrophins are Ca(2+)-activated Cl(-) channels, definitive proof is lacking partly because mice with the bestrophin-1 gene deleted have normal Ca(2+)-activated Cl(-) currents. Here, we provide compelling evidence to support the idea that bestrophin-1 is the pore-forming subunit of a cell volume-regulated anion channel (VRAC) in Drosophila S2 cells. VRAC was abolished by treatment with RNAi to Drosophila bestrophin-1. VRAC was rescued by overexpressing bestrophin-1 mutants with altered biophysical properties and responsiveness to sulfhydryl reagents. In particular, the ionic selectivity of the F81C mutant changed from anionic to cationic when the channel was treated with the sulfhydryl reagent, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) (P(Cs)/P(Cl) = 0.25 for native and 2.38 for F81C). The F81E mutant was 1.3 times more permeable to Cs(+) than Cl(-). The finding that VRAC was rescued by F81C and F81E mutants with different biophysical properties shows that bestrophin-1 is a VRAC in S2 cells and not simply a regulator or an auxiliary subunit. F81C overexpressed in HEK293 cells also exhibits a shift of ionic selectivity after MTSES(-) treatment, although the effect is quantitatively smaller than in S2 cells. To test whether bestrophins are VRACs in mammalian cells, we compared VRACs in peritoneal macrophages from wild-type mice and mice with both bestrophin-1 and bestrophin-2 disrupted (best1(-/-)/best2(-/-)). VRACs were identical in wild-type and best1(-/-)/best2(-/-) mice, showing that bestrophins are unlikely to be the classical VRAC in mammalian cells.

Show MeSH
Related in: MedlinePlus