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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 Cs+/Cl− permeability in HEK cells transfected with dBest1. Whole cell current was measured in wild-type dBest1 or dBest1-F81C–overexpressed HEK cells. Changes in Erev (ΔErev) as a function of extracellular CsCl concentration were plotted for cells with and without MTSES− treatment. Each data point represents the mean Erev ± SEM of two to seven cells. Gray dashed line was calculated from the GHK equation assuming that the channel is exclusively permeable to Cl− (PCs/PCl = 0). Filled symbols: MTSES−-treated; •, F81C (n = 2–7); ▪, wild-type dBest1 (n = 3–4). Open symbols: without MTSES− treatment; ○, wild-type dBest1 (n = 5); □, F81C (n = 3).
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fig6: Quantification of relative Cs+/Cl− permeability in HEK cells transfected with dBest1. Whole cell current was measured in wild-type dBest1 or dBest1-F81C–overexpressed HEK cells. Changes in Erev (ΔErev) as a function of extracellular CsCl concentration were plotted for cells with and without MTSES− treatment. Each data point represents the mean Erev ± SEM of two to seven cells. Gray dashed line was calculated from the GHK equation assuming that the channel is exclusively permeable to Cl− (PCs/PCl = 0). Filled symbols: MTSES−-treated; •, F81C (n = 2–7); ▪, wild-type dBest1 (n = 3–4). Open symbols: without MTSES− treatment; ○, wild-type dBest1 (n = 5); □, F81C (n = 3).

Mentions: The role of dBest1 in forming the pore of the channel was further tested in HEK cells transfected with either wild-type dBest1 or F81C. The dilution potential experiment was repeated in transfected HEK cells either with or without MTSES− treatment (Fig. 6). Wild-type dBest1 in the presence or absence of MTSES− and F81C dBest1 in the absence of MTSES− exhibited similar PCs/PCl ratios (∼0.1). In contrast, the MTSES−-modified F81C channel exhibited a significantly elevated cation permeability (PCs/PCl = 0.50). Despite the observation that the change in ionic selectivity is smaller in HEK cells than in S2 cells, MTSES− modification still caused a significant shift of ionic selectivity. We do not understand why there is a quantitative difference between HEK cells and S2 cells. It appears that there may be different factors or subunits in these two cell types that contribute to channel selectivity.


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

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

Quantification of relative Cs+/Cl− permeability in HEK cells transfected with dBest1. Whole cell current was measured in wild-type dBest1 or dBest1-F81C–overexpressed HEK cells. Changes in Erev (ΔErev) as a function of extracellular CsCl concentration were plotted for cells with and without MTSES− treatment. Each data point represents the mean Erev ± SEM of two to seven cells. Gray dashed line was calculated from the GHK equation assuming that the channel is exclusively permeable to Cl− (PCs/PCl = 0). Filled symbols: MTSES−-treated; •, F81C (n = 2–7); ▪, wild-type dBest1 (n = 3–4). Open symbols: without MTSES− treatment; ○, wild-type dBest1 (n = 5); □, F81C (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Quantification of relative Cs+/Cl− permeability in HEK cells transfected with dBest1. Whole cell current was measured in wild-type dBest1 or dBest1-F81C–overexpressed HEK cells. Changes in Erev (ΔErev) as a function of extracellular CsCl concentration were plotted for cells with and without MTSES− treatment. Each data point represents the mean Erev ± SEM of two to seven cells. Gray dashed line was calculated from the GHK equation assuming that the channel is exclusively permeable to Cl− (PCs/PCl = 0). Filled symbols: MTSES−-treated; •, F81C (n = 2–7); ▪, wild-type dBest1 (n = 3–4). Open symbols: without MTSES− treatment; ○, wild-type dBest1 (n = 5); □, F81C (n = 3).
Mentions: The role of dBest1 in forming the pore of the channel was further tested in HEK cells transfected with either wild-type dBest1 or F81C. The dilution potential experiment was repeated in transfected HEK cells either with or without MTSES− treatment (Fig. 6). Wild-type dBest1 in the presence or absence of MTSES− and F81C dBest1 in the absence of MTSES− exhibited similar PCs/PCl ratios (∼0.1). In contrast, the MTSES−-modified F81C channel exhibited a significantly elevated cation permeability (PCs/PCl = 0.50). Despite the observation that the change in ionic selectivity is smaller in HEK cells than in S2 cells, MTSES− modification still caused a significant shift of ionic selectivity. We do not understand why there is a quantitative difference between HEK cells and S2 cells. It appears that there may be different factors or subunits in these two cell types that contribute to channel selectivity.

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