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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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dBest1-F81C–rescued cells respond differently to MTSET+ modification than native S2 cells. Whole cell VRAC currents were established in hyposmotic solutions (I340/E300, Δ40 mosmol kg−1) and were recorded with voltage ramps from −100 to 100 mV. 1 mM MTSET+ was applied to the bath solution (E300) after the volume-sensitive current was fully activated. The bath was then replaced with 5 mM DTT to test if the effect of MTSET+ was reversible. (A and B) Current–voltage relationships in native (A) and dF81C-rescued (B) S2 cells before and after MTSET+ modification. (C and D) Time course of the effect of MTSET+ modification on native (C) and F81C-rescued (D) VRAC currents. These time course data were collected from the same cells shown in A and B.
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fig2: dBest1-F81C–rescued cells respond differently to MTSET+ modification than native S2 cells. Whole cell VRAC currents were established in hyposmotic solutions (I340/E300, Δ40 mosmol kg−1) and were recorded with voltage ramps from −100 to 100 mV. 1 mM MTSET+ was applied to the bath solution (E300) after the volume-sensitive current was fully activated. The bath was then replaced with 5 mM DTT to test if the effect of MTSET+ was reversible. (A and B) Current–voltage relationships in native (A) and dF81C-rescued (B) S2 cells before and after MTSET+ modification. (C and D) Time course of the effect of MTSET+ modification on native (C) and F81C-rescued (D) VRAC currents. These time course data were collected from the same cells shown in A and B.

Mentions: Additional evidence that dBest1 forms the VRAC pore was provided by the finding that MTSET+ had opposite effects on the native VRAC current and the rescued dBest1-F81C current. I-V curves and the time course of current development are shown in Fig. 2. MTSET+ caused a mean ∼35% reduction in the amplitude of the native dBest1 VRAC currents (Fig. 3, A and B) over 8–10 min. The effect of MTSET+ on native cells was not reversible by 5 mM DTT. In contrast, MTSET+ caused a dramatic augmentation in the F81C current. On average, the current was transiently increased 15-fold, followed by a gradual decline to a level that was still elevated approximately four- to sevenfold compared with the F81C currents before MTSET+ (Fig. 3, A and B). The stimulation by MTSET+ was at least partly reversible by DTT. In addition to stimulating the current, MTSET+ converted the F81C current from slightly inwardly rectifying to slightly outwardly rectifying and shifted Erev 1.9 ± 0.7 mV in the positive direction (Fig. 3 C). Although this shift was small, it was in the opposite direction to the shift produced by MTSES− (see below).


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

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

dBest1-F81C–rescued cells respond differently to MTSET+ modification than native S2 cells. Whole cell VRAC currents were established in hyposmotic solutions (I340/E300, Δ40 mosmol kg−1) and were recorded with voltage ramps from −100 to 100 mV. 1 mM MTSET+ was applied to the bath solution (E300) after the volume-sensitive current was fully activated. The bath was then replaced with 5 mM DTT to test if the effect of MTSET+ was reversible. (A and B) Current–voltage relationships in native (A) and dF81C-rescued (B) S2 cells before and after MTSET+ modification. (C and D) Time course of the effect of MTSET+ modification on native (C) and F81C-rescued (D) VRAC currents. These time course data were collected from the same cells shown in A and B.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: dBest1-F81C–rescued cells respond differently to MTSET+ modification than native S2 cells. Whole cell VRAC currents were established in hyposmotic solutions (I340/E300, Δ40 mosmol kg−1) and were recorded with voltage ramps from −100 to 100 mV. 1 mM MTSET+ was applied to the bath solution (E300) after the volume-sensitive current was fully activated. The bath was then replaced with 5 mM DTT to test if the effect of MTSET+ was reversible. (A and B) Current–voltage relationships in native (A) and dF81C-rescued (B) S2 cells before and after MTSET+ modification. (C and D) Time course of the effect of MTSET+ modification on native (C) and F81C-rescued (D) VRAC currents. These time course data were collected from the same cells shown in A and B.
Mentions: Additional evidence that dBest1 forms the VRAC pore was provided by the finding that MTSET+ had opposite effects on the native VRAC current and the rescued dBest1-F81C current. I-V curves and the time course of current development are shown in Fig. 2. MTSET+ caused a mean ∼35% reduction in the amplitude of the native dBest1 VRAC currents (Fig. 3, A and B) over 8–10 min. The effect of MTSET+ on native cells was not reversible by 5 mM DTT. In contrast, MTSET+ caused a dramatic augmentation in the F81C current. On average, the current was transiently increased 15-fold, followed by a gradual decline to a level that was still elevated approximately four- to sevenfold compared with the F81C currents before MTSET+ (Fig. 3, A and B). The stimulation by MTSET+ was at least partly reversible by DTT. In addition to stimulating the current, MTSET+ converted the F81C current from slightly inwardly rectifying to slightly outwardly rectifying and shifted Erev 1.9 ± 0.7 mV in the positive direction (Fig. 3 C). Although this shift was small, it was in the opposite direction to the shift produced by MTSES− (see below).

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